SCH 2358 Sample preparation Methods

Course objectives

- Discuss the importance of sample preparation

- Describe the principles of the various sample preparation methods

Course description

Importance of sample preparation in analytical chemistry. Sample handling and preservation.

Principles of extraction. Liquid-liquid extraction (LLE), Soxhlet extraction, Headspace
extraction, Solid-phase extraction (SPE), Supercritical fluid extraction (SFE). Acid digestion
for metal analysis, Microwave-assisted acid digestion, Hydride generation techniques.
Membrane based sample handling techniques, flow injection analysis. Recent advances in
sample preparation techniques.

Learning outcomes;

At the end of this course, the learner should be able to;

- Demonstrate competence in the use of various sample preparation methods

- Describe the various parameters to be optimized in a sample preparation method
- Explain the advantages and disadvantages of various methods

Teaching Methodology:

Lectures, Tutorials and practicals

Instructional Materials

Whiteboard/ smart board, white board markers, computer and projector, course notes, Duster

1
Course assessment

Written CATS 15%, assignments 5%, Practicals 10%, final written examination 70%

Course Journals

1. Journal of Analytical Sciences, Methods and Instrumentation Published/Hosted

by Scientific Research Publishing. ISSN (printed): 2164-2745. ISSN (electronic): 2164-
2753
2. International Journal of Analytical Chemistry ISSN: 1687-8760
3. Journal of Chromatography A, ISSN: 0021-9673

Reference Journals

1. International Journal of Analytical Chemistry ISSN: 1687-8760

2. Talanta Imprint: ELSEVIER ISSN: 0039-9140
3. The Annual Review of Analytical Chemistry ISSN: 19361335, 19361327
4. Trends in Analytical Chemistry ISSN: 0165-9936

REFERENCES

1. Quantitative Chemical Analysis, Daniel C. Harris, W. H Freeman and company, 5 th

Edition, 2007. ISBN-10: 0-7167-7041-5, ISBN-13: 978-0-7167-7041-1

2. Analytical Chemistry, Garey D. Christian, John Wiley and Sons, 6 th edition, 2004. ISBN
978-0-471-21472-4

3. Instrumental Methods of analysis, Douglas A. Skoog, ISBN-13: 978-0495558286

4. Modern Analytical Chemistry, D. Harvey, McGraw-Hill Publishers, 2000, ISBN; 0-07-

237547-7.

2
Sample preparation (analytical chemistry)
From Wikipedia, the free encyclopedia

In analytical chemistry, sample preparation refers to the ways in which a sample is treated
prior to its analysis. Preparation is a very important step in most analytical techniques,
because the techniques are often not responsive to the analyte in its in-situ form, or the results
are distorted by interfering species. Sample preparation may involve dissolution, extraction,
reaction with some chemical species, pulverizing, treatment with a chelating agent (e.g.
EDTA), masking, filtering, dilution, sub-sampling or many other techniques.

References
 "Sample Preparation". Retrieved 2007-11-09.

Encyclopædia Britannica
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Sample preparation
chemistry
Written By:
Thomas R. Dulski
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Sample preparation, in analytical chemistry, the processes in which a representative piece
of material is extracted from a larger amount and readied for analysis. Sampling and sample
preparation have a unique meaning and special importance when applied to the field of
analytical chemistry. Analytical chemistry in all its diverse forms can be looked upon as a
multistep endeavour with the measurement phase but one link near the end of a chain of
operations. That chain begins with sampling, an essential process that underlies all
subsequent work and imparts relevance to what would otherwise be a meaningless exercise.

Viking 1’s sample scoop, poised to extract material from the surface of Mars.

NASA

Sampling is critically relevant everywhere that analytical chemistry has a role to play.
Ambient sampling of the atmosphere is used to provide analytical data on seasonal or other
trends that can be correlated with natural or societal processes. For example, the extent of the
Antarctic ozone hole and its relation to fluorocarbon use were confirmed by this means. Near
ground level, monitoring sites provide data for air-quality assessment, for the design of
pollution-control strategies, and for regulatory enforcement. Groundwater-monitoring wells
are used to sample aquifers in order to ensure water quality. Rivers and streams are sampled
to track pollution from industry, agriculture, sewers, and cities. The ocean is sampled to study
the carbon cycle budget for Earth, and seafloor hydrothermal vents are sampled to obtain
clues about geochemistry deep in Earth’s crust.

5
 

Antarctic ozone hole, September 17, 2001.

NASA/Goddard Space Flight Center

Analytical chemistry that studies other worlds follows upon careful sampling. The
Apolloastronauts who explored the Moon were trained in geological sampling. Various
robotic probes have sampled Mars and Halley’s Comet for automated onboard analyses. The
European Space Agency’s Huygens probe sampled the atmosphere and surface of Saturn’s
moon Titan in 2005.

6
 

Artwork depicting the Cassini orbiter, centre, after having released the Huygens
probe, left, …

NASA/JPL

Back on Earth, manufactured products are sampled to ensure consumer safety; foods are
sampled to assay nutrients and to monitor pesticide residues and other potentially harmful
contaminants. Sampling methods are also used in connection with forensic analyses,
chemical analyses in customs work, and industrial processes.

Following close upon sampling is sample preparation, the entire process whereby the sample
is readied for measurement. The sample that arrives at the laboratory is commonly called the
laboratory sample. This is then converted by a set of operations to the test sample, from
which an analyst selects a test portion for an analytical determination. If the test portion is a
particulate solid, it may be necessary to convert it to a solution. If the analyte (i.e., the species
being determined) is present at low concentration, or if interfering substances are present, it
may be necessary to isolate or concentrate the analyte by one or more separation and
purification steps. In some cases additives are required to mask interference, or the analyte
must be chemically converted to another form to facilitate its measurement.

7
Sampling
Theory
The sampling plan is the strategy employed to represent the distribution of one or multiple
analytes in the object of study. The object of study may encompass objects with only spatial
dimensions, such as a mineral deposit, or it may be a dynamically changing system, such as a
river, which has a temporal component. In both cases the success of the sampling plan
depends upon how accurately a much larger system is represented in the microcosm of the
laboratory sample.

Materials vary widely in the degree of large- and small-scale uniformity that they exhibit. It is
most useful to speak of the heterogeneity of a material as a scalar function that approaches
perfect homogeneity in its limit. It is also essential to speak in terms of a given analyte or
suite of analytes, since some components in a material may be much more heterogeneously
distributed than others.

The most comprehensive sampling theory was formulated by French chemist Pierre Gy in the
second half of the 20th century. Gy defined two types of material heterogeneity: constitution
heterogeneity, which is the intrinsic heterogeneity of the material’s components, and
distribution heterogeneity, which is the heterogeneity that derives from the spatial mixing of
the components. While this dichotomy can be usefully applied to many material types, it is
best described and understood in reference to particulate solid mixtures. For example, if one
considers a mixture of silt and sand to be sampled for the presence of calcium, the variation
of that analyte among the silt and sand particles represents two forms of its constitution
heterogeneity. The degree of uniformity in the spatial arrangement of silt and sand particles
then determines the distribution heterogeneity of calcium. Appropriate grinding of such a
mixture to reduce the average particle size may diminish the constitution heterogeneity, and
the correct blending of such a mixture may lower its distribution heterogeneity.

Test Your Knowledge

Metals: Fact or Fiction?

Gy developed another concept that involves the likelihood that all a material’s constituents
have a high and equal probability of being included in the sample. Many commonly
employed sampling practices are seriously flawed in that some constituents have a zero
probability of being sampled. “Grab sampling,” in which one movement of a sampling device
is used to select the sample, most often falls into this category, which is called
nonprobabilistic sampling. Such methods can never satisfactorily represent highly
8
heterogeneous material. In contrast, probabilistic sampling methods are techniques in which
all constituents of the material have some probability of being included. However, it is only
in a correctly designed sampling plan that probabilistic sampling achieves true representation.

Sampling solids, liquids, and gases

In a discussion of sampling it is useful to distinguish two forms of solids, monolithic and
particulate, as well as liquids and gases and to treat each material type as a separate category.
At the same time, it is important to recognize that mixed phases also frequently need to be
sampled; gases dissolved in liquids and solids, particles suspended in liquids, and solid and
liquid aerosols are some examples. Sometimes the object of study is in one phase form, but
the sample must be in another. Thus, molten steel is sampled by casting solid forms for
analysis.

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Monolithic solids, even those with a very low order of heterogeneity, are very difficult to
sample rationally. However, as with all sampling, understanding the physical nature of the
object of study can significantly improve the sampling plan. For example, a large ore body
may extend for great distances underground in three dimensions, but mineralogical clues can
direct sampling for the mapping effort. Steel castings are commonly sampled at their cross-
sectional mid-radius, where they are known to be free of edge effects and centre porosity.

Sampling of particulate solids provides the model for much sampling theory. In general,
particulate system heterogeneity tends to be much greater than that of other phase systems.
Thus, the single-grab sample is nearly always inadequate. For this reason the sampling of
contaminated soil, for instance, may employ random, systematic, or judgment-based
sampling plans in order to achieve a given set of objectives (e.g., mapping concentration
gradients and locating “hot spots”). In industry a particulate commodity may be either
continuously or randomly sampled as it is being transported on a conveyer belt.

Very heterogeneous materials may need to be sampled in great bulk, amounting to 1 percent
or more of the total. The resulting sample then needs to be reduced in size by some means
that preserves its representative character. “Coning and quartering” is one approach. The
original sample is formed into a cone-shaped pile and then flattened into a disk. The disk is
divided into four quadrants. Two opposite quadrants are shoveled into a second pile, mixed
together, and then coned and quartered again. This sequence continues until the selected
material has been reduced to a size small enough for a useful laboratory sample.

9
Sampling liquids, such as groundwater from wells, may involve the use of specialized
“down-hole” sampling devices, with valves that can be remotely opened and closed, or of
pneumatic or electrical pumps of various designs. Similar approaches are applied in river and
ocean studies, and current and depth information are simultaneously recorded. Chemical
streams in pipes need to be sampled with specially designed diverter probes that avoid
turbulence and wall effects. Liquid samples often require the immediate addition of analyte-
specific preservatives. For certain trace-level analyses the sample collection vessel must be
composed of high-purity materials and rigorously cleaned before use.

Earth’s atmosphere at great heights is sampled with aircraft, unmanned balloons, and
sounding rockets. At ground level, automated monitoring sites are carefully located to avoid
adventitious spikes from human activity and to obtain the most representative samples.
Atmospheric samples are also obtained manually with glass vessels using some displacement
medium, such as water or mercury, or with a sealable airtight sampling syringe. Sometimes a
syringe is used to fill a fluoropolymer gas-sampling bag. Smokestack gases or room air is
sampled by pumping the atmosphere through a liquid or particulate-solid medium that
absorbs and collects the gaseous analyte. Solid and liquid aerosols are often collected by
drawing the atmosphere through microporous filters. Pressurized gases can be sampled by
means of a metal gas-sampling cylinder. Extreme care and special procedures are required in
the case of asphyxiating, flammable, toxic, and corrosive gases.

Preparing the test sample

The laboratory sample usually needs to be further reduced and processed to what is
frequently called the test sample. This is a much smaller, but still representative, subsample
with an often finer particle size, from which test portions are selected for specific analyte
determinations.

With a particulate material, if the analyte is associated with one or more constituents, it is
possible to grind the laboratory sample to reduce the average particle size until the analyte
can be regarded as a pointlike component of the entire laboratory sample. This particle
diameter is called the liberation size and varies with the analyte and the type of material.

Grinding (more generally called comminution) can be accomplished by various means,

ranging from simple manual approaches to fully automated techniques. Ground material is
often sieved, but for chemical analysis purposes the retained fraction is always returned to the
grinder until it all passes the desired mesh size.

Excess grinding of some materials can lead to contamination from or analyte loss to the
grinding tool. Also, overzealous grinding can result in the absorption of atmospheric gases
(including moisture) by the sample and in the loss of fines. In addition, very finely ground
material is sometimes impossible to mix adequately.

Mixing of the laboratory sample is another critical operation. If the particle size is reduced in
a series of steps, generally each step is followed by an interval of mixing. This can be
accomplished by hand with small laboratory samples, but other samples require some form of
automation.

10
The effectiveness of any given mixing operation will be related to the particle size, shape,
and density, as well as to external influences such as electrostatic or magnetic fields and air
turbulence. If the material at a given stage represents a broad range of particle sizes and
shapes, mixing must circumvent the tendency for fine particles to collect in the center of a
pile while rounded particles collect at a pile’s edge.

Reducing the volume of the ground and mixed laboratory sample while keeping the sample
representative is another concern. The sample can be poured through a set of riffles that
uniformly splits it into two (or more) streams. One is selected for further processing, with the
other(s) discarded or archived for future reference. If the laboratory sample is very large, the
riffling process may be repeated several times. At the end of this process, the final selected
stream is the test sample. The simplest riffles design is a stationary arrangement of alternating
chutes surmounted by a wide hopper, fabricated from sheet metal. Spinning rifflers use either
a rotating carousel of collection vessels and a stationary vibratory feeder or a ring of
stationary collection vessels and a rotating feeder. Another approach to sample size reduction
involves a tabletop version of the coning and quartering operation (seeSampling), usually
conducted on a large sheet of glazed paper.

Monolithic solids also need to be converted to a suitable test sample. In the case of metal,
surface oxides may need to be ground off or dissolved by acid. The piece may need to be cut
to size for spectrometric work, or millings or drillings may need to be obtained. Liquid
samples, even gases, require thorough blending. Liquids can be stirred or otherwise agitated.
Gases can be mixed by gently warming one end of the storage vessel.

Selecting the test portion

The selection of a test portion from the test sample is the first step in any specific analytical
determination. In many cases the analyst has the freedom to weigh a mass or transfer a
volume. This allows him to optimize the analyte response while controlling background and
interference effects. However, there is more to selecting the test portion than “fine tuning” the
analytical methodology. The test portion size also bears a critical relationship to the
subsampling error for a test sample with a given level of analyte heterogeneity. This means
that, for any given test sample and analyte, there exists a minimum test portion size that
achieves a truly representative sample.

For analytical methods whose precision with low-heterogeneity samples is good, it is possible
to calculate a laboratory sampling constant (K). Using a test portion size (w) that is known to
yield good accuracy with a similar analyte level in a low-heterogeneity sample, several
replicate analyses of the test sample are made, and the relative standard deviation, R, is
calculated fromR = 100s/x,where s is the standard deviation and x is the mean. The laboratory
sampling constant, K, is then calculated:K = R2w,where K is the sample weight that produces
a 1 percent subsampling error at a confidence interval of 68 percent. If a different test portion
weight, wo, is used, then the expected subsampling error, Ro, is given byRo = (K/wo)1/2.These
relationships, while linked to the specific analyte and test sample, are independent of the test
methodology employed.

Unfortunately, the analyst who works with solid samples has little choice in selecting the test
portion size. With solid samples, spectrometric methods are used in which the test portion is
that minute portion of a solid sampled by a spark, arc, or glow discharge to create a sample

11
plasma. In X-ray fluorescence spectrometry the test portion is typically only a few atomic
layers. In the trace concentration realm the use of such small test portions on even moderately
heterogeneous solids can lead to a high subsampling error. Thus, in arc and spark atomic
emission it is always prudent to average a series of “burns” at diverse locations on a solid. In
glow discharge and X-ray fluorescence work it is similarly wise to regrind and repolish the
sample and repeat the measurement several times.

When a trace-level analyte is concentrated in a very heterogeneously distributed constituent

of a test sample, a unique situation prevails. Here, replicates of a selected test portion size
that contain six or fewer particles of analyte-rich constituent will not produce a normal
distribution of results. The value for a single result, xi, derives fromxi = H + cz,where H is the
average concentration of analyte in the test sample matrix, c is the average concentration of
analyte in the analyte-rich particles, and z is the number of analyte-rich particles in the test
portion. At a test portion size where z is between 1 and 6, the data behave erratically, with a
large number of test results producing a skewed Poisson distribution. However, at a test
portion size where z is greater than 6, the data produce a normal (or Gaussian) distribution
and an accurate mean. Unfortunately, at very minute test portion sizes, where z is zero, the
data are also Gaussian because only the matrix analyte is being measured. In this case the
mean is precisely wrong. This suggests a warning to trace analysts: very reproducible results
at very small test portion sizes may be erroneous.

Dissolution of inorganic samples

The dissolution of inorganic samples nearly always means the preparation of an acidic
aqueous solution from the test portion. There are a number of ways to accomplish this, but
the most common approach is the direct application of one or more mineralacids.
Nonoxidizing acids, such as hydrochloric, hydrofluoric, and sulfuric acid, are particularly
useful when oxidizing conditions would produce a protective oxide film on the sample
surfaces.

Commonly employed oxidizing acids are nitric acid and perchloric acid. Nitric acid is not a
strong complex former, but it does dissolve many metals, and it forms an impervious passive
film on many others. Perchloric acid is completely noncomplexing. It is nonoxidizing at room
temperature but becomes extremely oxidizing at elevated temperatures. Oxidizing acid
mixtures are generally useful dissolution media for many inorganic samples. For example,
aqua regia is three parts hydrochloric acid and one part nitric acid. It is an effective solvent
for platinum, palladium, and gold as well as a host of other metals and ores.

Despite the variety of acids and acid mixtures available, there are many sample types that
require alternative measures. The material itself may be too acid resistant even for high-
pressure acid bomb or sealed-vessel microwave approaches. The time for an acid dissolution
may be excessive, or the only feasible acid approach may add interference or result in analyte
loss. In these cases molten salt fusion is frequently the answer. In this technique the finely
ground test portion is mixed with a powdered flux in a crucible and heated until molten. The
cooled melt is then dissolved in water or an aqueous acidic solution.

Dissolution of organic samples

12
With organic materials there are two distinct methodologies, depending on whether the
analyte is inorganic or organic. For inorganic analytes the dissolution process generally
requires the complete destruction of the organic matrix, and no single approach is universally
applicable. Ignition in a high-temperature laboratory furnace is the simplest technique, but it
results in the loss of many elements.

Ignition in a sealed oxygen atmosphere is a better approach when dealing with a volatile
analyte. Schöniger flask combustion involves a special Erlenmeyer flask to which is added a
small volume of a suitable solution (often a dilute sodium carbonate or sodium hydroxide
solution) that absorbs and retains the inorganic analytes. The organic sample test portion is
either directly applied to a filter paper or loaded into a gelatin capsule, which is then wrapped
in a filter paper. The wrapped paper is secured in the platinum gauze clip attached to the flask
stopper. The flask is flushed with pure oxygen. The filter paper is ignited, and the stopper is
plunged into the flask. The flask is inverted and held securely until the combustion is
complete. The flask is then shaken for several minutes. Water or additional absorbing
solution is added to the collar to aid in rinsing the flask neck when the stopper is withdrawn.

Combustion in an armoured metal oxygen bomb is another alternative. A small test portion is
weighed into a sample cup suspended above a small volume of absorbing solution. An igniter
wire lies across the sample. The lid is attached; the bomb is pressurized to 25 atmospheres
with pure oxygen; and the sample is ignited electrically. The bomb is then cooled and is
shaken periodically. The pressure is then slowly released, and the lid is removed.

In a plasma dry asher the sample remains below about 200 °C (400 °F), while its organic
content is destroyed. This technique uses a low-pressure oxygen plasma generated by high-
frequency induction coils to remove organic matter from the test portion.

There are also several important wet ashing techniques. Digestion with nitric and sulfuric
acid and digestion with nitric and perchloric acid are approaches that are used safely and
routinely with some sample types; however, with certain materials there is a severe danger of
an explosion. These techniques must be avoided for high-boiling or temperature-resistant
organic materials, including fats, oils, greases, and waxes. In general, these procedures, like
all wet digestions of organic matter, should be attempted only with small test portions of
known material, strictly following well-established procedures designed for the specific
sample type.

When organic samples are dissolved for the determination of organic analytes, of course,
much milder conditions are employed. Perhaps the simplest, dissolution in water, is
sometimes useful for short-chain-length alcohols, aldehydes, anhydrides, ketones, esters,
ethers, organic acids, and simple carbohydrates. There are a few general rules concerning
organic compound solubilities, although there are many exceptions. A solvent is considered
inert in a dissolution if it can be quantitatively distilled or evaporated away from the solute.
All others are considered reaction solvents. The compounds in a homologous series tend to
show decreasing solubility in inert solvents with increasing molecular weight. Thus,
methanol, ethanol, and n-propanol are completely miscible with water; n-butanol and n-
pentanol show diminishing solubility; and the longer-chain normal alcohols are all insoluble
in aqueous solution. Chain branching tends to moderate this effect. For example, isobutanol is
more soluble in water than is n-butanol.

13
A compound tends to be most soluble in the solvent that it most closely resembles
structurally. For example, n-octane is insoluble in water but completely soluble in high-
molecular-weight straight-chain alcohols. The presence of two or more hydroxyl groups tends
to favour solubility in water.

Polymeric materials, both natural and man-made, present special problems if the analyst
wishes to preserve their complex features in solution. Sometimes this can be achieved only
by a simultaneous dissolution and derivatization, which preserves some gross structural
features. Some polymers, however, can be dissolved and reconstituted intact from solvents.
Examples are polyvinyl chloride from dimethylacetamide and polystyrene from
methylisobutyl ketone.

Isolation and preconcentration

In many modern analytical procedures, once the test portion has been dissolved, it is diluted
to some fixed volume and measured. However, this is not always the case. Sometimes it is
necessary to remove or mask interferences, perhaps even to completely isolate the intended
analyte from its sample matrix and dissolution medium.

Many isolation techniques involve the use of heat to change the analyte into a gaseous
species and are conveniently referred to as thermal evolution techniques. For inorganic
analytes it is possible to distinguish those that can sometimes be isolated directly from the
undissolved test portion. This category includes the instrumental methods that use reactive
gases and those that use inert gases. The most common reactive gas is oxygen, which is used
to convert carbon to carbon monoxide and sulfur to sulfur dioxide as the test portion is
rapidly heated in either a resistance or an induction furnace. Noble gases, such as helium and
argon, are commonly used as carriers to transport nitrogen, oxygen, and hydrogen from the
test portion as it is rapidly heated.

The distillation of inorganic elements from an aqueous solution is applicable to a number of

different species, both for the removal of interferences and for the collection of analytes. The
volatilization of interferences can be accomplished from a beaker on a hot plate. The
distillation and isolation of volatile analytes, however, requires a special enclosed apparatus,
which usually includes a water-cooled condenser.

In organic work, thermal evolution techniques include fractional distillation and gas
chromatography. In the former, careful temperature control allows the collection of constant-
boiling species from simple mixtures.

In general, gas chromatography allows much higher resolution separations and is widely used
in both preparation and analysis. Analytical gas chromatography is normally a combined
separation and measurement approach. There are also methods that use a “pre-column” to
remove the bulk of the sample matrix while passing the analyte components onto a separating
column. For example, in headspace analysis the equilibrated atmosphere above the surface of
a liquid is sampled and injected into a gas chromatograph. It is a thermal evolution method in
the sense that the distribution of analyte between the liquid and gas phases is temperature
dependent.

14
Solvent extraction is another isolation technique widely used in both inorganic and organic
analysis. When two immiscible or partially miscible solvents are agitated together, a
dissolved chemical species may preferentially migrate to one of the two liquid phases. Use of
this phenomenon is practical only for electrically neutral species. One of the two solvents is
usually water.

It is also often critical that the analyte be adjusted to a specific oxidation state prior to
extraction. Similarly, it may be necessary to adjust the oxidation state of a potential
interferent to ensure that it is not extracted. Masking agents are frequently added to convert
interferents to nonextractable species.

Ion exchange can also be used to isolate the analyte. It is most frequently applied as a form of
column chromatography in which ions in a (usually aqueous) solution passed through a resin-
packed glass column switch places with ions bound on ion-exchange resin beads. Ion-
exchange separations are based on the fact that different ions will have different affinities for
the resin. Thus, an ion with lower affinity will be displaced by an ion of greater affinity. The
displaced ion is then washed off the column and perhaps collected. Often most of the sample
matrix ions are initially bound to the resin, and then each type of ion is selectively removed
by washing the column with various electrolytes.

A common means of partially or completely isolating the analyte is the precipitation reaction,
which requires the formation of a low-solubility, easily filterable product. Complete
precipitation of the analyte may require the addition of a “carrier” species that “co-
precipitates” with the analyte under the same reaction conditions. The carrier is chosen to
have no effect in subsequent manipulations with the analyte. Sometimes, however, co-
precipitation occurs with an interferent element from the sample matrix. In this case some
chemical strategy must be applied to avoid co-precipitation.

Sometimes the analyte precipitates in such finely divided form that it is impossible to filter by
ordinary means. In this case warming, boiling, or adding reagents may be necessary to
agglomerate the analyte to filterable size. The quantitative transfer of a precipitate to a filter
is a manipulative art that becomes more critical at higher analyte concentrations.

Masking
Sometimes it is not necessary to isolate the analyte chemically in order to deal with
interferences. Masking agents are additives that undergo some reaction in the sample solution
that complexes (or precipitates) potential interfering elements and converts them to a form
that does not interfere with subsequent analyte manipulation or measurement. Masking agents
are used in molecular absorption spectrophotometry, gravimetry, titrimetry, and voltammetry.

In a larger sense, any additive that facilitates the analytical process by removing some
impediment to an accurate measurement may be considered a masking agent. Matrix
modifiers are substances added to prevent analyte loss and volatilize away interferences
during the char cycle of electrothermal atomic absorption measurement. Ionization

15
suppressors are additives that are readily ionized in flame atomic absorption measurement;
they reduce the ionization of the analyte and thus enhance sensitivity. Commonly used
ionization suppressors are lanthanum, sodium, and strontium compounds.

Derivatization
In many analytical procedures it is necessary to convert the analyte chemically to another
form to make its measurement possible. While infrared absorption techniques for organic
analytes are usually direct methods, nearly all quantitative ultraviolet and visible absorption
spectrophotometric methods are derivatizations in which the nonabsorbing analyte reacts with
a reagent to form a strongly absorbing complex. Many gas chromatographic procedures
involve the conversion of nonvolatile analytes into thermally stable, volatile derivatives. For
example, fats can be transesterified into fatty acid methyl esters, which can be readily
separated and measured.

Thomas R. Dulski

Additional Reading
External Links
 Sigma Aldrich - Sample Preparation and Handling
 University of Massachusetts Amherst - Information Technology - Sampling and Data
Analysis

 Introduction
 Sampling
o Theory
o Sampling solids, liquids, and gases
 Preparing the test sample
 Selecting the test portion
 Dissolution of inorganic samples
 Dissolution of organic samples
 Isolation and preconcentration
 Masking
 Derivatization

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18
acid–base reaction, a type of chemical process typified by the exchange of one or more
hydrogen ions, H +, between species that may be neutral (molecules, such as water, H 2 O; or
acetic acid, CH3CO 2 H) or electrically...
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 Table Of Contents

 Introduction

 Sampling

 Preparing the test sample

 Selecting the test portion

 Dissolution of inorganic samples

 Dissolution of organic samples

 Isolation and preconcentration

 Masking

 Derivatization

19
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Methods for taking, preserving and analyzing samples to

monitor the water quality of pools and other artificial
reservoirs
Official Version ( pdf format, 87 Ko)

 Introduction
 General precautions
 On-site analysis
 Laboratory analysis
o Legend
o Table 1: Preservation methods for chemical parameters (on-site analysis)
o Table 2: Preservation methods for microbiological and chemical parameters
(laboratory analysis)

Introduction
This guide contains information that details methods for the collection, preservation and analysis of
samples required for the monitoring of analytical parameters prescribed under the Regulation
respecting the water quality of pools and other artificial reservoirs (Q-2, r. 39*) in order to ensure
that water for bathers is safe, sanitary and stable. It is therefore important to ensure good
cooperation between reservoir managers, samplers and the personnel assigned to laboratory
analysis. According to the Regulation, the person responsible for a reservoir describes any owner or
operator of a pool or other artificial reservoir concerned by the said regulation.

This document has been prepared for all managers of reservoirs to explain the general preparations
necessary and the safety measures that must be implemented at the time of sampling. It also
outlines methods for sample collection in pools and other artificial reservoirs of water. The
document contains a section concerning on-site analysis of parameters required for the chemical
analysis of swimming water and a section relating to collection and preservation methods for
samples intended for laboratory analysis. It also details recommended sample volumes,

21
preservatives, types of containers and the timeframes that must be respected between sampling
and analysis.

General precautions

The manner in which samples are collected has a direct impact on the quality of analytical
findings. To minimize the risks of sample contamination by the sample collector and to
ensure sample integrity, basic precautions must be taken to obtain a representative sample.
Samples can become contaminated through careless sampling techniques. It is therefore the
responsibility of the sample collector or of an artificial reservoir manager to ensure the
quality of sample collection, preservation and suitable transportation of samples that are sent
to a laboratory accredited by the Ministère du Développement durable, de l’Environnement et
de la Lutte contre les changements climatiques. The sample collector or of an artificial
reservoir manager is also responsible for ensuring a representative sampling where on-site
analyses are conducted.

On-site analysis

Specific Precautions

The following precautions are necessary to prevent sample contamination:

 closely follow the instructions provided by the manufacturer of the chemical test kits
used at the sampling site;

 always use contaminant-free containers and where possible, prepare containers and
testing equipment at sampling sites;

 keep an accurate record of each sample collected using the correct form;

 always use devices or instruments that have been calibrated at the required frequency
(thermometer and pH-meter);

 reagents that are used for analysis must be kept in a clean, dry, well-ventilated and
dark location until use;

 always take measurements with reagents in a suitable location. Avoid leaving reagents
in prolonged sun exposure;

 seal reagent bottles correctly after use;

 never place wet fingers on reagent bottles; this may lead to inaccurate results;

 verify product expiry dates. Once the date indicated is past, you cannot be certain that
results are reliable;

 discard expired products, in accordance with regulations in effect

22
Preservation Methods

The methods of preserving the analytical parameters prescribed in the Regulation respecting
the water quality of pools and other artificial reservoirs are closely tied to analysis methods
and are described in Table 1.

Table 1 - Preservation methods for chemical parameters (on-site analysis)

Recommended Time between sample

Parameter Preservative* Container*
volume (ml) collection and analysis

Alkalinity N P or G 10 30 minutes

Hardness N P or G 10 30 minutes

Total residual
N P or G 10 30 minutes
bromine

Chloramines N P or G 10 30 minutes

Free residual
N P or G 10 30 minutes
chlorine

Total residual
N P or G 10 30 minutes
chlorine

pH N P or G 10 2 hours

Water
N P or G 125 3 minutes
temperature

* See legend

Sampling Method

Samples intended for chemical analysis must be collected during normal operating hours, 15
to 30 cm below the surface of the water or, where a reservoir is less than 30 cm deep, halfway
between the surface of the water and bottom of the reservoir.

Samples must be collected in an area that is not very frequented by bathers at the time of
sampling. It is also important to collect the sample in an area between the filtration system
intake and water return. In the case of whirlpool baths, samples can be collected anywhere
below the surface of the water.

It is important to carefully follow the instructions provided by the manufacturer of the

chemical test kits used. It is also essential that the hands of the person who is collecting the

23
samples be extremely clean to prevent subsequent contamination (by sweat, chemical
residues, etc.) when handling samples.

The measuring cells of the kit must remain clean and transparent. Cells must be rinsed with
the sample before their filling up and the level of liquid adjusted. They must then be wiped
with a soft clean cloth before making colour comparisons, so that outer surfaces remain clean
and dry. Always fill cells to the required level to prevent discrepancies in measurements.

Analysis Methods

Water temperature

For a water temperature reading, the following is required:

 collect a sample of water in a 125 ml wide-mouth glass or plastic bottle;

 dip a thermometer into the sample and wait at least 3 minutes for the temperature to
stabilize;
 with the thermometer still in the sample, hold the bottle and thermometer at eye level and
read the temperature;
 record the temperature to the nearest 0.5ºC (adequate marking). Use only calibrated
thermometers.

A water temperature reading can also be taken directly in the artificial reservoir.

Other Parameters

It is possible to obtain different types of test kits for on-site analysis of chemical parameters
(pH; free, total and combined residual chlorine; alkalinity; etc.). These kits are very useful for
verifying the principal parameters that can be used to analyze water quality and therefore
ensure that treatment systems are operating correctly.

These kits generally include test containers, measuring cells and reactive products that help to
determine concentrations of the products tested by using colorimetric comparators. Analyses
must be conducted using reliable equipment and appropriate and not expired reagents.

The reservoir manager must at least have a test kit capable of measuring free residual
chlorine to between 0.2 mg/1 and 5.0 mg/1 with a precision of 0.2 mg/l. If using brome as a
disinfectant, it is also necessary to have a test kit capable of measuring total brome to
between 1.0 mg/l and 5.0 mg/ with a precision of 0.5 mg/l.

Test kits must also be capable of measuring pH with a precision of 0.2 units, hardness and
total alkalinity of the water with a precision of 10 mg/l.

Laboratory analysis

Specific Precautions

24
  all samples sent for microbiological analyses must always be collected in sterile containers
provided by a laboratory accredited by the Ministère du Développement durable, de
l’Environnement et de la Lutte contre les changements climatiques;
 always begin a sampling campaign with microbiological samples, after, collect the chemical
samples;
 always leave an air space of at least 2.5 cm between the surface of the liquid and the
container lid. This helps to produce a homogenized sample for laboratory analysis;
 the necessary aseptic conditions must be respected when a sample is collected (e.g. avoid
inserting fingers or other objects in the mouth or on the lid of the container and minimize
exposure of the container to air at the time of sampling);
 samples should never be collected in unknown origin containers (always use laboratory-
supplied containers);
 never rinse containers provided by a laboratory because some contain preservatives
required for analysis;
 store sampling equipment in a clean and well-ventilated location;
 carefully close all containers tightly after sampling;
 where possible, precool samples in a refrigerator before sending them (particularly in
summer);
 make a detailed record of all samples as soon as possible after sampling, using the
appropriate forms, if samples are shipped to a laboratory;
 carefully pack samples to prevent breakage or leakage and use labeled shipping containers
designed for sample shipment;
 use a reliable transport service to ensure sample integrity and delivery within the prescribed
analytical timeframe.

Preservation Methods

The preserving methods for the analytical parameters prescribed in the Regulation respecting
the water quality of pools and other artificial reservoirs are closely tied to laboratory analysis
methods. In fact, the desired sensitivity and quantification limits can be used to determine the
volume and type of sample to be collected. The type of container and sample preservation
technique are also determined on the basis of the analysis method. It is therefore vital to work
in close cooperation with laboratory staff to obtain the proper additional information. In
addition to the specific provisions detailed in Table 2, the following general considerations
apply:

 from the moment samples are collected to the time they are received by a laboratory, all
samples must be preserved at a temperature of approximately 4°C (use ice boxes or
refrigerants) or chilled in an environment where an ambient temperature of approximately
4 °C is maintained;
 if ice boxes are used, they must remain clean and, where possible, reserved exclusively for
water analysis of pools and other artificial reservoirs;
 preservation and transportation are the responsibility of the sample collector or reservoir
manager. Working in close cooperation with the laboratory is essential.

Table 2 -  Preservation methods for microbiological and chemical parameters

(laboratory analysis)

25
 Recommended Time between sample
Parameter Preservative* Container*
volume (ml) collection and analysis

MICROBIOLOGY

Fecal coliforms ST3 PPS or GS 100 48 hours

Escherichia coli ST3 PPS or GS 100 48 hours

Pseudomonas
ST3 PPS or GS 100 48 hours
aeruginosa

Staphylococcus
ST3 PPS or GS 100 48 hours
aureus

CHEMISTRY

Turbidity N/A P or G 125 48 hours

* See legend

Sampling Method

Samples sent for microbiological and turbidity analysis must be collected during normal
operating hours, 15 to 30 cm below the surface of the water or where the reservoir is less than
30 cm deep, halfway between the surface of the water and the bottom of the reservoir.
Samples must be collected in an area that is not very frequented by bathers at the time of
collection and in an area between the filtration system intake and water return. In the case of
whirlpool baths, samples can be collected anywhere below the surface of the water. Bottles
must be filled to the rim, leaving an air space of at least 2.5 cm and the lid must be sealed
immediately after collection.

For water samples from an artificial reservoir destined for microbiological analysis, sampling
containers contain a reagent. This reagent neutralizes the residual disinfectant present in the
water when a sample is collected. To preserve the reagent, ensure that you tip the container
towards the bottom of the reservoir at a 45° angle in one motion. This preventive measure is
necessary, otherwise results may be negatively distorted. It’s also possible to collect sample
by adding immediately and aseptically a sterile solid tablet of sodium thiosulfate in the
sample. Samples for microbiological analysis must be collected with very particular attention
paid to avoiding hand contamination, even when a collector has washed his or her hands
before. If more than one sample is required, always begin with microbiological samples, and
after with the chemical samples, to avoid dipping a microbiological analysis container in
water that has been contaminated by the collector.

Legend
CONTAINERS

P Bottles and lid linings are made of the following plastics: high- or low-density

26
 polyethylene, polypropylene, polystyrene, polyvinyl chloride or teflon

PPS Sterile polypropylene bottle

G Glass bottle

GS Sterile glass bottle

PRESERVATIVES

N No preservative required

ST3 Sodium thiosulfate at a final concentration of 0.01 % (p/v)

OTHER

N/A Not applicable

*Due to a change to regulation numbering that followed adoption of the Act respecting the
Compilation of Québec Laws and Regulations, RSQ, c R-2.2.0.0.2, regulation # Q-2, r. 39
henceforth replaces former regulation # Q-2, r. 18.1.02.

|  Accessibility | Access to information (French) |

© Gouvernement du Québec, 2017

STANDARD OPERATING PROCEDURES

SOP:
2003
PAGE:
1
of 7
REV:
0.0

27
DATE:
08/11/94
SAMPLE STORAGE, PRESERVATION AND HANDLING
CONTENTS
1.0
SCOPE AND APPLICATION
2.0
METHOD SUMMARY
3.0
SAMPLE PRESERVATION, CONTAINERS, HANDLING AND STORAGE
3.1
Sample Storage and Preservation
3.2
Special Analytical Requests
4.0
INTERFERENCES AND POTENTIAL PROBLEMS
5.0
EQUIPMENT/APPARATUS
6.0
REAGENTS
7.0
PROCEDURES
8.0
CALCULATIONS
9.0
QUALITY ASSURANCE/QUALITY CONTROL*
10.0
DATA VALIDATION
11.0
HEALTH AND SAFETY
12.0
REFERENCES
13.0
APPENDICES
A
-
Table*
*These sections affected by Revision 0.0.
SUPERCEDES: SOP #2003; Revision 2.0; 01/09/92; U.S. EPA Contract
EP-W-09-0031.

STANDARD OPERATING PROCEDURES

SOP:2003PAGE:2of 7REV:0.0
DATE:08/11/94

SAMPLE STORAGE, PRESERVATION AND HANDLING

1.0SCOPE AND APPLICATION

28
The purpose of this Standard Operating Procedure (SOP) is to provide general
guidelines for the storageand preservation of water and soil/sediment samples.
Requirements for sample volume, matrixspike/matrix spike duplicate (MS/MSD)
sample volume, container type, and preservation techniques are presented for
both individual parameters and groups of parameters. Specific requirements for
sample storage, preservation and handling must be established in the Quality
Assurance (QA) Work Plan prior tosample collection.
These are standard (i.e., typically applicable) operating procedures which may be
varied or changed as required, dependent upon site conditions, equipment
limitations or limitations imposed by the procedure.
In all instances, the ultimate procedures employed should be documented and
associated with the final report.
Mention of trade names or commercial products does not constitute U.S. Enviro-
nmental Protection Agency (U.S. EPA) endorsement or recommendation for use.

2.0METHOD SUMMARY
This SOP is applicable to all water or soil/sediment samples collected by ERT/
SERASpersonnel. For handling, storage and preservation requirements for waste
and air samplesrefer to the specific SOPs forwaste and air sampling techniques.
3.0SAMPLE PRESERVATION, CONTAINERS, HANDLING AND STORAGE
3.1Sample Storage and Preservation
Samples should be collected using equipment and procedures appropriate to
the matrix, parameters and sampling objective. The volume of the sample
collected must be sufficient to perform the analysis requested. Sample containers
must not be pre-rinsed with the sample prior tosample collection.
Table 1 (Appendix A) contains alist of parameters which are typically of interest in
ERT/SERASactivations. Table 1 also indicates sample volumes to be collected
by matrix and parameter.
Samples must be stored in the proper types of containers and preserved in a
manner appropriate tothe analysis to be performed. This information is also
provided in Table 1. To prevent leakageduring shipping, sample containers
should be no more than 90% full. If air space would affect sample integrity (i.e.,
samples for VOA analysis), fill the sample container completely and placethe
container in a second container to meet the 90% requirement.All samples must
be cooled to 4OC from the time of collection until analysis. When a preservative
other than cooling is used, the preservative is generally added after the sample is
collected, unless the sample container has been pre-preserved by the laboratory.
If necessary, the pH must be adjusted to the appropriate level and checked with
pH paper in a manner which will not contaminate the sample.
Depending on the arrangements for sample analysis and the amount of sample
required for the analysis, it is possible that aliquots for several analyses may be
taken from the same sample container. This should be verified with the laboratory
performing the analyses prior to sample collection.

STANDARD OPERATING PROCEDURES

SOP:2003
PAGE:3of 7REV:0.0DATE:08/11/94
SAMPLE STORAGE, PRESERVATION AND HANDLING
29
All sample containers must be labelled appropriately. The exterior of the sample
containers must be wiped clean and dry prior to sample packaging. All samples
must be packaged according to the requirements of U.S. Department of
Transportation (U.S. DOT) or International Air Transportation Association (IATA).
3.2Special Analytical Requests
If a parameter or group of parameters is not included in Table 1 (Appendix A), the
laboratory performing the analysis should be contacted to determine
theappropriate sample containers, volumes and preservatives. This information
shall be documented in the QA Work Plan.
4.0INTERFERENCES AND POTENTIAL PROBLEMS
The following are interferences or potential problems associated with sample
storage, preservation and handling:
1.Samples should be protected from sunlight which may initiate photodegradation
of sample
components.
2.Delaying sample preservation may cause chemical reactions to occur, altering
original sample composition.
3.Improper sample preservation may adversely affect sample results.
4.Inadequate sample volume may prohibit the appropriate analyses from being
performed.
5.0EQUIPMENT/APPARATUS
The equipment/apparatus required to collect samples must be determined on a
site specific basis. Due to the wide variety of sampling equipment available, refer
to the specific SOPs for sampling techniques which include lists of the
equipment/apparatus required for sampling.
The following specific equipment/apparatus may be required for proper sample
preservation:
-pipettes (various sizes)
-bulb
-pH paper
-safety equipment
6.0REAGENTS
Reagents required for preservation of samples are specified in Table 1 (Appendix
A). The preservatives required are specified by the analyses tobe performed.
Decontamination solutions are specified in ERT/SERASSOP #2006, Sampling
Equipment Decontamination.
7.0PROCEDURES
STANDARD OPERATING PROCEDURES
SOP:2003PAGE:4of 7REV:0.0DATE:08/11/94
SAMPLE STORAGE, PRESERVATION AND HANDLING
Once aqueous samples are collected, add the appropriate preservative to reach
the desired pH. For non-aqueous samples, cool samples to 4 OC immediately after
collection. For handling, storage and preservation requirements for waste and air
samplesrefer to the specific SOPs.
8.0CALCULATIONS
This section is not applicable to this SOP.
9.0QUALITY ASSURANCE/QUALITY CONTROL

30
Refer to the specific SOPs for the type and frequency of QA/Quality Control (QC)
samples to be analyzed, the acceptance criteria for the QA/QC samples, and any
other QC activities which are associated with sampling techniques. All data
associated with sampling must be documented on Field Data Sheets or within
site logbooks.

10.0DATA VALIDATION
Refer to the specific SOPs for data validation activities that are associated with
sampling techniques.
11.0HEALTH AND SAFETY
When working with potential hazardous materials, follow U.S. EPA, OSHA and
corporate health and safety procedures.
12.0REFERENCES
This section is not applicable to this SOP.
STANDARD OPERATING PROCEDURES
SOP:2003PAGE:5of 7REV:0.0DATE:08/11/94
SAMPLE STORAGE, PRESERVATION AND HANDLING
APPENDIX A
TableSOP #2003
August, 1994
STANDARD OPERATING PROCEDURES
SOP:2003PAGE:6of 7REV:0.0DATE:08/11/94

SAMPLE STORAGE, PRESERVATION AND HANDLING

Assignment 2: W.R.T.: Sample Containers, Volumes to be Collectedand
Preservatives by Parameter and Matrix
_________________________________________________________
Question: With respect to the above characteristics, describe in terms of
Sample Storage, Preservation and Handling of water or soil/sediment
samples, for the determination of the following parameters:
- Acidity/Alkalinity
- BOD and COD
- Cyanide and Creosotes
- Dioxin/Furans
- Herbicides
- Metals
- Oil & Grease
- Petroleum Hydrocarbons
- Pesticides/PCBs
- Phenols

31
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International Journal of Analytical Chemistry
Volume 2017 (2017), Article ID 4870671, 7 pages
https://doi.org/10.1155/2017/4870671

Research Article
Effectiveness of Liquid-Liquid Extraction, Solid Phase Extraction, and Headspace Technique
for Determination of Some Volatile Water-Soluble Compounds of Rose Aromatic Water
Hale Seçilmiş Canbay

Department of Bioengineering, Faculty of Engineering and Architecture, Mehmet Akif Ersoy

University, 15030 Burdur, Turkey

Correspondence should be addressed to Hale Seçilmiş Canbay

Received 16 March 2017; Revised 5 June 2017; Accepted 14 June 2017; Published 16 July
2017

Academic Editor: Alberto Chisvert

Copyright © 2017 Hale Seçilmiş Canbay. This is an open access article distributed under the
Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Abstract
Steam distillation is used to isolate scent of rose flowers. Rose aromatic water is commonly
used in European cuisine and aromatherapy besides its use in cosmetic industry for its
lovely scent. In this study, three different sampling techniques, liquid-liquid extraction (LLE),
headspace technique (HS), and solid phase extraction (SPE), were compared for the
analysis of volatile water-soluble compounds in commercial rose aromatic water. Some
volatile water-soluble compounds of rose aromatic water were also analyzed by gas
chromatography mass spectrometry (GCMS). In any case, it was concluded that one of the
solid phase extraction methods led to higher recoveries for 2-phenylethyl alcohol (PEA) in
the rose aromatic water than the liquid-liquid extraction and headspace technique. Liquid-
liquid extraction method provided higher recovery ratios for citronellol, nerol, and geraniol
than others. Ideal linear correlation coefficient values were observed by GCMS for
quantitative analysis of volatile compounds (). Optimized methods showed acceptable
repeatability (RSDs < 5%) and excellent recovery (>95%). For compounds such as α-
pinene, linalool, β-caryophyllene, α-humulene, methyl eugenol, and eugenol, the best
recovery values were obtained with LLE and SPE.
1. Introduction

Aromatic waters are clear and saturated solutions of volatile oils, aromatic or volatile
substances, and some water-soluble compounds of essential oil. Aromatic waters are
generally produced by distillation of aromatic plants such as rose, thyme, and rosemary.
Water acquired by concentration of steam during steam distillation to isolate the scent of
aromatic plant flowers includes low amounts of essential oil. Rose aromatic water is liquid
preparation obtained by hydrosol portion of the distillate of fresh rose petals and contains
aromatic compounds in the form of either solution or suspended particles [1–4].

33
 Rose aromatic water is a commercially important commodity since it is commonly used in
European cuisine and aroma therapy. Due to its fragrance, rose aromatic water is used in
cosmetic industry, food flavoring, soaps, and toiletry. It is also used in traditional medicine as
antiseptic facial tonic, fever reducer, cooling assist, pain killer, astringent, mild laxative, and
antibacterial and in treatment of sore throat, enlarged tonsils, cardiac troubles, eye diseases,
gall stones, and gut troubles [3, 5–9].

In water distillation method, rose aromatic water contains very low amounts of (below 0.1%)
essential oil and its main component is phenylethyl alcohol [4, 10–16]. Double distillation
method is used as a traditional method. In this method, rose aromatic water is obtained in
the final step of rose oil production, which is retained in large-capacity copper/stainless steel
distillation devices [17].

Due to the complexity of rose aromatic water and relatively low concentration of some
terpenes, its analyses require isolation/preconcentration steps. The low concentration of
volatile compounds makes enrichment necessary as the basis for identification and
quantification, and among them liquid-liquid extraction (LLE), solid phase extraction (SPE),
solid phase microextraction (SPME), simultaneous distillation extraction (SDE), supercritical
fluid extraction (SFE), headspace solid phase microextraction (HS-SPME), solid phase
extraction (SPE), and stir bar sorptive extraction (SBSE) have been extensively studied [2–4,
10–16, 18–36].

This study is aimed at selecting the best extraction technique for studying the volatile
composition of rose water, and LLE, SPE, and HS technique were used for quantitative
determination of volatile compounds of aromatic rose water. Numerous studies have been
carried out on chemical composition of rose aromatic water [3, 4, 10–16, 19] from different
countries but analytical values (extraction efficiency, detection limit, etc.) were not included
in these studies. To our knowledge, this present study is the first involving these parameters.
2. Materials and Methods
2.1. Chemicals and Reagents

Methanol (HPLC grade), n-hexane (HPLC grade), n-pentane (HPLC grade), chloroform (GC
grade), dichloromethane (HPLC grade), and ethyl acetate (HPLC grade) were obtained from
Merck (Germany) and Sigma Aldrich (USA). Sep Pak Plus C18 cartridges (Waters, USA)
and Isolute ENV+ (Biotage, USA) were used for solid phase extraction. Chemical standards
of α-pinene, linalool, α-humulene (α-caryophyllene), nerol, 2-phenylethyl alcohol (PEA), β-
caryophyllene, citronellol, geraniol, methyl eugenol, and eugenol were supplied from Sigma
Aldrich (USA) while NaCl was purchased from Merck (Germany).
2.2. Aromatic Rose Water Samples

Commercial rose water samples were purchased from a local store.

2.3. Isolation and Preconcentration Techniques

Three different methods were used for the isolation and concentration of volatile compounds
from rose aromatic water as explained below.
2.3.1. Liquid-Liquid Extraction Procedure

The volatile components were extracted by the methods of Hernanz et al. [33] and Cabredo-
Pinillos et al. [34]. Briefly, 200 mL of sample containing 4 g of NaCl was placed in a 250 mL
glass flask. The extraction was performed with 5 mL of chloroform, dichloromethane, n-
hexane, and ethyl acetate. The flasks were introduced into the ultrasonic bath (Bandelin
Sonorex, Germany) and sonicated for 30 min at 25°C. The organic layer was then separated

34
 via pipetting. All samples were extracted in duplicate. Finally, 1 μL of aliquots was injected
into a GCMS system.
2.3.2. Solid Phase Extraction Procedure

Two different solid phase cartridges (Sep Pak Plus C18 cartridges and Isolute ENV+) were
tested for the isolation and concentration of volatile compounds from rose aromatic water
(Table 1). The solid phase extractions were carried out in a Visiprep SPE vacuum manifold
(12-port model) from Supelco (Supelco, USA). Solid phase extractions were performed
according to the method developed by López et al. [20] and Piñeiro et al. [21]. Cartridges
were placed in the manifold system and activated with 4 mL dichloromethane, 4 mL of
methanol, and finally rinsing 4 mL of water. Then 100 mL rose aromatic water samples were
passed through the cartridges by vacuum manifold, after which the sorbents were dried.
Volatile compounds were eluted from the cartridges using an organic solvent (n-pentane, n-
hexane, dichloromethane, and methanol).
Table 1: Main properties of solid phase cartridges used in isolation and concentration.
2.3.3. Headspace Extraction

Isolation and preconcentration of volatile compounds were achieved using a 7697A

headspace sampler apparatus (Agilent, USA). 4 mL volume of sample was used for analysis.
Table 2 shows the headspace sampler setup parameters for the headspace sampler
apparatus. The headspace transfer line was directly passed through the 7890A GC injector
port and connected to the GC CP-Wax 52 CB column using a universal capillary column
connector.
Table 2: Headspace sampler setup parameters.
2.4. Standard Solutions, Calibration Curves, and Recovery Studies

Stock standard solutions of 10 mg/mL of each compound were prepared in methanol and
stored at −20°C. In both cases, different working standard solutions were prepared by
dilution in the same solvent. Six concentrations were used for calibration curves of volatile
compounds. The average recoveries of the analytes were determined by comparing the
peak areas obtained from each volatile compound.
2.5. Chromatography and Apparatus

Apparatus GCMS was Agilent 7890A gas chromatograph equipped with a 5975 mass
detector (MSD), a 7693B automatic sampler, and a MSDCHEM (Agilent, USA) data system.
Analytes were separated in a fused silica capillary column CP-Wax 52 CB stationary phase
(50 m × 0.25 mm; film thickness 0.2 μm) (Agilent, USA). Oven temperature program was as
follows: initial temperature 60°C, held for 1 min, increased to 220°C at 2°C/min, and held at
20 min. The carrier gas (helium) flow rate was 15 psi. Splitless injection of a 1 μL volume was
carried out at 250°C. Temperature of the detector was 250°C. MSD conditions were ion
source temperature, 230°C; electron energy, 70 eV; mass scan range, 30–500 amu. The
same system and temperature program were used in HS analysis. A 7697A headspace
sampler was used instead of automatic sampler [3].
2.6. Statistical Analyses and Validation Procedures

Limit of detection (LOD), limit of quantification (LOQ), linearity of calibration, intraday and
interday accuracy, precision, and recovery were estimated for the validation of this method.
Six concentrations of standard solutions were prepared. Each standard solutions (volatile
compounds) concentration was measured in five replicates. Calibration curves for the
studied volatile compounds were preprepared by plotting peak areas versus concentrations
for GCMS and HS-GCMS. We defined the LOD was defined as three times the background
noise of the chromatographic instrument. The extraction recovery was determined by spiking

35
 blank rose water with each compound (standard addition method) in three replicates; they
were extracted as previously described. Standard solutions of target compounds were
analyzed five times in a day and once a day on three consecutive days for intraday and
interday precisions, respectively.
3. Results and Discussion

Ten volatile water-soluble compounds in rose aromatic water were used as target
compounds during the LLE, SPE, and HS method development: α-pinene, linalool, α-
humulene, nerol, PEA, β-caryophyllene, citronellol, geraniol, methyl eugenol, and eugenol.
3.1. Optimization of SPE and Eluting Solvent

Two different kinds of solid phase sorbents were studied, one of them with reversed solid
phase (C18) and the other one with hydroxylated polystyrene-divinyl benzene copolymer
(ENV+) solid phase. 2 mL of each eluting solvent was used to recover terpenoid ingredients
from the solid phase sorbents. n-Pentane and n-hexane were the worst sorbents eluting
solvents. Recoveries < 50% were obtained for all volatile water-soluble compounds for two
kinds of solid phase sorbents. Methanol was selected as the best eluting solvent for
compound elution from the Sep Pak Plus C18 cartridge. Also dichloromethane was the best
eluting solvent for volatile water-soluble compounds elution from the Isolute ENV+. Piñeiro et
al. [21] studied the effectiveness of four different eluting solvents (n-pentane, methanol,
dichloromethane, and ethanol) for the SPE of wine terpenoids and concluded that the
highest isolation and concentration efficiency was achieved by using dichloromethane as the
eluting solvent.
3.2. Optimization of LLE and Extraction Solvent

Complete extraction from rose aromatic water was achieved by using chloroform,
dichloromethane, ethyl acetate, and n-hexane. LLE of rose aromatic water with chloroform,
dichloromethane, and ethyl acetate provided target compounds in rose aromatic water
extract in quantitative determination. n-Hexane was worst LLE solvents.

Agarwal et al. [4] studied the effectiveness of different eluting solvents (dichloromethane,
chloroform, hexane, and benzene) for the LLE of rose water terpenoids and concluded that
the highest isolation and optimum results were achieved by using dichloromethane as
extraction solvent.
3.3. Linearity of Calibration Curves and Limits of Detection and Quantification

The retention time (), linearity (), LOD, and LOQ were summarized in Table 3. A regression
equation was obtained with good linearity () for GCMS and for HS-GCMS.
Table 3: Retention time () and performance characteristic obtained by GCMS and HS-
GCMS.

The values ≥0.999 for target compounds. Lei et al. [15] reported the PEA in rose water and
the acquired value was 1.0000. Piñeiro et al. [21] studied SPE methods and found values
were >0.999. Vila et al. [35] reported volatile compounds in wine and linear correlation
coefficients were ≥0.994. Won et al. [36] indicated PEA values in Bulgarian rose and
Provence lavender oil and value for PEA was 0.9814.

The LOD values were between 0.360 and 1.600 μg/L for studied compounds for GCMS and
between 1.143 and 4.650 μg/L for studied compounds for HS-GCMS. The relatively low
values indicated that the method possessed good sensitivity. In the HS-GCMS technique,
the LOD values were slightly higher because the noise ratio was slightly higher than the
GCMS technique. Piñeiro et al. [21] used SPE methods and obtained LOD values between

36
0.33 and 3.37 μg/L. Sánchez-Palomo et al. [27] evaluated the LLE, SPE, and SDE methods,
and the obtained LOD values were within 0.01–0.02 μg/L, 0.02–0.04 μg/L, and 0.01–0.08 
μg/L, respectively. Won et al. [36] reported PEA values in Bulgarian rose and Provence
lavender oil and LOD value for PEA was 0.77 ng/mL.

Precision and repeatability are summarized in Tables 4 and 5. The intraday and interday
relative standard deviations (RSDs) for target compounds were within 0.10%–0.23% and
0.13%–0.24%, respectively, for GCMS and within 0.29%–0.43% and 0.32%–0.47%,
respectively, for HS-GCMS. The RSD values were less than 5% in all experiments. Lei et al.
[15] studied the PEA in rose water and obtained intraday and interday RSDs of PEA 0.15%
and 0.19%, respectively.
Table 4: Intraday and interday precisions for GCMS.
Table 5: Intraday and interday precisions for HS-GCMS.
3.4. Recoveries

Recovery test was carried out using the method of standard addition. Rose water samples
were spiked with three different concentrations of standard solutions and analyzed. The
recovery of PEA ranged from 32.22% to 69.45%, with RSDs less than 5.00% for LLE.
Recovery values ranged from 25.76 to 98.86% for chloroform as an extraction solvent while
they varied from 32.07 to 98.81% for dichloromethane. For ethyl acetate, the range of
recovery values was between 57.49 and 95.29%, which was generally higher than that of n-
hexane (32.71 to 67.35%) (Table 6).
Table 6: LLE average recovery.

When the C18 cartridge was used, the recovery values were between 80.44 and 99.75%.
For ENV+ recoveries ranged from 32.96 to 87.22%. For HS-GCMS recovery ranged
between 33.53 and 116.90% (Table 7).
Table 7: SPE average recovery.

Lei et al. [15] reported that recovery values were within 99.3–101.0% for PEA in rose water.
Piñeiro et al. [21] found that recovery values were within 96.8–100.8% for optimized method.
Hernanz et al. [33] evaluated the effectiveness of different extraction methods (LLE and
SPE), and the recovery values for PEA, linalool, citronellol, nerol, and geraniol were 92.6%,
109.6%, 97.2%, 97%, and 99.3% for LLE 1, 18.6%, 19.7%, 19.4%, 19.3%, and 20.7% for
LLE 2, and 96.5%, 98.7%, 88.9%, 88.2%, and 97.4% for SPE, respectively.
3.5. Chemical Profiles of Rose Aromatic Water

The predominant components of rose water volatiles are PEA, citronellol, geraniol, nerol,
and methyl eugenol [3, 4, 10–16, 19]. In addition to these components, α-pinene, linalool, β-
caryophyllene, and α-humulene components were also included in our study. These
components are also found in rose oil and rose water [3, 4, 10–19]. In rose aromatic water
samples, PEA ( μg/g), citronellol ( μg/g), nerol ( μg/g), and geraniol ( μg/g) were found as
main compounds followed by α-pinene ( μg/g), linalool ( μg/g), α-humulene ( μg/g), eugenol ( 
μg/g), and methyl eugenol ( μg/g). β-Caryophyllene was not detected in rose aromatic water
samples.
4. Conclusions

This study was carried out to quantify the volatile water-soluble compounds of rose aromatic
water samples using different isolation and preconcentration techniques. The recoveries
obtained using samples spiked with standard target compounds ranged within 80.44 and
99.75% for C18 cartridge, 32.96 and 87.22% for ENV+, 33.53 and 116.90% for HS, 25.76
and 98.86% for chloroform, 32.07 and 98.81% for dichloromethane, 57.49 and 95.29% for

37
ethyl acetate, and 32.71 and 67.35% for n-hexane. The RSD values were less than 5% in all
experiments. The method showed good recoveries and high repeatabilities. PEA was the
main volatile water-soluble constituent of aromatic rose water.

The SPE method is a faster technique than LLE and HS but the HS technique is achieved
without any solvent. Different solvents such as chloroform, dichloromethane, ethyl acetate,
n-pentane, and n-hexane are used in SPE and LLE techniques, which may cause
contamination of the environment at different levels.
Conflicts of Interest

The author declares no conflicts of interest regarding the publication of this paper.
Acknowledgments

The author thanks Professor Dr. Yusuf Yilmaz for technical evaluation of the study and the
Scientific and Technology Application and Research Center of Mehmet Akif Ersoy
University, Burdur, for providing facilities for analyses.
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International Journal of Analytical Chemistry

Volume 2010 (2010), Article ID 398381, 9 pages
http://dx.doi.org/10.1155/2010/398381

Review Article
Extraction Techniques for Polycyclic Aromatic Hydrocarbons in Soils
E. V. Lau, S. Gan, and H. K. Ng

Faculty of Engineering, The University of Nottingham Malaysia Campus, Jalan Broga,

Semenyih, Selangor Darul Ehsan 43500, Malaysia

41
Received 25 August 2009; Accepted 10 March 2010

Academic Editor: Peter S. Haglund

Copyright © 2010 E. V. Lau et al. This is an open access article distributed under the
Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Abstract

This paper aims to provide a review of the analytical extraction techniques for polycyclic
aromatic hydrocarbons (PAHs) in soils. The extraction technologies described here include
Soxhlet extraction, ultrasonic and mechanical agitation, accelerated solvent extraction,
supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase
extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-
bed extraction. The influencing factors in the extraction of PAHs from soil such as
temperature, type of solvent, soil moisture, and other soil characteristics are also discussed.
The paper concludes with a review of the models used to describe the kinetics of PAH
desorption from soils during solvent extraction.
1. Introduction

Polycyclic aromatic hydrocarbons or polynuclear aromatic hydrocarbons (PAHs) are

compounds produced through incomplete combustion and pyrolysis of organic matter. Both
natural and anthropogenic sources such as forest fires, volcanic eruptions, vehicular
emissions, residential wood burning, petroleum catalytic cracking, and industrial combustion
of fossil fuels contribute to the release of PAHs to the environment [1]. The presence of PAH
compounds in soils is an issue of concern due to their carcinogenic, mutagenic, and
teratogenic properties. In 2008, 28 PAHs have been identified as priority pollutants by the
National Waste Minimization Programme, a project which is funded by US Environment
Protection Agency [2].

PAHs which consist of fused benzene rings are hydrophobic in nature with very low water
solubility and high octanol-water partition coefficient (). Hence, they tend to adsorb tightly to
organic matter in soil rendering them less susceptible to biological and chemical
degradation. Prolonged aging time in contaminated soil promotes the sequestration of PAH
molecules into micropores and increases the recalcitrance of PAHs towards treatment [3].
Thus the extraction process of PAHs from soil for analysis is made more complicated due to
these factors. In this paper, various analytical extraction techniques for PAHs in soils will be
reviewed, ranging from more widely applied methods such as Soxhlet extraction, sonication,
mechanical agitation, and accelerated solvent extraction to alternative ones such as
supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase
extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-
bed extraction. The influencing factors in the extraction of PAHs from soil such as
temperature, type of solvent, soil moisture and other soil characteristics are also discussed.
Finally, a review of the models used to describe the kinetics of PAH desorption from soils
during solvent extraction will be provided.
2. Extraction Techniques
2.1. Soxhlet Extraction

The Soxhlet extraction has been vastly used as a benchmark technique in the extraction of
PAHs from soils and sediments. Basically, in the Soxhlet extraction technique, the solid
sample is placed into an extraction thimble which is then extracted using an appropriate

42
solvent via the reflux cycle. Once the solvent is boiled, the vapour passes through a bypass
arm into the condenser, where it condenses and drips back onto the solvent in the thimble.
As the solvent reaches the top of the siphon arm, the solvent and extract are siphoned back
onto the lower flask whereby the solvent reboils, and the cycle is repeated until all the
sample is completely extracted into the lower flask.

The main disadvantage of this extraction process is the use of large volumes of solvent,
which can be more than 150 mL for the extraction of PAHs from a mere 10 g of soil sample.
In addition to that, this method is very labour intensive and time consuming, as the solvent
has to be refluxed up to 24 hours to achieve considerable extraction efficiencies [4, 5]. The
Soxhlet extraction too has been shown to have relatively poor selectivity for PAHs compared
to bulk soil organic matter, with approximately a quarter to one third of bulk soil organic
matter removed during extraction [6]. Studies have indicated that the chromatograms of
extracts produced via Soxhlet using GC-MS and GC-FID yielded more artefact peaks with
branched alkane “humps,” demonstrating that compounds such as n-alkanes and humic
substances other than PAHs are coextracted using the Soxhlet technique [6, 7]. Other minor
drawbacks of using the Soxhlet apparatus include the likelihood of sample carryover, the
need to fractionise extracts to avoid heavy contamination of GC injection port, and the
unfeasibility of redissolving dried Soxhlet extracts [8, 9].

Nonetheless, the Soxhlet extraction is still the preferred method because of its comparative
extraction results despite the nature of matrix sample. Not only does the Soxhlet extraction
yields similar results with methods such as the supercritical fluid extraction (SFE),
microwave-assisted extraction (MAE), accelerated solvent extraction (ASE), and ultrasonic
methods, but the results also show small variations with low relative standard deviations
[10–12]. Statistically, Berset et al. [12] showed that the Soxhlet method resulted in median
values which corresponded to the overall mean of other extraction procedures including
ASE, SFE, MAE and sonication. The efficiency of the Soxhlet extraction increases with
molecular weight, reaching an efficiency range of 84–100% for PAHs with more than 4 rings
[13].

To further improve the Soxhlet extraction technique, Edward Randall patented the
automated Soxhlet extraction method in 1974. This is a two-step procedure which combines
boiling and rinsing such that the total extraction time is reduced while the evaporated solvent
condenses rapidly for reuse, reducing the amount of total solvent required. In this improved
technology, the extraction thimble is initially lowered directly into the flask containing the
boiling solvent to remove residual extractable material while the extractable materials pass
readily from the sample and dissolve into the solvent simultaneously. The level of solvent is
then reduced to a level below the extraction thimble such that the configuration mimics the
traditional Soxhlet extractor whereby the PAH is extracted by refluxing condensed solvent
and collected in the solvent below the extraction thimble. With this improvisation, the PAH
extraction efficiencies and precisions were statistically improved, with almost 100% recovery
rates [14]. In addition to that, the compact design of the automated system also allows
several samples to be extracted simultaneously with its multiple extraction cells assembly
while being run unattended [4, 5].
2.2. Ultrasonic Agitation/Sonication

The ultrasonic agitation, also known as sonication, is a technique which engages the
acoustic energy of ultrasonic waves with a minimum frequency of 16 kHz in fluid, causing
rapid compression and rarefaction of fluid movement which results in the cavitation
phenomenon, that is, the reoccurring formation and collapse of microbubbles. This agitation
can be performed either by immersing a sonicator transducer also known as an ultrasonic

43
horn into the sample solvent mixture or placing the sample solvent mixture directly into a
sonication bath. The desired ultrasound is generated by means of piezoelectric ceramic
attached either to the ultrasonic horn or the walls of the sonication bath.

Sun et al. [15] claimed that sonication was better than the Soxhlet because it provided higher
extraction efficiencies, was more economical and easily operated. Likewise, Guerin [4] noted
that similar levels of extraction efficiency to the Soxhlet extraction method could be attained
through vigorous sonication. However, the level of extraction efficiency was observed to be
highly dependent on the sample matrix and concentration of contaminants in the sample.
Contrary to these observations, other studies have indicated that sonication was less
efficient than the Soxhlet with relatively low recoveries particularly for lower molecular weight
PAHs (44–76%) [13, 16].

The power amplitude and duration of sonication need to be carefully controlled in order to
avoid extensive exposure to the irradiation which may degrade the contaminants in the
sample and reduce the extraction rates of PAHs. The decrease in efficiency during
excessive sonication is due to an increase in broken carbonaceous particles and additional
contact surface area which adsorbs the PAHs more readily, causing a reversed adsorption
cycle of PAHs [16]. Additionally, further separation techniques such as centrifugation or
filtration are required after the extraction process.
2.3. Mechanical Agitation

This simple, low-cost method uses agitation or mixing action to extract the PAHs from
samples in a shake-flask placed onto a rotary shaker, or with a magnetic stirrer submersed
into the flask directly. Although it is an easy handling method with minimal glassware and
smaller volumes of extraction solvent, this method has not been as widely used as the
Soxhlet and sonication due to the lower extraction efficiency and unsatisfactory quantitative
results [5, 7]. Although some studies reported that this method was comparable to the
Soxhlet technique, the results obtained using mechanical shaking showed larger variations
and less selectivity due to the difficulty in quantifying the PAH extracts [12, 17]. Comparable
results were only attainable with long shaking times to extend the contact time with solvent
[18, 19].
2.4. Accelerated Solvent Extraction (ASE)/Pressurised Fluid Extraction (PFE)

Accelerated solvent extraction (ASE) or pressurised fluid extraction (PFE) is a fairly new
technology which raises the solvent temperature above its boiling point but maintains it in the
liquid phase by elevating the pressure. As a result, the high pressure aids in the
solubilisation of air bubbles, thereby exposing more of the sample to the extraction solvent
while increasing the capacity of the heated solvent to impart better solubility.

Today, ASE systems are commercially available for extracting organic compounds from a
variety of solid samples. The ASE system is built up of several extraction cells on a loading
tray proximate to an oven. During extraction, organic solvent is pumped into the extraction
cells preloaded with soil samples while increasing the temperature and pressure to the
desired values. Once extraction is completed, a nitrogen cylinder is used to purge the
samples of residual solvent.

With the usage of the ASE system, the recovery of PAHs from soils and sediments was
reported to be two times higher than using the Soxhlet extraction method [20], while the
accuracy was also improved with a relative standard deviation of less than 10% [21]. Other
benefits of ASE include reduction of solvent consumption and total time required due to the
use of high pressures. The extraction procedure can be fully automated with an online

44
purification column, preventing loss of the volatile PAHs, avoiding tedious preparation and
potential contamination as in the case of mechanical shaking [21, 22].

2.5. Supercritical and Subcritical Fluid Extraction

Supercritical fluids exhibit a continuum of both gaseous and liquid phase properties. Their
physical characteristics including liquid-like density, low viscosity, high diffusivity and zero
surface tension enable them to penetrate almost anything and dissolve most materials into
their components. Carbon dioxide which has a supercritical temperature and pressure of
31°C and 74 bar, respectively, is widely employed in SFE as an environmentaly friendly
solvent in its supercritical state [23].

In a study by Miége et al. [9], comparisons between Soxhlet and SFE extraction revealed
that the recoveries of PAHs for both methods were almost similar. Although the SFE
technique was more difficult to optimise, the technique provided extraction results with lower
relative standard deviation and better selectivity, due to cleaner extracts. Other studies [6,
23] also indicated that SFE removed only 8% of the bulk organic matrix in comparison with
Soxhlet extraction or ASE which extracted a quarter to one third of bulk soil organic matter.
Furthermore, integrated SFE systems allow concentrated extracts to be directed
straightaway into the cleanup column, reducing the need to remove the eluate manually. In
certain SFE systems, the extracts may also be analysed directly by GC without any cleanup.
This prevents extra contamination that may occur during manual handling [12, 24]. However,
the high complexity of the SFE process may contribute to inconsistent results this system
should be carried out in different laboratories [23].

In the development of SFE, water has also been considered as the extraction fluid. However,
the use of supercritical water is limited because of the high temperature (>374°C) and
pressure (>218 atm) requirements which creates a highly corrosive environment [25]. Thus,
subcritical water extraction (SWE) also known as pressurised hot-water extraction is used
instead. As the temperature of water is raised from 100°C to 274°C under pressure, the
hydrogen bonding network of water molecules weakens resulting in a lower dielectric
constant and simultaneously decreasing of its polarity. Thus, subcritical water becomes
more hydrophobic and organic-like than ambient water, promoting miscibility of light
hydrocarbons with water [26]. In contrast to SFE which extracts mostly non polar organic
compounds, it has been reported that SWE gives better preference to more polar analytes,
therefore providing a higher extraction efficiency of PAHs with less or almost no extraction of
other alkanes [6]. Wet oxidation or SWE combined with oxidation using oxidising agents
such as air, oxygen, or hydrogen peroxide was reported to remobilise bound organic
residues, providing a higher extraction capability [27, 28]. In one study, SWE combined with
oxidation resulted PAH soil extraction efficiencies within the range of 99.1–99.99%
compared to extraction efficiencies within 79–99+% using SWE alone [28].
2.6. Microwave-Assisted Extraction (MAE)

Another highly instrumental extraction technique is the MAE whereby both solvent and
samples are subjected to heat radiation energy attained from electromagnetic wavelengths
between 1 m and 1 mm, with frequencies of 300 MHz to 300 GHz. Microwave radiation is
preferred compared to conventional heating due to its rapid heating which is reproducible
and has less energy losses. Modern designs of the microwave ovens include carousels
which can hold at least twelve extraction vessels allowing simultaneous multiple extractions.
The main advantages of the MAE method are the reductions in solvent usage and time. In
comparison to SFE, the cost of MAE is moderately lower [20]. Additionally, this unique

45
heating mechanism provides selective interaction with polar molecules which greatly
enhances the extraction efficiency of PAHs [29, 30].

The major drawback of this method however is that the solvent needs to be physically
removed from the sample matrix upon completion of the extraction prior to further analysis.
In certain cases whereby samples are pretreated with activated copper bars to assist the
extraction process, the removal of this copper is necessary for a cleaner extract [10].
Although a subsequent purification step can be implemented to rectify this problem, there
may be possibilities of losing analytes or inducing contaminants with additional cooling time
for this extra handling. Furthermore, the sample allowance for analysis is limited to 1.0 g
which is insufficient for a homogenous analysis [31].
2.7. Alternative PAH Extraction Techniques

Solid phase extraction (SPE), a method that is generally used to clean up a sample has
been used for rapid and selective extraction of PAHs from soil samples. Soil samples are
washed with solvent to leach away undesired components before extraction of PAHs with a
different solvent into a collection tube [32]. When this extraction technique is employed,
filtering over an empty SPE column or using purified sand prior to extraction is usually
recommended to prevent soil samples clogging the SPE column. A variation to the SPE of
PAHs from soils is the solid phase microextraction (SPME). Ouyang and Pawliszyn [33]
described the application of the technique on PAH extraction from soils. This solvent free
approach utilises a small diameter fused-silica fibre coated with the extracting phase and
mounted in a syringe-like device for protection and ease of handling. The depth of injected
needle is adjusted for headspace sampling before exposing the fibre which adsorbs the
PAHs from the soil. The exposed SPME fibre is then transferred directly to the injection port
of an analytical instrument such as a GC for quantitative analysis. The major advantage of
the SPME is its fast, simple and convenient extraction which can be done on-site. The
configuration of the solid-phase microextractor offers solutions to sampling problems
because it allows extraction of small volume of samples which can then be analysed without
any pretreatment. When stored properly, the fibre on the needle can also be analysed
several days later in the laboratory without significant loss of volatiles. The capability of the
SPME device to extract such small volumes of samples requires extreme precision during
manufacturing to achieve homogeneity in the construction of the fibre (extraction phase
surface) to provide consistency in extraction outcomes and qualities [34]. One study using
SPME revealed that only volatile compounds such as lower molecular weight (LMW) PAHs
(less than 4 rings) were detected [35].

Another alternative PAH extraction technique is thermal desorption which does not use
solvents or high-pressure extraction equipments. The thermal desorption technique is
commonly coupled with GC by direct injection of solid sample onto the cold injector. The
carrier gas is temporarily halted while the injector is rapidly heated to the desired
temperature approximately within 200–500°C to volatilise targeted compounds from soil. The
carrier gas is then resumed and the isothermally extracted compounds are swept onto the
GC column, providing a direct and rapid analysis of the contaminated soil. Thermal
desorption and online GC analysis technique has been widely employed in the analysis of
PAHs in various matters including fly ash, ambient air particulate matter as well as creosote
and petroleum contaminated soil [36–39]. The technique, however, requires prior calibration
to allow for nonlinear response to sample size and concentration of contaminants [39].

Contrary to thermal desorption, pyrolysis (Py) or high temperature distillation (HTD)

extraction technique employs high rate temperature ramping or flash pyrolysis at high
temperatures. In flash pyrolysis, the sample is heated in a very short time using either

46
inductive heating (also known as Curie point pyrolysis) or Ohmic heating using platinum foil.
The significant increase in heat energy in the system causes thermal cracking of larger
macromolecules into simpler monomers which are more volatile. Due to its high heating
velocity, accurate temperature reproducibility and wide temperature range, the Py has
successfully been applied to various nonvolatile compounds and matrices such as synthetic
plastics, rubbers and paints. Buco et al. [40] have demonstrated its novel application in the
analysis of PAHs in contaminated soil. Here, induction heating of the soil sample is carried
out in a ferromagnetic foil called pyrofoil in an oven equipped with a radio frequency field to
reach the Curie point temperature (160–1040°C) whereby the pyrofoil loses its magnetic
properties and simultaneously adopts the specific property of a heated alloy. As such, the
soil sample which is wrapped inside the pyrofoil is desorbed of the PAHs and the PAH
bearing pyrolysates are transferred immediately into an online GC column for further
analysis. Pyrolysis methods have been a more popular choice than thermal desorption due
to their capabilities in providing greater temperature control. With high temperature Py
method, the extraction speed is also significantly reduced, permitting a higher number of
samples to be analysed. The main advantages of thermal desorption or pyrolysis with online
GC is the exclusion of reconcentration and clean-up steps necessary for some other
extraction methods. Therefore, the contamination risks are lower with higher sensitivity and
specificity when these methods are employed. Similar to SPME, the use of solvents are also
eliminated, which subsequently reduces cost. Nonetheless, the small sample size used
(approximately 30 mg) may result in insignificant data analysis errors since it does not
provide a good representative of the entire field soil. In addition, the temperature program
used has to be carefully optimised to avoid the decomposition of the cellulose filter itself,
which may result in formations of undesirable byproducts.

Fluidised-bed extraction (FBE) has also been reported in the specialised literature to extract
PAHs from soils [41]. The system is analogous to the automated Soxhlet extraction
apparatus whereby the soil sample is loaded into an extraction tube secured with a filter at
the bottom while the extraction solvent is filled into the basic vessel beneath the soil sample.
The heating block of the device is first heated up to evaporate the extraction solvent through
the filter which then condenses when in contact with the cooling bar above the soil sample.
The condensed solvent then drips back into the soil sample and further down into the
collected solvent. The constant penetrating flow of solvent vapour heats up and agitates the
soil mixture, causing it to be fluidised. The collected solvent in the basic vessel is then
concentrated for further analysis. In comparison with the conventional Soxhlet extraction, the
extraction duration and solvent used is reduced under optimised conditions.
3. Influencing Factors
3.1. Temperature

In the majority of analytical studies using ASE, SFE, and MAE, the PAH extraction
efficiencies were observed to generally increase with increasing temperatures, as can be
seen in Table 1 [9, 31, 42–46]. Elevated temperatures reduce both fluid density and
viscosity, resulting in lower surface tension and improved contact between the solvent and
targeted PAH analytes. The diffusion of PAHs through the soil as well as the diffusion of
solvent into the interior of the soil matrix is enhanced. Likewise, the desorption of PAHs from
the solid matrix and their solubilities in the extraction solvent are improved by increased
temperatures. As such, the time to achieve equilibrium is significantly shortened.
Unfortunately, 2- and 3rings PAHs are highly volatile and more susceptible to evaporation
instead of extraction at higher temperatures. Thus, the reported extraction efficiencies for
LMW PAHs were less than the higher molecular weight (HMW) PAHs. A few papers
reported that increasing temperatures caused a general decrease in the PAH extraction

47
efficiencies and recovery yields [44, 47]. While there is no certain explanation for this
behaviour, it has to be noted that these studies were using SFE.
tab1
Table 1: Bibliographic compilation of studies on extraction temperature.
3.2. Solvent Type

Table 2 is a bibliographic compilation of PAH extraction studies from soils using various
solvents. Generally, the choice of extraction solvent is dependent on several factors, with
one of them being the degree of PAH concentration in the soil. For lowly polluted soil ( dry
weight sample), PAHs are mainly found on the surface, therefore a more polar solvent such
as acetone is preferred to break up the soil aggregates and to allow intensive contact
between particles. For highly polluted soil ( dry weight sample) however, a relatively
nonpolar solvent such as toluene or cyclohexane would be a better solvent [12]. Since the
principles of solvent extraction are based on the theory of like dissolves like, the polarity of
solvent with respect to the polarity of PAH contaminants also plays a role in determining the
extent of solubility. For instance, it was shown that dichloromethane as an extraction solvent
for PAHs resulted in low recoveries for all compounds, whereas hexane-acetone (1 : 1) was
an effective extraction solvent for PAHs [48, 49]. Apart from PAH concentration and polarity
of solvents, extraction efficiencies vary from one technique to another. In MAE, for example,
solvents are chosen based on their dissipation factor (dielectric constant) which determines
the degree of absorption of microwave energy [31, 49].
tab2
Table 2: Bibliographic compilation of solvents used in the extraction of PAHs.
3.3. Soil Moisture and Other Soil Characteristics

The effects of soil moisture on PAH extraction efficiencies are dependent on the type of
extraction technique employed as shown in Table 3. With MAE studies, PAH extraction
efficiencies were generally observed to increase with increasing soil moisture. This is mainly
due to the ability of the localised superheating to form gas bubbles from existing water
residues in soil and cause expansion of pores, allowing solvent penetration into the matrix.
Additionally, the high dielectric constant of water allows more microwave absorption which in
turn provides more heating [29, 30]. Similarly, a study using SFE showed that for water
content less than 10 wt. %, the water in soil acted as a modifier to the extraction solvent
which increased the fluid’s capability to penetrate further into the soil particles [50]. Other
SFE and Soxhlet experiments revealed that the presence of soil moisture decreased or did
not significantly affect the efficiency of PAH removal from soil. Soil drying is therefore carried
out in some cases to eliminate the influence of moisture on the PAH extraction efficiency.
Comparisons between various drying methods showed that thermal drying of soil between
temperatures of 25°C and 40°C for several days was best for prevention of losses of volatile
PAHs while air drying was reasonably sufficient and freeze drying was least preferable due
to partial loss of highly volatile PAHs such as naphthalene [12].
tab3
Table 3: Bibliographic compilation of studies on soil moisture.

Apart from soil moisture, the composition of soil affects the extraction of PAHs. The
extraction process of PAHs was observed to be significantly more difficult from high clay
content soil (>40%) due to the fact that 32% of the total carbon content where most of the
HMW PAHs resided in was concentrated in the clay fraction [17]. Strong adsorption of PAHs
to clay surfaces also result in reduced desorption during thermal extraction and less
detectable hydrocarbons [39]. The size of soil particles also impacts the efficiency of PAH
extraction. It has been demonstrated that PAHs accumulate preferentially on smaller
particles [41]. As such, PAHs are more easily extracted from fine soil fractions such as fine

48
silts and clays than larger aggregate size fractions. Reduced particle sizes allow ample
diffusion and better accessibility of solvent through the matrix, thus increasing the flow rate
of solvent and rate of extraction [17, 51].
4. Kinetics Models of PAH Desorption from Soils
4.1. First-order Mass Transfer with Single Equilibrium Desorption

The dissolution and desorption of PAHs can be fitted to a first-order mass transfer coefficient
model [52]:

where is the liquid-phase concentration at any point in time, is the lumped mass transfer
coefficient, is the equilibrium liquid-phase concentration and is the contact time with the
extraction solvent.
4.2. First-order Mass Transfer with Dual Equilibrium Desorption

The desorption process in sediments and soils contaminated with hydrophobic contaminants
can be classified as a biphasic process, with a fast and a slow component [53–55]. This two-
site kinetic model is described by

where is the liquid-phase concentration at any point in time, is the equilibrium liquid-phase
concentration, is the equilibrium liquid-phase concentration of the first stage (rapid), is the
mass transfer coefficient of first stage, is the equilibrium liquid-phase concentration of
second stage (slow), is the mass transfer coefficient of second stage, and is the contact time
with the extraction solvent.

This model treats the process as a combination of two kinetically controlled reactions
occurring simultaneously, whereby the first stage is governed by a rapid partitioning between
the solid and liquid phases while the latter stage is which generally slower than the first is
kinetically controlled by other processes. Equation (2) can also be employed in its fractional
form whereby the rapidly desorbing fraction is while the slowly desorbing fraction is ():

where is the fraction of the PAH extracted after time .

5. Conclusions

Of the PAH extraction technologies discussed here, Soxhlet extraction, ultrasonic and
mechanical agitation can be implemented easily since the processes are carried out with
minimal instruments or glassware and at ambient pressures. In comparison, ASE and MAE
provide a faster extraction with lesser solvent consumption albeit at higher capital costs and
possibly operating costs. PAH extraction using supercritical carbon dioxide or subcritical
water is an environmentaly friendly technique but entails the use of high pressure
equipment. SPE and SPME, thermal desorption and flash pyrolysis, as well as fluidised-bed
extraction are novel alternatives which require further in-depth studies prior to wide-scale
adoption in laboratories. It has to be recognised that no single extraction technology can be
the solution for all extractions of PAHs in soils and sediments. Costs, the required accuracy
and precision in results, analysis time, as well as technical competence are factors to be
considered in deciding the right extraction technique.
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Research article

54
Open Access

Comparative study of three digestion

methods for elemental analysis in
traditional medicine products using atomic
absorption spectrometry
 ABM Helal UddinEmail author,
 Reem Saadi Khalid,
 Mohamed Alaama,
 Abdualrahman M. Abdualkader,
 Abdulrazak Kasmuri and
 S. A. Abbas

Journal of Analytical Science and Technology20167:6

https://doi.org/10.1186/s40543-016-0085-6

©  Uddin et al. 2016

Received: 2 October 2015

Accepted: 20 January 2016

Published: 30 January 2016

Abstract
Background

Traditional medicine mainly of herbal origin is widely used all around the world. Heavy
metal contamination in such products is frequently reported. Accumulation of heavy metals
in the human body leads to various health hazards. Thus, precise determination for such
contaminants is required for safety assurance. Sample preparation is a significant step in
spectroscopic analysis to achieve reliable and accurate results. Wet digestion methods are
basically used for the dissolution of herbal product samples prior to elemental analysis.

Methods

This study has been designed to evaluate the efficiency of three acid digestion methods using
different solvents. Five samples were digested with three different acid digestion methods
namely method A (a combination of nitric-perchloric acids HNO3–HClO4 in a ratio 2:1),
method B (only nitric acid HNO3), and method C (a mixture of nitric-hydrochloric acids
HNO3–HCl in a ratio 1:3), to recommend the most efficient digestion method that gains the
highest analyte recovery. The analysis of arsenic (As), cadmium (Cd), lead (Pb), nickel (Ni),

55
zinc (Zn), and iron (Fe) was conducted using various techniques of atomic absorption
spectrometry (AAS).

Results

The statistical analysis revealed that method C which represented the combination of nitric-
hydrochloric acids HNO3–HCl in a ratio 1:3 was the most efficient digestion method for
herbal product samples as it had given a significant high recovery (p < 0.05) for all metals
compared to method A and method B. Accuracy of the proposed method was evaluated by
the analysis of standard reference material (SRM) 1515 Apple Leaves from the National
Institute of Standards and Technology (NIST) which presented good recoveries for all metals
ranging from 94.5 to 108 %.

Conclusion

Method C provides highest recovery for all the analytes under investigation using AAS in
herbal medicine samples.

Keywords
Traditional medicineAcid digestionHeavy metalsNitric acid

Background
Traditional medicine (TM) has a significant contribution to the global health care system
(Chan 2003). A significant proportion of the world’s population relies on TM to support their
basic health care needs (Jayaraj 2010). Therefore, safety and quality of such products become
a major concern (Igweze et al. 2012). Inorganic contaminants such as heavy metals are often
present in herbal medicine in various concentration levels (Saeed 2010; Hina et al. 2011;
Qing-hua et al. 2001). The presence of heavy metals in such products is either referred to the
ingredients itself or they might arise during the processing part (Sharma and Dubey 2005).
Arsenic, cadmium, lead, and nickel are toxic heavy metals that might be present in TM
(Uddin et al. 2012). Prolonged exposure to these metals may cause many adverse health

56
effects including cancer (Ray and Ray 2009). Although zinc and iron are essential metals for
the human body at trace concentrations yet, they are toxic if present in higher concentrations
(Vaikosen and Alade 2011). Consequently, heavy metal content in TM products must be
accurately determined. Highly sensitive spectroscopic techniques such as flame (FAAS),
graphite furnace (GFAAS), and hydride generation atomic absorption spectrometries
(HGAAS) are mainly applied for elemental analysis in various samples. Such techniques
require aqueous samples. Thus, solid samples need to be regularly converted into solutions
using an appropriate dissolution method (Charun and John 2006). Acid digestion methods are
generally used for the dissolution of herbal product samples prior to elemental analysis
(Duyusen and Görkem 2011). In a spectroscopic elemental analysis sample preparation, acid
digestion is an important step of the entire analytical procedure. It has a substantial effect on
the recovery of various analyte contents in highly complex matrices such as herb and plant
materials. Therefore, it requires further improvement to provide a standard technique that is
able to gain accurate results (Nabil 2010). It is essential to assess the digestion efficiency of
various digestion methods to achieve the optimal sample preparation method with clearer
background (low noise level). Majority of samples are dissolved by various acids prior to
spectroscopic elemental analysis. Wet/acid digestion has the benefits of being effective on
both organic and inorganic substances as it has the ability to destroy the sample matrix and
consequently minimize the interference. However, at this preliminary stage of the analytical
processes, there are still some sources of potential errors such as incomplete digestion.
Rational selection of the acid combinations used for various sample digestions is very
important to achieve the reliable analytical method. Nitric acid is often utilized for this
purpose as an oxidant reagent either individually or mixed with other digestion reagents such
as acids and/or hydrogen peroxide. The oxidizing capacity, accessibility, and the affordability
of nitric acid make it prevalent in this respect (Sastre et al. 2002). This study was aimed to
assess the digestion efficiency of three acid digestion methods namely A, B, and C which
represented a combination of nitric-perchloric acids HNO3–HClO4 in a ratio of 2:1, only
nitric acid HNO3, and a mixture of nitric-hydrochloric acids HNO3–HCl in a ratio of 1:3,
respectively. Five TM samples of herbal origin were digested with the abovementioned
methods. The digestion processes were conducted using the conventional open vessel heating
system as it provided the advantage of low equipment cost (Güler and Arzu 2006). The
analysis of heavy metals was conducted using various techniques of atomic absorption
spectrometry (AAS).

Methods
Traditional medicine samples

Finished products of traditional medicine samples were collected from three different states
in the East Coast region of Peninsular Malaysia, namely Pahang, Terengganu, and Kelantan,
from various commercial places of the sampling area. Finished herbal products used for
medical purposes are herbal preparations that underwent all stages of production including
packaging. They might consist of different herbs/plants, various parts of the same plants, and
plant extracts. Five samples of herbal origin in capsule and tablet dosage forms were used to
perform the optimization of acid digestion method.

57
Chemicals and sample preparation

All chemicals and reagents used in this study were of analytical and trace metal grades. Trace
metal grades 65 % HNO3, 37 % HCl, and 70 % HClO4 were obtained from Fisher Malaysia.
Stock standard solutions for each metal arsenic (As), cadmium (Cd), lead (Pb), nickel (Ni),
zinc (Zn), and iron (Fe) with a concentration of 1000 ppm were supplied by Perkin Elmer
USA. Deionized water was used throughout the study. Sodium borohydride (NaBH4), sodium
hydroxide (NaOH), l-ascorbic acid (C6H8O6), and potassium iodide (KI) were from Merck
(Germany). A standard reference material (SRM) 1515 Apple Leaves was obtained from the
National Institute of Standards and Technology (NIST, USA). All glassware were soaked in
5 % (v/v) HNO3 overnight then rinsed with deionized water and dried using lab dryer FDD-
720 prior to use.

Methods of digestion

Samples were accurately weighed (0.5 g each) and placed in a 100-mL PTFE beaker. The
samples were subjected to three different acid digestion methods, as will be explained, to
identify the most appropriate digestion method to determine the contents of As, Pb, Cd, Ni,
Zn, and Fe in TM samples by AAS.

Method A (nitric-perchloric acid digestion 2:1)

To the sample, 5 mL of 65 % HNO3 was added, and then the mixture was boiled gently for
30–45 min. After cooling, 2.5 mL of 70 % HClO4 was added, and the mixture was gently
boiled until dense white fumes appeared. Later, the mixture was allowed to cool, and 10 mL
of deionized water was added followed by further boiling until the fumes were totally
released (Hseu 2004).

Method B (nitric acid digestion)

To the sample, 5 mL of 65 % HNO3 was added, and then the mixture was boiled gently over
a water bath (90 °C) for 1–2 h or until a clear solution was obtained. Later, 2.5 mL of 65 %
HNO3 was added, followed by further heating until total digestion (Zheljazkov and Nielson
1996).

Method C (nitric-hydrochloric acid digestion 1:3)

To the sample, 9 mL of freshly prepared acid mixture of 65 % HNO3 was added, and 37 %
HCl was added. Then, the mixture was boiled gently over a water bath (95 °C) for 4–5 h (or
until the sample had completely dissolved) (Ang and Lee 2005).

During the digestion procedures, the inner walls of the beakers were washed with 2 mL of
deionized water to prevent the loss of the sample, and at the last part of the digestion
processes, the samples were filtered with Whatman 42 (2.5-μm particle retention) filter paper.
Then, a sufficient amount of deionized water was added to make the final volume up to
50 mL.

58
Analytical procedure
Heavy metals were measured using a Perkin Elmer atomic absorption spectrometer (AAnalyst 800).
In this study, three different AAS techniques were used for elemental measurements in certified and
real samples of finished herbal products. The instrument is designed to analyze the sample using
three different atomization techniques: FAAS for Zn and Fe; GFAAS for Cd, Pb, and Ni; and HGAAS for
As. In FAAS, the aqueous sample is aspirated in the flame atomizer by the nebulizer to measure the
analyte concentration at a parts per million (ppm) concentration level with good precision. Pb, Cd,
and Ni were analyzed by GFAAS. It is an efficient measurement system for a number of elements at
relatively low levels of concentration with the use of several matrix modifiers (Shah et al. 2009). In
GFAAS, the instrument is equipped with a transverse heated graphite atomizer (THGA) which
provides uniform temperature distribution across the entire length of the graphite tube atomizer to
overcome the potential chemical interference effects; also, the instrument offers an auto-sampler
system and provides an accurate background correction (Zeeman correction). Arsenic was detected
by the HGAAS method which is based on the reaction of NaBH4 with acidified sample results in total
separation of the analyte as hydride from the matrix before measurement which reduces the matrix
interferences. In this technique, standards and samples were pre-reduced from an arsenate
pentavalent (V) to an arsenite trivalent (III) state. This was achieved by adding a reducing solution
containing 5 % (w/v) KI, 5 % (w/v) ascorbic acid, and 10 % HCl. The treated samples and standards
were allowed to stand at room temperature for approximately 40 min prior to analysis. Table 1
shows the instrumental parameters of FAAS, GFAAS, and HGAAS for all metals.

Table 1

Instrumental parameters for FAAS of Zn and Fe analysis; GFAAS for Cd, Pb, and Ni
analysis; and HGAAS for As analysis

Instrumental parameters FAAS Zn/Fe GFAAS Cd/Pb/Ni HGAAS As

Wavelength (nm) 213.9/248.3 228.8/283.3/232.0 193.7
Slit (nm) 0.7/0.2 0.7/0.7/0.2 0.7
Lamp type HCL/HCL HCL/EDL/HCL EDL
Atomization temp. (°C) 2300/2300 1400/1500/2300 900
Analysis of the standard reference material

The accuracy of the optimize method was verified by the analysis of SRM 1515 Apple
Leaves.

Statistical analysis

Results were expressed as the mean of triplicates ± standard deviation (SD). The data were
analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for
multiple comparisons using SPSS.

Results and discussion

59
The results obtained from all experiments indicated that method C which represented the mixture of
HNO3–HCl had given the highest analyte recovery for As, Cd, Pb, Ni, Zn, and Fe in TM samples.
Method A using the HNO3–HClO4 mixture and method B using HNO3 only gave convergent recoveries
for all elements. The mean values of As, Cd, Pb, Ni, Zn, and Fe with standard deviation for each
method are shown in Table 2. The differences were found statistically significant with a p value <0.05
for all TM samples digested with method C for all elements (Figs. 1 and 2).

Table 2

Concentration of different metals (μg/g) (±SD) in traditional medicine samples (TM 1–TM 5)
using different acid digestion methods

Sample ID As Cd Pb Ni Zn Fe
TM 1            

 A 0.25 (±0.02)a 0.25 (±0.004) 4.177 (±0.09) 6.26 (±0.07) 51.7 (±0.1) 219 (±0.7)
 B 0.24 (±0.02) 0.19 (±0.009) 4.16 (0.04) 6.30 (±0.02) 46.9 (±0.3) 154 (±0.7)
 C 0.3 (±0.04) 0.33 (±0.004) 4.66 (±0.02) 7.02 (±0.9) 56.8 (±1.3) 306 (±1.6)
TM 2            

 A 0.25 (±0.03) 0.19 (±0.004) 2.545 (±0.1) 4.54 (±0.05) 37.6 (±0.02) 213 (±1.2)
 B 0.27 (±0.02) 0.15 (±0.02) 2.41 (±0.09) 4.26 (±0.02) 36.2 (±0.01) 100 (±0.7)
 C 0.33 (±0.03) 0.26 (±0.002) 2.95 (±0.01) 4.70 (±0.16) 41.5 (±0.2) 303 (±1.1)
TM 3            

 A 0.29 (±0.04) 0.12 (±0.001) 4.94 (±0.04) 4.60 (±0.01) 11.9 (±0.1) 114 (±0.5)
 B 0.73 (±0.04) 0.11 (±0.001) 4.94 (±0.04) 4.64 (±0.01) 11.4 (±0.1) 93 (±1.9)
 C 1.2 (±0.06) 0.14 (±0.004) 5.39 (±0.05) 4.89 (±0.02) 14.3 (±0.1) 168 (±0.5)
TM 4            

 A 1.2 (±0.06) 0.15 (±0.001) 2.48 (±0.3) 4.40 (±0.1) 20.4 (±0.1) 227 (±2.0)
 B 0.85 (±0.01) 0.12 (±0.001) 2.32 (±0.07) 4.28 (±0.07) 19.5 (±0.3) 198 (±1.7)
 C 1.9 (±0.12) 0.24 (±0.001) 2.93 (±0.03) 4.80 (±0.12) 23.1 (±0.3) 688 (±2)
TM 5            

 A 1.3 (±0.06) 0.18 (±0.03) 1.95 (±0.09) 5.32 (±0.09) 44.2 (±0.3) 108 (±0.1)
 B 0.88 (±0.03) 0.18 (±0.03) 1.88 (±0.10) 5.00 (±0.03) 42.3 (±0.3) 92 (±0.7)
 C 1.6 (±0.01) 0.25 (±0.003) 2.50 (±0.3) 5.80 (±0.29) 52.3 (±1) 127 (±0.5)

TM traditional medicine, A nitric-perchloric acid, B nitric acid, C nitric-hydrochloric acid

a
Results presented as the mean of triplicates (±SD)

60
Fig. 1

Concentration of As, Cd, and Pb (μg/g) in traditional medicine samples using methods A, B,
and C

61
Fig. 2

Concentration of Fe, Ni, and Zn (μg/g) in traditional medicine samples using methods A, B,
and C

62
The accuracy of the proposed method was checked by the analysis of SRM 1515 Apple Leaves
obtained from NIST. The results indicate good agreement between measured and certified values,
and the recovery percentage for all metals was in the range 94.5–108 % within the specification limit
of AOAC guidelines which verifies the accuracy of the method. Table 3 shows the results for metals
content in SRM.

Table 3

Recovery percentages and concentrations of As, Cd, Pb, Ni, Zn, and Fe in SRM (1515)

Analyte SRM conc. μg/g (±SD) Measured conc. μg/g (±SD) Recovery (%)
As 0.038 (±0.007) 0.039 (±0.002) 102
Cd 0.013 (±0.002) 0.012 (±0.009) 96
Pb 0.47 (±0.02) 0.5 (±0.02) 106
Ni 0.91 (±0.12) 0.86 (±0.004) 94.5
Zn 12.5 (±0.3) 13.5 (±0.3) 108
Fe 83 (±5) 83.3 (±4) 100

All the TM samples were contained heavy metals at different concentrations. Highest analyte
recoveries for all TM samples were gained using method C that ranged 0.3–1.9; 0.14–0.33;
2.5–4.66; 4.7–7.02; 14.3–56.8; and 127–688 μg/g for As, Cd, Pb, Ni, Zn, and Fe,
respectively. Based on the fact that TM are highly consumed worldwide, a significant
concern from many health institutes in different countries had imposed permissible limits of
heavy metals in raw/finished herbal product. In Canada, the maximum limits for As, Pb, and
Cd are 5, 10, and 0.3 ppm, respectively; in India, 10, 10, and 0.3 ppm for As, Pb, and Cd,
respectively (Gupta et al. 2010). It is equitable to assume that heavy metal intake through
such products has significant influence on human’s health. Therefore, an adequate method for
their determination is of importance.

Previous studies suggested various methods for digesting different samples for metal analysis
(Nabil 2010). Aqua regia has been proposed as the best digestion method for samples with
low carbonate or organic matter contents such as sediments and agricultural soils (Sastre et
al. 2002). Another study reported that there was no significant differences between the
digesting capacity of HNO3 acid and HNO3–HClO4 acid mixture in the measurement of
phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn),
zinc (Zn), and copper (Cu) contents of barley (Hordeum vulgare L. cv. Minorimugi) and rice
(Oryza sativa L. cv. Akihikari) seedlings (Shaibur, et al. 2010). Nitric acid digestion was
proposed as the most efficient method for recovering Cd, Mn, and Ni in the majority of
composts samples (Hseu 2004). Another study recommended the combination of HNO3–HCl
in a ratio of 1:2 as the most efficient digestion method which yielded the highest recovery of
Pb, Zn, and Fe in canned sardines samples (Fong et al. 2006). However, TM samples have
complex matrices as they are made from either one herb or a mixture of herbs from any part
of a plant such as leaves, roots, seeds, and flowers that might have different chemical
properties. The digestion capacity of hydrochloric-nitric acids HNO3–HCl in a ratio of 1:3
mixture had proven to be the best acid combination suitable for the decomposition of TM
samples due to the ability of such mixture to release the metal ions from such complex
matrices of herbal materials and subsequently to minimize the noise level during the
detection procedure.

63
Conclusions
Sample preparation is a crucial step in spectroscopic elemental analyses as it can
considerably affect the accuracy of results. Significant differences between the digesting
capacities of different methods were identified. The digestion capacity using a mixture of
hydrochloric-nitric acids HNO3–HCl in a ratio of 1:3 was the most efficient method in terms
of the recovery of As, Cd, Pb, Ni, Zn, and Fe in herbal medicine samples.

Abbreviations
AAS: 

atomic absorption spectrometer

AOAC: 

American Organization of Analytical Chemistry

ANOVA: 

analysis of variance

As: 

arsenic

Cd: 

cadmium

EDL: 

electrodeless discharge lamp

FAAS: 

flame atomic absorption spectrometer

Fe: 

iron

GFAAS: 

graphite furnace atomic absorption spectrometer

HCL: 

64
 hollow-cathode lamp

HCl: 

hydrochloric acid

HClO4 : 

perchloric acid

HNO3 : 

nitric acid

KI: 

potassium iodide

NaBH4 : 

sodium borohydride

NaOH: 

sodium hydroxide

HGAAS: 

hydride generation atomic absorption spectrometer

Mg(NO)3 : 

magnesium nitrate

NaOH: 

sodium hydroxide

Ni: 

nickel

NIST: 

National Institute of Standards and Technology

Pb: 

65
 lead

SD: 

standard deviation

SPSS: 

Statistical Package for Social Sciences

SRM: 

standard reference material

Zn: 

zinc

Declarations
Acknowledgements

The authors would like to express their gratitude to all the academic and technical staff of the
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, IIUM, for their support.
They are also thankful to the Research Management Centre (RMC), IIUM, and Ministry of
Higher Education (MOHE), Malaysia, for funding this study.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution
4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made.

Competing interests

The authors declare that they have no competing interests.

Authors' contribution

RSK, ABM HU, MA has conducted the experimental part and manuscript preparation. AA,
SAA has contributed for the statistical analysis and AK and SAA has edited the paper for
technical improvement.

Authors’ Affiliations
66
(1)

Analytical and Bio Analytical Research Laboratory, Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, International Islamic University Malaysia (IIUM)

(2)

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, International Islamic University

Malaysia (IIUM)

(3)

Department of Basic medical Science, Faculty of Pharmacy, International Islamic University Malaysia
(IIUM)

(4)

Taylors University, 1, Jalan Taylor’s

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Table of Contents

 Abstract
 Background
 Methods
 Results and discussion
 Conclusions
 Declarations
 References

68
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Microchim. Acta 148, 1–20 (2004)

DOI 10.1007/s00604-004-0246-y
Fundamental Review
What Flow Injection has to Offer in the Environmental
Analytical Field
Manuel Miro
́
1
;
and
Wolfgang Frenzel
2
1
Department of Chemistry, Faculty of Sciences, University of the Balearic Islands, Carretera de
Valldemossa,
Km. 7.5, 07122 Palma de Mallorca, Illes Balears, Spain
2
Institut f
€
uur Technischen Umweltschutz, Technische Universit
€
aat Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany
Received December 5, 2003; accepted February 16, 2004; published online July 19, 2004
#
Springer-Verlag 2004
Abstract.
Flow injection analysis (FIA) is presented
as a powerful tool to meet the analytical requirements
of environmental and agricultural assays in terms of
automation, monitoring, microsampling, speciation
and sample processing. Its potentials and limitations
are critically discussed and demonstrated in the body
of the text with relevant examples devoted to water, air,
soil and plant analysis. The features of this continuous
flowing stream technique are classified according to
the particular role that FIA plays as a universal han-
dling system interfacing wet chemical reactions with
analytical instrumentation. Special attention is paid
to its assets as a liquid delivery system, chemical de-
rivatization method as well as on-line sample pre-
treatment tool.
Key words:
Flow injection; environment; agriculture; solution
handling system; on-line sample pre-treatment; monitoring.
Since the late sixties, the acceptance of a correlation
between environmental protection and standard of liv-
ing as well as quality of agricultural products has

70
increased people’s sensitivity towards environmental
problems. This led to the need for environmental
surveillance and routine control of a large number of
relevant parameters in an increasing number of sam-
ples within short periods of time. Aiming to overcome
the high workload, analytical laboratories started to
perform the classical wet-chemical methods in an au-
tomatic fashion. Air segmented flow analysis (SFA)
introduced by Skeegs [1] in the late fifties, and ini-
tially applied to the clinical field under the tradename
of Technicon, was the first automatic flowing tech-
nique widely employed in environmental laboratories
[2]. In spite of the massive acceptance of SFA auto-
analysers in the various analytical fields, the presence
of air bubbles in the flow conduits gives rise to a series
of drawbacks which were thoroughly presented by
Valca
́
rcel and Luque de Castro [3], and detailed as
follows: the air-segmented system becomes tech-
nically complicated, hindering its miniaturisation;
the compressibility of the gas phase results in some
degree of pulsation, which affects the signal profile;
the efficiency of continuous separation systems in-
corporated into the flow network is decreased as a
consequence of the reduction of the effective transfer
surface, and finally, the flow velocity is not easily
controlled.
Flow injection analysis (FIA) proposed by Hansen
and Ruzicka [4] in the mid-seventies has been consoli-
dated as a non-segmented flow alternative to SFA. The
outstanding features of FIA for analytical applications
Author for correspondence. E-mails: [email protected],
[email protected]
are its extremely straightforward configuration, easy
operation, low costs and compatibility with almost
any detection principle [5, 6]. Automated analyses
with high sample throughput are attained using rela-
tively simple instrumentation. The micro-format of
FIA results in a considerable reduction of sample
and reagent consumption, typically one or two orders
of magnitude in comparison with the manual counter-
parts, which is especially desirable in the case of
environmentally harmful chemicals. Furthermore, as
opposed to SFA methodologies, the reproducible tim-
ing and readily controllable dispersion of the analyte
zone allows the straightforward performance of ki-
71
netic assays [7]. Other differential features of FIA in
relation to its forerunner SFA are the injection of
smaller sample volumes (of the order of 10–100
m
L),
the suppression of the washing cycles, the increase of
the sample throughput up to 120 injections h
1
,
repeatability improvement as well as the ease of mani-
fold construction and flexibility. Changing over from
one determination method to another generally only
takes a few minutes and makes FIA cost-effective
even when a limited number of samples are to be
analysed for several constituents. Moreover, due to
the inherent miniaturisation of FIA and the high ana-
lytical throughput, a considerable decrease in waste
generation, and hence also in costs for waste disposal,
is achieved. In this context, the impact exerted by
flow-based analytical chips on the development of
miniaturised lab-on-chip instrumentation is also worth
mentioning [8].
FIA has attracted the attention of many researchers
owing to its ability to fulfil the current demands of
environmental analysis. The fast response of FIA
makes the analytical information available in real-
time, which is especially valuable in monitoring
schemes. FIA has also been exploited for in-field anal-
ysis aiming to obtain high temporal and spatial reso-
lution data without the need for discrete sample
collection and storage [9]. This is of great interest
Fig. 1.
Schematic representation of (a) Flow
Injection and (b) Sequential Injection mani-
folds. The merging of reagent and sample
zone in the reaction coil of FIA systems,
and stacking of segments in the holding coil
of SIA assemblies are also illustrated.
C
Car-
rier;
R
Reagent;
S
Sample;
D
Detector;
PP
Peristaltic Pump;
SP

72
Syringe Pump;
RC
Re-
action Coil;
HC
Holding Coil;
MV
Multiposi-
tion Valve;
W
Waste
2
M. Mir
oo and W. Frenzel
whenever off-line analysis or excessive sample han-
dling is unacceptable due to the rapid transformation
of the target analyte.
Though FIA is basically a sequential method, i.e. a
single parameter is determined in a series of samples,
multichannel-FIA analysers and multi-analyte detec-
tors have been successfully applied to simultaneous
determinations. In various environmental applications
use is made of the different kinetic behaviour of the
analytes with a common reagent [10, 11], the multi-
detection capability of a single detection system, such
as a diode-array spectrophotometer, in combination
with chemometric multivariate methods [12, 13], the
serial or parallel arrangement of several detection sys-
tems within a single flow manifold [14–17] or the
microfluidic manipulation of the sample plug to allow
for different chemical reactions for the individual ana-
lytes and conventional detection of the reaction prod-
ucts [15, 18, 19]. As a consequence of the flexibility
for accommodating simultaneous determinations, FIA
is an excellent tool for speciation analyses which are
of increasing concern in environmental studies [20].
Thus, specific oxidation states of species as well as
inorganic and organic forms of elements are acces-
sible from a single sample injection [18, 20, 21]. In
this respect, particular advantages also result from the
fact that dynamic equilibria in the environmental sys-
tems under exploration are less distorted because of
the fast response and short residence times typical for
FIA procedures.
In the context of flowing techniques applied to envi-
ronmental surveillance, Sequential Injection Analysis
Fig. 2.
Flow injection analysis conceived as
an analytical tool for interfacing sample and
detector

73
What Flow Injection has to Offer in the Environmental Analytical Field
3
(SIA) proposed in 1990 [22] as a new concept for
handling solutions, alternatively to FIA, should be
highlighted. As opposed to FIA, SIA methodology is
a fully software-controlled discontinuous technique
based on the sequential stacking of precise volumes
of sample and reagent(s) into the holding coil, which
afterwards are dispensed by flow reversal into the
reaction coil, and further towards the flow-through
detector. Therefore, considerable sample and reagent
saving is achieved – of the order of 20 times in rela-
tion to FIA. The replacement of the common rotary
injection valve used in FIA with a multiposition valve
has enabled the incorporation of additional reactors
and modules, the performance of backward-forward
flow movements, the exploitation of stopped-flow
schemes, and the simple development of multipara-
metric determinations by coupling different detectors
and appropriate reagents to the selection valve
[23]. The use of syringe pumps as liquid drivers has
allowed the manipulation of sample and reagent vol-
umes at low
m
L levels with high precision.
The microanalytical features of SIA along with the
handling of the exact volumes required for the particu-
lar assays have opened new avenues in the monitoring
of environmentally relevant parameters [24]. For
example, atmospheric precipitates are often only pres-
ent in limited quantities, or long collection times are
required, so that microsampling is extremely benefi-
cial. The main drawback of SIA for analytical applica-
tions is the lower sampling rate achieved in relation to
FIA as a consequence of periodical filling of the liquid
driver and stacking the entire set of segments in the
holding coil prior to detection. Besides, the sequential
propelling of the solutions which takes place in SIA
results in longer residence times for proper mixing of
adjacent zones. As a consequence of their different
features, FIA and SIA can be considered complemen-
tary techniques. Figure 1 shows typical flow injection
and sequential injection arrangements assembled for
on-line determinations using a single reagent. The
basic differences between both concepts regarding
the introduction of sample and reagent solutions into

74
the flow network are also highlighted.
According to the above considerations, flow tech-
niques can, in a more general view, be regarded as
universal solution handling systems interfacing sam-
ple with detection instruments [25, 26], whose virtues
for environmental applications are presented in detail
in the body of the text. The potentials and drawbacks
are critically discussed and exemplified with relevant
examples devoted to water, air, soil and plant analysis.
The applications related to aqueous solutions of envi-
ronmental origin are classified in accordance with the
particular role that flow systems play in sample-detec-
tor hyphenation, as presented schematically in Fig. 2.
Flow Injection as a Liquid Delivery System
to the Detector
Flow injection analysis can be simply described as an
automated system for reproducible sample presenta-
tion to the detector [27, 28] (see Fig. 3a). In contrast
to chromatographic techniques relying on separation
schemes, flow systems need to be hyphenated with
high discrimination instruments. The potentials of
coupling FIA with continuously operating atomic
spectrometric detectors, such as flame atomic absorp-
tion spectrometers (FAAS) and inductively coupled
plasma optical emission (ICP-OES) or ICP-mass
spectrometers (ICP-MS) for trace metal determina-
tions, have been highlighted in several monographs
[29, 30]. Particularly remarkable is the enhanced
reliability owing to sample processing in an inert
closed system, which minimizes contamination risks
and additionally prevents technicians coming into
contact with hazardous chemicals. The major attrac-
tion of FIA-ICPMS coupling is its multi-elemental
capability combined with high speed of analysis and
excellent sensitivity [31, 32]. In addition, the possibil-
ity of performing isotope and on-line isotope dilution
analysis offers unique applications in the environmen-
tal field [33, 34]. The capabilities of FIA-time-of-
flight-MS as a fast transient detector for simultaneous
determination of a large number of isotopes in an FIA
peak have recently been demonstrated by Adams et al.
[35].
Flow rates of FIA systems for sample introduction
are usually compatible with the continuous working
mode of the aforementioned techniques and permit
on-line operation using relatively simple interfaces.

75
The interfacing of FIA with ICP-MS, for example, is
readily attained by connecting the outlet of the FIA
manifold with the liquid flow inlet of the nebuliser
through a short length of narrow-bore tubing [35].
Besides, improvement of the nebulisation efficiency
and facile adjustment of the signal magnitude is
accomplished via optimised automatic methods. It is
also reported that the injection of sample into a con-
tinuously pumped carrier solution at a constant flow
rate (instead of the conventional aspiration) minimizes
4
M. Mir
oo and W. Frenzel
transport interferences due to differences in viscosity
of the samples and eliminates carry-over effects be-
tween consecutive samples owing to the periodical
rinsing of the nebuliser [36]. The ability of handling
sample volumes at the
m
L level also prevents some
interfering processes, such as matrix deposition in
the nebuliser, and enables the use of organic solvents,
which may be incompatible with the detector (e.g.
ICP) in a continuous fashion. The microanalytical fea-
tures of FIA with a typical sample consumption of
10–200
m
L are particularly important when a limited
amount of sample is available and can also be of
advantage in the analysis of solid samples since
microanalytical procedures with smaller sample mass
and simplified sample pre-treatment schemes can be
adopted [37].
Although custom-built interfaces have been designed
for coupling FIA with electrothermal atomic absorption
spectrometry (ETAAS) [38], it should be emphasised
that the discontinuous operation of the SIA technique
is better suited than FIA systems for hyphenation
with ETAAS as a consequence of the discrete, non-
continuous operation of the detector [39]. Furthermore,
the minute volumes of solutions able to be accommo-
dated in the graphite tube (
<
50
m
L) are easily manipu-
lated with the automated SIA assembly, and generally
76
injected into the furnace via air-segmentation.
Flow injection also has much to offer in combina-
tion with electrochemical techniques such as voltam-
metry and potentiometry. In FIA measurements, it is
easier to avoid incidental contaminations or those
resulting from the solution leaking from the reference
electrode. Moreover, deactivation of the sensing sur-
face of the working or indicator electrode due to
adsorption, precipitation or corrosion is greatly mini-
mized as a consequence of the short contact time with
the sample plug [40]. An additional advantage result-
ing from the continuous flowing of the sample zone is
the improved selectivity attained by exploiting the
mechanism of kinetic discrimination between the ana-
lyte and the interfering species for potentiometric
measurements [40, 41]. The response time limitation
of potentiometric detectors under dynamic regime for
low analyte concentrations, which is observed mainly
as a slow return of the potential value to the baseline
level, can be readily overcome in FIA by adding a
Fig. 3.
Basic flow injection configurations
exploited for (a) sample presentation to
high discrimination detectors (b) homoge-
neous and (c) heterogeneous reactions.
C
Carrier;
PP
Peristaltic pump;
OS
Optional
stream;
MC
Mixing coil;
HDD
High dis-
crimination detector;
R
Reagent;
D
Detector;
W
Waste
What Flow Injection has to Offer in the Environmental Analytical Field
5
small concentration of the sensed ion to the carrier
stream [42]. Flow systems are also useful schemes
for proper sample presentation to the selective sensor
since common strategies such as buffering, ionic-
strength adjustment, pH control and addition of mask-
ing agents are easily performed [43].

77
Flow injection
=
Sequential injection anodic strip-
ping voltammetry should be highlighted as a suitable
alternative for the determination of metal ions at trace
levels owing to the straightforward change of chemi-
cal conditions during the different stages of the ana-
lytical procedure as well as the contamination control
through the automation of the analyses [44].
It should be stressed in this section that unseg-
mented flow systems have also played an outstanding
role in the enhanced performance of probe-type (bio)
sensors applied to environmental monitoring. The
salient assets resulting from the implementation of a
(bio)sensor in a continuously flowing stream are the
facile system handling, high analytical throughput,
and the possibility of automating both the probe (or
sample) conditioning and regeneration steps. In addi-
tion, the short contact time of the sample zone with
the active microzone yields a remarkable tolerance
improvement to matrix interferents and enlarges the
sensor lifetime [45].
Flow Injection for Chemical Derivatization
Since the early development of flow injection analy-
sis, many environmental applications have been based
on its role as an automatic chemical derivatization
system. FIA has proven to be an ideal vehicle for
the conversion of non-detectable analytes into detect-
able species through kinetically controlled chemical
reactions. In fact, as depicted in Fig. 3b, flow tech-
niques can be defined as analytical tools able to perform
in a continuous fashion the basic stages of any deriv-
atization reaction in homogeneous phase (i.e. pour-
ing sample with reagent, mixing, and measuring) via
installation of T-connectors, reaction coils and on-line
detectors into the flow network. Molecular spectro-
scopic detection principles such as spectrophotometry,
turbidimetry, spectrofluorimetry and chemilumines-
cence are fully compatible with FIA, as demonstrated
by the multitude of environmental applications for
inorganic species and organic compounds reported
to date and recently reviewed [46–49]. It should
be stressed that controlled chemical conditions can be
created in a highly repeatable manner and that
measurements are performed with the cuvette always
left in place with rapid alternate readings of baseline

78
and peak signal rather than permanent exchange
of cuvettes to empty, fill, and measure in sequence.
With respect to fluorescence determinations, additional
benefits are taken from the minimization of oxygen
quenching due to the closed environment provided by
the automated technique. FIA should also be high-
lighted as a unique strategy for accommodation of
fast chemiluminescence reactions, most often involv-
ing catalysed-luminol oxidation [50, 51], wherein light
emission with half-lives of only a few milliseconds
occurs instantaneously upon merging the sample plug
and the chemiluminogenic reagent. The highly repro-
ducible mixing of streams and controllable timing
makes possible novel applications never thought
of before. The monitoring of unstable intermediate
reaction products clearly illustrates the powerful ca-
pabilities of FIA. For example, the classic spectro-
photometric determination of cyanide is based on
halogenation of the target species with chloramine-
T, whereafter it reacts with a mixture of pyrazolone
or barbituric acid to form a violet polymethine dye.
Yet the determination of the metastable red-coloured
intermediate product allows significant sensitivity im-
provement since it exhibits higher molar absorptivity
[52].
As a consequence of the aforementioned advan-
tages of FIA for homogeneous chemical derivatiza-
tion, several flowing stream methodologies applied
to the determination of inorganic ions (viz., chloride,
nitrite, nitrate, ammonium, orthophosphate, silicate
and cyanide) and organic compounds such as phenolic
derivatives as well as surfactants in environmental
water samples have already been accepted as official
standard methods (see Table 1 for further details),
gradually replacing the manual counterparts.
The use of solid-phase reactors for heterogeneous
chemical derivatization should be considered as one
of the most rapidly developing and challenging areas
in FIA research [53]. Installing packed-bed microcol-
umns in the flow manifold for on-line analyte reduc-
tion or oxidation has proven an excellent avenue for
speciation studies in the environmental field [18, 20].
As shown in Table 1, the standard method for nitrate
determination relies on the heterogeneous reduction
with copperized cadmium beads, and full benefit is
taken from the precise kinetic control aiming to pre-

79
vent overeduction phenomena [54]. A worthwhile
virtue of FIA as opposed to batch procedures is the
6
M. Mir
oo and W. Frenzel
likelihood of using unstable reagents in solution
via entrapment into suitable matrices, thus obtaining
highly stable reagent sources, or in-line generation
of the active species at a solid-phase redox reactor
[55]. Displacement reactions based on the detection
of the delivered species ensuing analyte passage
through a packed column containing an insoluble salt
have attracted the interest of many researchers
[56, 57] owing to the feasibility of reagent reutilisa-
tion, precipitate collection in the reactor as well as on-
line hyphenation with various detection instruments
such as spectrophotometers or atomic absorption
spectrometers.
The coupling of catalytic heterogeneous reactions
using packed-bed or open-tubular enzyme reactors
with numerous detection techniques such as chemilu-
minescence, spectrophotometry, potentiometry, spec-
trofluorimetry or amperometry has been presented as
a useful on-line approach for indirect determinations
[58]. These FIA methods offer the inherent selectivity
of the enzymatic reactions together with the economy
gained by the immobilisation of the often costly
catalysts. Though a wealth of flow systems have
been devised for monitoring substrates in biological
matrices, the inhibition of enzymatic reactions caused
by pesticide residues (e.g. organophosphate, carbamate
Table 1.
Analytical performance of currently available EN
=
ISO standard methods for monitoring relevant environmental parameters in
water samples using flow injection analysis
Parameter
Official method
Detection
Method
=
reaction
Dynamic
linear range
(mg L
1
)
Water matrices
Ammonium
EN
=

80
ISO
11732 (1997)
photometry
gas-diffusion
(colour change
of pH indicator)
0.1–10
ground, drinking,
surface and
wastewater
Nitrite
EN
=
ISO
13395 (1996)
photometry
Griess-Ilosvay
0.01–1.0
drinking, surface,
ground and
wastewater
Oxidised
nitrogen
EN
=
ISO
13395 (1996)
photometry
Cu-Cd reduction
=
Griess-Ilosvay
0.2–20
ground, drinking,
surface and
wastewater
Orthophosphate
EN
=
ISO
15681 (2001)
photometry
molybdenum blue
0.01–1.0
drinking, surface,
ground and
wastewater
Dissolved
organic
phosphorous
EN
=
ISO
15681 (2001)
photometry
UV-photooxidation
=
molybdenum blue

81
0.1–10
drinking, surface,
ground and
wastewater
Chloride
EN
=
ISO
15682 (2000)
potentiometry
and photometry
mercury(II)thiocyanate
(photometry)
ion-selective
electrode (potentiometry)
1.0–1000
surface, ground,
drinking, wastewater
Free and
total cyanide
EN
=
ISO
14403 (2000)
photometry and
amperometry
gas-diffusion
=
chloramine-T
method (photometry)
gas-diffusion
=
direct
detection at a Pt working
electrode (amperometry)
UV-irradiation for total
cyanide
0.01–0.1
industrial effluents,
waste, ground
and surface
Silicate
EN
=
ISO
16264 (2000)
photometry
molybdenum blue
=
masking
agents (tartaric or
oxalic acids)
0.1–10
ground, surface,
waste and drinking
Phenol index
EN

82
=
ISO
14402 (1999)
photometry
4-aminoantipyrine
=
in-line solvent extraction
0.01–1.0
industrial effluents,
surface, ground
and wastewater
Anionic
surfactants
EN
=
ISO
16264 (2000)
photometry
methylene blue
=
solvent extraction
0.1–10
surface, waste,
ground
Similar flow injection methods have been standardised by US-EPA, Japanese Authorities as well as
Regulatory Authorities from other
countries.
What Flow Injection has to Offer in the Environmental Analytical Field
7
and organochlorine pesticides) [59] and heavy metal
ions [60] has been proven effective for the deter-
mination of the above pollutants in environmental
matrices [61]. While the monitoring of residues is
based mainly on their inhibition action on cholines-
terases, for the determination of heavy metals various
immobilised enzymes, namely urease, glucose oxi-
dase, alkaline phosphatase, butyrylcholinesterase and
xanthine oxidase, are commonly exploited. Enzyme re-
actors have also been successfully implemented in spe-
ciation protocols. As an example, the determination of
specific phosphorous fractions through enzymic reac-
tions involving immobilised alkaline phosphatase [62]
or 3-phytase [63] coupled to the molybdenum blue
method has potential as an indicator of the pool of
biologically available and unavailable dissolved or-
ganic phosphorous, respectively.
The optimisation of heterogeneous reactions has
ultimately been linked to the increasing interest
in the development of electrochemical
=
optical flow-
through sensors based on monitoring the properties of
83
a reagent phase that change on exposure to the analyte
or an active derivative. In contrast to probe-type
designs, most of the optical flow-through sensors
reported to date use a dedicated thin layer as a sensing
zone which is implemented in the flow cell [64]. It is
generally prepared by immobilisation of an indicator,
chromogenic ligand, organic ionophore or catalyst in
a PVC matrix and supported by a cellulose filter paper
or quartz glass plate. These reversible bulk membrane
based sensors, which operate in a thermodynamic or
kinetic regime, can be regarded as simple and inex-
pensive alternatives to certain conventional analytical
methods in homogeneous phase [65]. Yet they usually
suffer from a lack of sensitivity concerning environ-
mental applications since, for example, the dynamic
working range for metal ions typically covers only the
mg L
1
level [65, 66]. Enrichment strategies capitalis-
ing on packed-bed flow-through cells containing
modified sorbents via adsorption and
=
or electrostatic
immobilisation of a reactive agent (mainly a chelator
or ligand) have been successfully explored for metal
trace analysis [67]. Sorbent extraction optosensing
using immobilised reagents features the integration
of separation, reaction, and enrichment with the detec-
tion process. Further benefits are obtained in spectro-
photometric measurements via minimization of the
Schlieren effect caused by refractive index differ-
ences, as well as the performance of multicomponent
determinations exploiting multivariate algorithms, dif-
ferences in sorption kinetics, or the sequential arrival
of analytes at the sensed microzone by on-line chro-
matographic or non-chromatographic separations
[68].
Figure 3c depicts a single flow injection manifold for
heterogeneous chemical derivatization, though it should
be borne in mind that the position of the solid reactor in
the flow network is directly related to the kind of solid
phase and intended analytical application.
Flow Injection as a Sample Pre-Treatment Tool
Flow injection and related flowing techniques play an
important role in the automation, acceleration and
miniaturisation of solution handling in sample pre-

84
treatment protocols [69, 70]. The on-line coupling of
continuous flow sample pre-treatment with chromato-
graphic systems (viz., HPLC, IC or GC) is an out-
standing venue for improving the overall efficiency
of methods based on column separation principles.
The main advantage of the FIA-HPLC
=
IC
=
GC hy-
phenation is the potential incorporation of various
on-line separation and preconcentration modules in
series into the flow assembly prior to chromatographic
resolution. As a result, the performance of chromato-
graphic separations applied to the determination of
pollutants at environmental levels in complicated
matrices is greatly enhanced, as recently demonstrated
by Luque de Castro’s and Valca
́
rcel’s groups [71–73].
These benefits are equally applicable to capillary elec-
trophoresis (CE). However, while the interfacing of
FIA with HPLC
=
IC is extremely simple since the
enriched, minute volume of solution containing the
isolated analytes directly feeds the injection loop
of the chromatograph, the FIA-CE coupling requires
specific split-flow interfaces as reported by Fang’s
[74] and Karlberg’s [75] groups. Hence, the flow-
through sample treatment may be readily hyphenated
with the electroosmotic flow driven CE system with-
out any further hindrance.
It should be emphasised that flow techniques are
powerful tools to fulfil the demands not only of
column separation systems but also of spectroscopic
detectors. Since flow injection can be defined as the
process of gathering information from a concentration
gradient formed by an injected zone of fluid dispersed
into a continuous unsegmented carrier stream, the
immediate consequence is the controlled sample
=
standard dilution which could, for example, be ex-
ploited for calibration purposes [76]. Higher dilution
8
M. Mir
oo and W. Frenzel

85
factors, mainly applied to the environmental determi-
nation of alkaline, alkaline-earth or transition metals
in combination with flame photometry or atomic
absorption spectrometry, were readily attained using
flow-splitting or zone sampling procedures [77], or by
the single or serial arrangement of flow-through par-
allel-plate dialysers [78] in the flow assembly.
Solvent extraction was the first sample processing
technique comprising analyte isolation and concentra-
tion adapted to automated flow systems. The foremost
asset was the minimization of the shortcomings that
had arisen from the batch counterparts, namely large
sample volumes, loss of analyte by manipulation,
low sampling frequency, contamination of laboratory
atmosphere with organic vapours and production of
large amounts of residual solvents. The essential steps
of a batch liquid–liquid extraction procedure, i.e.
introduction of the aqueous and organic phases, ana-
lyte transfer by shaking and physical separation of the
phases, are carried out in continuous flow systems
using a phase segmentor, an extraction coil and a
phase separator (shown schematically in Fig. 4a),
being – as stated by Kuban [79] – as many existing
designs as practical applications described. Despite its
Fig. 4.
Flow injection configurations used for
on-line sample pre-treatment involving either
separation
=
preconcentration techniques or diges-
tion schemes: (a) Liquid–liquid extraction. (b)
Sorbent microcolumn extraction. (c) Membrane
separation. (d) On-line digestion.
C
Carrier;
R
Reagent;
Org
Organic solvent;
PP
Peristaltic
pump;
Seg
Segmentor;
Sep
Separator;
EC
Extrac-
tion coil;
RC
Reaction coil;
D

86
Detector;
DS
Donor stream;
RS
Recipient stream;
MW
Microwave;
Usd
Ultrasound;
W
Waste
What Flow Injection has to Offer in the Environmental Analytical Field
9
initial progress concerning the separation and precon-
centration of heavy metals from a variety of environ-
mental samples [79, 80] and the establishment
of official standard methods for phenol index and
surfactant monitoring, its further development has
been rather slow since the repeatability, sensitivity
and accuracy of the methods are drastically limited
by the existence of the segmentor and phase separator.
Although overall performance of solvent extraction in
flowing systems can be improved exploiting novel
separator designs [81, 82], many efforts have been
made to the development of various alternatives pre-
venting the use of the former classic elements. In this
context, the exploitation of liquid membranes [83],
hydrophobic chromatomembranes [84] and the devel-
opment of the iterative reversed-flow technique [85]
should be emphasized. The ‘‘on-tube’’ detection prin-
ciple based on the software-controlled discrimination
between segments and determination of the extracted
analyte without phase separation has been success-
fully applied to the photometric monitoring of anionic
surfactants in environmental waters by ion-pairing
with methylene blue or rhodamine B [86, 87].
A noteworthy advance in terms of reliability, sensi-
tivity and avoidance of carryover between samples
was achieved with the advent of the novel sequential
injection
=
wetting-film methodology without segmen-
tation or separation of immiscible phases [88]. It is
based on the formation of a coating on the inner walls
of a PTFE tubular reactor with a thin organic layer as
a result of the hydrophobic interactions between the
solvent and reactor which delay the organic phase
with respect to the aqueous solutions. The enhanced
aqueous
87
=
organic phase volume ratio in comparison
with classical FIA solvent extraction renders high
concentration factors. This film is also responsible
for high extraction efficiencies, avoiding the axial
dispersion of the analyte occurring in segmented FIA
extraction. Wetting-film extraction has been demon-
strated suitable for the preconcentration of metal
traces (e.g. Cu, Cr) [89] in hyphenation with atomic
spectrometric detection, for metal speciation analysis
(viz., Cr(VI)
=
Cr(III) and V(V)
=
V(IV)) [90, 91] and
for multicomponent determination of various phenolic
compounds [92]. Particularly remarkable is the ca-
pability to process environmental samples for further
monitoring of anthropogenic radionuclides at low
activity levels [93]. The most serious shortcoming of
wetting-film extraction is the pseudo-stationary nature
of the organic phase [92] which requires careful opti-
misation of the hydrodynamic variables for each spe-
cific analytical application.
Despite the numerous applications of solvent
extraction in FIA
=
SIA systems, the predominating
sample processing method that has been rapidly grow-
ing in recent years as a consequence of the improved
concentration factors and considerable reduction of
organic solvent consumption is sorbent extraction.
Most often it is employed by using microcolumns
packed with appropriate bead materials (shown sche-
matically in Fig. 4b) and placed into the manifold
prior to detection [94]. This technique plays an im-
portant role in combination with atomic spectro-
metric detection, as has been demonstrated in several
recent reviews and monographs [35, 69, 70, 94–96].
The aim is to improve the sensitivity and selectivity of
the analytical methodology and
=
or overcome the in-
herent low tolerance of the detection system to sample
constituents via analyte pre-concentration and
=

88
or
removal of interfering components from complex
environmental matrices. Whenever trace analysis is
pursued, matrix separation concomitantly occurrs
with analyte enrichment.
A multitude of solid-phase reactors with different
designs have been successfully implemented in flow-
ing stream assemblies [97]. The temporary retention
of low levels of individual metal ions, nutrients, or
ionic complexes onto ion-exchanger packed-bed col-
umns or chelating reactors (generally containing imi-
nodiacetic acid or 8-hydroxyquinoline moieties) and
derivatised non-polar chelates (mainly dithiocarba-
mate or dithiophosphate derivatives) on reversed-
phase materials (e.g. octadecyl-chemically modified sil-
ica gel, PTFE beads or co-polymeric sorbents) have been
reported for environmental applications [94, 97, 98].
The sorption
=
elution strategy has commonly been ex-
ploited in hyphenation with various detection schemes.
Yet optical sensing at solid surfaces, involving direct
measurement of light attenuation of the sorbent phase
following the sorptive preconcentration of the target
analyte conveniently derivatised, is an excellent alter-
native to conventional procedures with eluate detection
[67]. The partial loss of the preconcentration capabil-
ities gained during the sorption step due to the mea-
surements in the eluate phase is thus circumvented.
The current state-of-the-art of the flow-through solid-
phase optosensing concept was reviewed very recently
[99], and potentialities and limitations of the various
optrode
=
sensor configurations for practical applications
have been thoroughly detailed.
10
M. Mir
oo and W. Frenzel

89
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Spectroscopy Letters
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Volume 42, 2009 - Issue 6-7: Green Spectroscopy and
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Original Articles

Multisyringe Flow Injection Technique for

Development of Green Spectroscopic
Analytical Methodologies
Fernando Maya , José Manuel Estela &Víctor Cerdà

91
Pages 312-319 | Received 26 Jun 2008, Accepted 14 Dec 2008, Published online: 01 Dec 2009

 Download citation
 http://dx.doi.org/10.1080/00387010903178715

In this article

 ABSTRACT
 INTRODUCTION
 GREEN ANALYTICAL CHEMISTRY (GAC)
 MULTISYRINGE FLOW INJECTION ANALYSIS (MSFIA) TECHNIQUE
 CONTRIBUTIONS OF THE MULTISYRINGE FLOW INJECTION TECHNIQUE AS A TOOL FOR
GREENING SPECTROSCOPIC ANALYTICAL METHODOLOGIES
 CONCLUSIONS
 References










Abstract
Since its creation, the Multisyringe Flow Injection Analysis (MSFIA) technique has proved
to be a highly useful tool for the automation of sample pretreatment procedures prior to
spectroscopic detection (UV-vis spectrophotometry, fluorescence, or chemiluminescence).
This article reviews the recent applications of the MSFIA technique and how they have
contributed to the Green Analytical Chemistry field. These contributions are mainly based on
the development of multicommutated analytical methodologies for the minimization of the
consumption of reagents, automation and miniaturization of solid phase extraction, reagent
immobilization, or in the automation and miniaturization of the required pretreatments prior
to separation techniques.

KEYWORDS: automated sample pretreatment, Green Chemistry, Multisyringe Flow Injection

Analysis, spectroscopic detection

INTRODUCTION
It is a well-known fact that the production of any chemical activity produces some impact just
by itself, while a huge number of chemical substances or chemical processes are widely used
in increasing the quality of life, but also producing an environmental impact, in some cases of
worldwide extension. Concerning these problems in 1990s emerged a new movement called
Green Chemistry (GC),[1Anastas , P. T.; Warner , J. C.Green Chemistry: Theory and Practice
; Oxford University Press : New York , 1998 . [Google Scholar]] promoting the use of
chemistry with the aim of the minimization of the feedstocks and by-products produced in the

92
use of any chemical activity. Green Analytical Chemistry (GAC) is a sub-area of GC,
recently reviewed by Armenta et al.[2Armenta , S.; Garrigues , S.; de la Guardia , M.Green
analytical chemistry . Trends Anal. Chem.2008 , 27 ( 6 ), 497 – 511 .[Crossref], [Web of
Science ®], [Google Scholar]] GAC is based on the improvement of analytical methodologies
for the determination of target compounds in any area of interest, turning on the classical
methodologies in a more environmentally friendly ones.[3Anastas , P. T.Green Chemistry and
the role of analytical methodology development . Crit. Rev. Anal. Chem.1999 , 29 ( 3 ), 167 –
175 .[Taylor & Francis Online], [Web of Science ®], [Google Scholar],4Keith , L. H.; Gron ,
L. U.; Young , J. L.Green analytical methodologies . Chem. Rev.2007 , 107 ( 6 ), 2695 – 2708
. [Google Scholar]] Here it should be pointed out that flow injection (FI)[5Trojanowicz ,
M.Flow Injection Analysis: Instrumentation and Applications ; World Scientific Publication :
Singapore , 2000 .[Crossref], [Google Scholar]] techniques are a powerful tool for greening
classical analytical methodologies. Since their creation (in the 1970s) until now, the first
Flow Injection Analysis (FIA) systems[6Ruzicka , J.; Hansen , E. H.Flow injection analyses:
Part I. A new concept of fast continuous flow analysis . Anal. Chim. Acta1975 , 78 ( 1 ), 145
– 157 .[Crossref], [Web of Science ®], [Google Scholar]] have been improved gradually by
the development of the Sequential Injection Analysis (SIA) systems,[7Ruzicka , J.; Marshall ,
G. D.Sequential injection: A new concept for chemical sensors, process analysis and
laboratory assays . Anal. Chim. Acta1990 , 237 , 329 – 343 .[Crossref], [Web of Science
®], [Google Scholar]] the Multicommutated Flow Injection Analysis (MCFIA) systems,
[8
Rocha , F. R. P.; Reis , B. F.; Zagatto , E. A. G.; Lima , J. L. F. C.; Lapa , R. A. S.; Santos ,
J. L. M.Multicommutation in flow analysis: Concepts, applications and trends . Anal. Chim.
Acta2002 , 468 ( 1 ), 119 – 131 .[Crossref], [Web of Science ®], [Google Scholar]] or the
Multisyringe Flow Injection Analysis (MSFIA) systems.[9-12Cerdà , V.; Estela , J. M.;
Forteza , R.; Cladera , A.; Becerra , E.; Altamira , P.; Sitjar , P.Flow techniques in water
analysis . Talanta1999 , 50 ( 4 ), 695 – 705 .
Miró , M.; Cerdà , V.; Estela , J. M.Multisyringe flow injection analysis: Characterization and
applications . Trends Anal. Chem.2002 , 21 ( 3 ), 199 – 210 .
Horstkotte , B.; Elsholz , O.; Cerdà , V.Review on automation using multisyringe flow
injection analysis . J. Flow Inject. Anal.2005 , 22 ( 2 ), 99 – 109 .
Segundo , M. A.; Magalhaes , L. M.Multisyringe flow injection analysis: State-of-the-art and
perspectives . Anal. Sci.2006 , 22 , 3 – 8 . ] The evolution of flow techniques produces an
improvement in the automated handling of fluids, providing an increase in efficiency,
accuracy, economy, and quickness, besides other relevant features. The use of these
techniques allows the approximation of the classic analytical methodologies, to the principles
stated by the GC movement.

This revision is focused in the recent contributions of the MSFIA technique in the automation
of analytical methodologies with spectroscopic detection, and how these improved
methodologies have contributed to the GC or GAC field.

GREEN ANALYTICAL CHEMISTRY (GAC)

It is a well-known fact that the amounts of waste products generated in a laboratory-scale
process are lower than those generated in an industrial-scale process. Nevertheless, it is an
important fact that requires consideration due to the high number of hazardous chemicals
used, starting for the sample preservation procedures and finishing in the cleaning procedure
of the instrumentation used once the analysis is completed. As a representative example, in
the use of analytical methodologies for the determination of hazardous substances for the

93
environment, ironically the chemicals used along the analytical methodology often contribute
to further environmental problems.

The Green Chemistry movement is based in 12 principles.[1Anastas , P. T.; Warner , J.

C.Green Chemistry: Theory and Practice ; Oxford University Press : New York ,
1998 . [Google Scholar]] Between them, the most applicable to analytical chemistry are the
following: (a) waste prevention; (b) use of safer chemicals; (c) design for energy efficiency;
(d) reduce derivatives; (e) real-time analysis for pollution prevention; and (f) inherent safer
chemistry for accident prevention. If we are talking about the development of analytical
methodologies, the expression “Green Chemistry” is usually related to the design of
improved methods in which the use or generation of hazardous substances is reduced or
completely removed.

The different contributions of the flow techniques to the development of greener analytical
methodologies can be established in a priority order[13Rocha , F. R. P.; Nóbrega , J. A.;
Fatibello Filho , O.Flow analysis strategies to greener analytical chemistry. An overview .
Green Chem.2001 , 3 , 216 – 220 . [Google Scholar]]: An ideal analytical methodology from
the point of view of GC should be a reagentless procedure. If it is not possible, the next step
should be the replacement of hazardous reagents for other more benign ones. Advantageous
characteristics of these two greener alternatives can be increased with the use of FI
techniques owing to their intrinsic advantages, such as the downscaling of the different
analytical operations that make up an analytical methodology. But when the use of reagents is
indispensable and the substitution of these is not a feasible option, the best alternative is the
minimization of its consumption. At this point is when the automation of analytical
methodologies by means of FI techniques plays its main role in the GC context.

Lastly, if any of the previous options is applicable in a concrete analytical methodology, the
use of FI techniques could facilitate the implementation of other alternatives such as the
waste recycling (with concomitant reutilization) or the automated treatment of the generated
waste products.

MULTISYRINGE FLOW INJECTION ANALYSIS

(MSFIA) TECHNIQUE
MSFIA technique is based on the use of a multisyringe burette for the automated handling of
fluids into a flow network (FN). The FN is specially designed for the implementation of each
concrete analytical methodology. As can be seen in Fig. 1, the multisyringe is composed of a
maximum number of 4 syringes of different volumes (usually between 1–10 mL). Syringes
are set between a metal bar common to all of them, and a three-way solenoid valve for each
one of them. The four syringes are displaced simultaneously and at the same flow rate (only
if all of them have the same volume). Each syringe, as can be seen in Fig. 1, achieves four
different movements: (a) Pickup in “Off” position (Load solution from a reservoir); (b)
Pickup in “On” position (Load solution from the FN); (c) Dispense in “Off” position (Unload
solution toward a reservoir); (d) Dispense in “On” position (Unload or inject solution toward
the FN). MSFIA technique presents a high flexibility in the automated handling of fluids due
to its 32 different combinations for flow management (if four syringes are used). The current
versions of the commercialized multisyringe modules have a piston pump of 40.000 steps,
which allows a perfectly reproducible flow management at the µL range (1 motor step = 25
nL of flow displacement, using a 1 mL syringe).

94
FIGURE 1 (Up) Schematic representation of a typical multisyringe burette used in MSFIA
systems. (Down) Different possibilities of fluid handling allowed by each syringe of a
multisyringe burette.

Display full size

This flexible and precise flow management provided by the MSFIA technique allows the
implementation of acknowledged analytical methodologies in a more environmentally benign
way. It is produced due to the efficient downscaling of the different pretreatment steps
required in each concrete analytical method, such as: sampling, development of chemical
reactions, solid- or liquid-phase extraction, membrane isolation (gas diffusion, pervaporation,
dialysis), mineralization (UV, chemical, microwave), or separation procedures (capillary
electrophoresis, liquid chromatography). Furthermore, the selection of the MSFIA technique
for the automation of analytical methods provides other advantageous features in comparison
with the other existent flow techniques, such as: (a) robustness similar to SIA technique,
avoiding the use of the Tygon® tubes commonly used in FIA and MCFIA systems, thus
allowing the use of aggressive reagents (concentrated acids or organic solvents); (b) as in a
multicommutated technique, the volumes are perfectly controlled, obtaining a saving of
reagents in comparison with classical FIA systems; (c) the required volumes of sample and
reagents can be injected toward the detector following a forward flow scheme as in FIA
technique, thus avoiding the usual problems in the development of the reaction product when
two or more reagents are used in SIA technique. By this way the injection throughput is
increased, and thus the efficiency of the system.

95
CONTRIBUTIONS OF THE MULTISYRINGE FLOW
INJECTION TECHNIQUE AS A TOOL FOR
GREENING SPECTROSCOPIC ANALYTICAL
METHODOLOGIES
Simple Multicommutated Procedures with Spectroscopic Detection

Several analytical methodologies have been implemented in the MSFIA technique through
the use of multicommutated procedures, minimizing by this way the consumption of reagents.
These methodologies are based in the precise injection of the minimum amounts of the
required reagents to carry out a concrete determination. Some examples that can be found in
the recent literature are: (a) the spectrophotometric determination of S2− in waters by the
methylene blue method, diminishing the consumption of N,N-dimethyl-p-phenylenediamine
(DMPD), NH4Fe(SO4)2 and HCl[14Ferrer , L.; De Armas , G.; Miró , M.; Estela , J. M.;
Cerdà , V.A multisyringe flow injection method for the automated determination of sulfide in
waters using a miniaturised optical fiber spectrophotometer . Talanta2004 , 64 ( 5 ), 1119 –
1126 . [Google Scholar]]; (b) the time-based determination using fluorimetric detection of
Al3+ in drinking waters using 8-hydroxyquinoline-5-sulphonic acid,[15De Armas , G.; Miró ,
M.; Cladera , A.; Estela , J. M.; Cerdà , V.Time-based multisyringe flow injection system for
the spectrofluorimetric determination of aluminium . Anal. Chim. Acta2002 , 455 ( 1 ), 149 –
157 .[Crossref], [Google Scholar]] or the (c) multicommutated procedure for the
spectrophotometric determination of Cl− in waters[16Maya , F.; Estela , J. M.; Cerdà ,
V.Spectrophotometric determination of chloride in waters using a multisyringe flow injection
system . Talanta2008 , 74 ( 5 ), 1534 – 1538 . [Google Scholar]] using Fe3+ and Hg(SCN)2. In
all the previous examples the consumption of reagents is diminished in comparison with FIA
systems and in similar levels than in SIA systems but achieving higher injection throughputs
than these last ones.

If we study more deeply the concrete case of the spectrophotometric determination of Cl− in
waters based on the Cl−/Hg(SCN)2/Fe3+ reaction system where the reaction product [Fe(NCS)
(H2O)5]2+ is followed at 470–480 nm, we can appreciate that it is a very useful methodology
(easy, cost-effective, sensitive, reproducible, and quick—overcoat if it is automated). By this
reason, the automated version of this methodology is an acknowledged reference method.
[17
APHA-AWWA-WPCF. 4500 Cl− E. In Standard Methods for the Examination of Water
and Wastewater ; Clesceri , L. S., Greenberg , A. E., Eaton , A. D., Eds.; American Public
Health Association : Baltimore , 1998 ; 4.83 – 4.85 . [Google Scholar],18U.S. Environmental
Protection Agency . Methods for Chemical Analysis of Water and Wastes1979 . EPA-600/4-
79-020, Method 325.2 . ] However, this method presents a serious drawback, that is, the use
of considerable amounts of Hg(SCN)2, a highly toxic reagent. Since the first direct FIA
system for this purpose[19Ruzicka , J.; Stewart , J. W. B.; Zagatto , E. A. G.Flow injection
analysis: Part IV. Stream sample splitting and its application to the continuous
spectrophotometric determination of chloride in brackish waters . Anal. Chim. Acta1976 , 81
( 2 ), 387 – 396 . [Google Scholar]] in the year 1976, the reagent consumption was improved
following alternative strategies based on automation such as membrane reagent introduction
in 1998,[20Chalk , S. T.; Tyson , J. F.Determination of chloride by flow injection
spectrophotometry with membrane reagent introduction . Anal. +Chim. Acta1998 , 366 ( 1–

96
3 ), 147 – 153 . [Google Scholar]] the Hg(SCN)2 immobilization in an epoxy resin in 2005,
[21
Silva , C. R.; Vieira , H. J.; Canaes , L. S.; Nóbrega , J. A.; Fatibello-Filho , O.Flow
injection spectrophotometric method for chloride determination in natural waters using
Hg(SCN)2 immobilized in epoxy resin . Talanta2005 , 65 ( 4 ), 965 – 970 . [Google Scholar]]
or the multicommutated MSFIA procedure in 2008.[16Maya , F.; Estela , J. M.; Cerdà ,
V.Spectrophotometric determination of chloride in waters using a multisyringe flow injection
system . Talanta2008 , 74 ( 5 ), 1534 – 1538 . [Google Scholar]] The consumption of Fe3+ and
Hg(II) for the previously commented systems can be seen in Table 1. We can appreciate a
progressive reduction in the consumption of Hg(II) with the concomitant increase of the
automation degree. With the MSFIA method[16Maya , F.; Estela , J. M.; Cerdà ,
V.Spectrophotometric determination of chloride in waters using a multisyringe flow injection
system . Talanta2008 , 74 ( 5 ), 1534 – 1538 . [Google Scholar]] the lowest reagent
consumption levels are achieved. The manifold correspondent to this MSFIA system for the
spectrophotometric determination of chloride in waters in schematically depicted in Fig. 2.

FIGURE 2 Schematic representation of the MSFIA manifold for chloride determination. C:

carrier (distilled water); R1: Hg(SCN)2 reagent; R2: Fe3+ reagent; E: empty (not used); S1–
S4: syringes; E1–E6: solenoid valves; On–Off: solenoid valves positions; SC: security coil;
HC: holding coil; Cp: confluence point; KR: knotted reactor; FC: flow cell.

Display full size

TABLE 1 Summarized Analytical Performance of Several Flow Systems for the

Spectrophotometric Cl− Determination Based on the Cl−/Hg(SCN)2/Fe3+ Reaction System
CSVDisplay Table

Another improvement is the combination of MSFIA technique with cold vapor atomic
absorption spectrometry (CVAAS)[22Leal , L. O.; Elsholz , O.; Forteza , R.; Cerdà ,
V.Determination of mercury by multisyringe flow injection system with cold-vapor atomic
absorption spectrometry . Anal. Chim. Acta2006 , 573–574 , 399 – 405 . [Google Scholar]]
and cold vapor atomic fluorescence spectrometry (CVAFS),[23Serra , A. M.; Estela , J. M.;
Cerdà , V.MSFIA system for mercury determination by cold vapour technique with atomic
fluorescence detection . Talanta2008 , 77 ( 2 ), 556 – 560 . [Google Scholar]] both for the
determination of mercury in water and fish samples (after manual mineralization of the solid
sample). In this line must be added the proposed MSFIA systems with hydride generation

97
atomic fluorescence spectrometry (HGAFS) for the determination of inorganic arsenic,
[24
Semenova , N. V.; Leal , L. O.; Forteza , R.; Cerdà , V.Multisyringe flow-injection system
for total inorganic arsenic determination by hydride generation-atomic fluorescence
spectrometry . Anal. Chim. Acta2002 , 455 ( 2 ), 277 – 285 . [Google Scholar]] inorganic
antimony,[25Semenova , N. V.; Leal , L. O.; Forteza , R.; Cerdà , V.Multisyringe flow-
injection system for total inorganic selenium determination by hydride generation-atomic
fluorescence spectrometry . Anal. Chim. Acta2003 , 486 ( 2 ), 217 – 225 . [Google Scholar]]
and for the speciation of inorganic selenium[26Semenova , N. V.; Leal , L. O.; Forteza , R.;
Cerdà , V.Antimony determination and speciation by multisyringe flow injection analysis
with hydride generation-atomic fluorescence detection . Anal. Chim. Acta2005 , 530 ( 1 ),
113 – 120 . [Google Scholar]] and inorganic arsenic.[27Leal , L. O.; Forteza , R.; Cerdà ,
V.Speciation analysis of inorganic arsenic by a multisyringe flow injection system with
hydride generation-atomic fluorescence spectrometric detection . Talanta2006 , 69 ( 2 ), 500
– 508 . [Google Scholar]] On the one hand, as seen in Table 2, the implementation of these
methodologies in the MSFIA technique provides a lower consumption of reagents than in
FIA systems and a concomitant high efficiency as can be seen in the injection throughput
values. On the other hand, the reagent consumption is similar or higher than in SIA systems,
but still maintaining a higher efficiency in terms of injection throughput.

TABLE 2 Comparison of MSFIA Systems with AFS or AAS Detection with Analogous FIA
and SIA Systems
CSVDisplay Table

Solid Phase Extraction

Another relevant alternative in the implementation of analytical methodologies in a cleaner

way, is the use of solid-phase extraction (SPE) instead of the classic liquid–liquid extraction
(LLE). An improved and greener way to perform SPE is based on its miniaturization and
consequent exploitation in automated FI systems, besides the direct sensing of the analytical
signal in the same surface of the solid support.

The use of the MSFIA technique for the in-line SPE procedures for analyte isolation and
preconcentration provides a reduction of the required amounts of solid sorbents and eluents.
Some examples of SPE–MSFIA systems presented in the literature are: the preconcentration
and fluorimetric determination of warfarin in waters using a packed column with a C18
resin[28De Armas , G.; Miró , M.; Estela , J. M.; Cerdà , V.Multisyringe flow injection
spectrofluorimetric determination of warfarin at trace levels with on-line solid-phase
preconcentration . Anal. Chim. Acta2002 , 467 ( 1–2 ), 13 – 23 . [Google Scholar]]; the
determination of arsenic in waters by HGAFS using a packed column with an anion exchange
resin,[29Leal , L. O.; Semenova , N. V.; Forteza , R.; Cerdà , V.Preconcentration and
determination of inorganic arsenic using a multisyringe flow injection system and hydride
generation-atomic fluorescence spectrometry . Talanta2004 , 64 ( 5 ), 1335 – 1342 . [Google
Scholar]] the smart analyzer for the determination and speciation of iron in waters using
chelating disks,[30Pons , C.; Forteza , R.; Cerdà , V.Expert multi-syringe flow-injection
system for the determination and speciation analysis of iron using chelating disks in water
samples . Anal. Chim. Acta2004 , 524 ( 1–2 ), 79 – 88 . [Google Scholar]] the
spectrophotometric determination of the total phenolic index based in the 4-aminoantipirine
method using a packed column with Amberlite XAD-4 resin[31Oliveira , H. M.; Segundo , M.
A.; Reis , S.; Lima , J. L. F. C.Multi-syringe flow injection system with in-line pre-
concentration for the determination of total phenolic compounds . Microchim. Acta2005 , 150

98
, 187 – 196 . [Google Scholar]]—in which LLE with chloroform is substituted by an in-line
SPE procedure; or the spectrophotometric determination of nitro-substituted phenol isomers
based on the use of sorbent disks made from co-polymeric poly(styrenedivinylbenzene)
modified with benzenesulphonic groups.[32Manera , M.; Miró , M.; Estela , J. M.; Cerdà ,
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Furthermore, if we are talking about efficiency in analytical methodologies, we have the

requirement to talk about the MSFIA methods and its combination with multipumping flow
systems (MPFS),[33Lapa , R. A. S.; Lima , J. L. F. C.; Reis , B. F.; Santos , J. L. M.; Zagatto ,
E. A. G.Multi-pumping in flow analysis: Concepts, instrumentation, potentialities . Anal.
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implemented for the sample pretreatment of radioactive species. The required pretreatment
for matrix isolation is accomplished in a completely automated fashion, saving reagents and
time per determination. Once the sample pretreatment has been completed, the target analytes
are determined off-line with a low background proportional counter. In the recent literature
appear two MSFIA systems for the pretreatment of the determination in waters of radioactive
strontium[34Fajardo , Y.; Gómez , E.; Mas , F.; Garcias , F.; Cerdà , V.; Casas ,
M.Multisyringe flow injection analysis of stable and radioactive strontium in simples of
environmental interest . Appl. Radiat. Isotopes2004 , 61 ( 2–3 ), 273 – 277 . [Google
Scholar]] and radioactive yttrium.[35Fajardo , Y.; Gómez , E.; Garcias , F.; Cerdà , V.; Casas ,
M.Multisyringe flow injection analysis of stable and radioactive yttrium in water and
biological simples. Anal. Chim. Acta2005, 539(1–2), 189–194.[Crossref], [Google Scholar]]
Furthermore can be found two hybrid MSFIA—MPFS systems for the pretreatment of the
determination of radium[36Fajardo , Y.; Gómez , E.; Garcias , F.; Cerdà , V.; Casas ,
M.Development of an MSFIA-MPFS pre-treatment method for radium determination in
water samples . Talanta2007 , 71 ( 3 ), 1172 – 1179 . [Google Scholar]] in waters and for the
separation and preconcentration of americium and plutonium in waters[37Fajardo , Y.; Ferrer ,
L.; Gómez , E.; Garcias , F.; Casas , M.; Cerdà , V.Development of an automatic method for
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Finally, a relevant fact in the contribution of SPE–MSFIA procedures in the sample

pretreatment prior to spectroscopic measurements is the accomplishment of reflectometric
measurements based on optical fiber flow-through optical sensors (optrodes).[38Miró , M.;
Frenzel , W.Flow-through sorptive preconcentration with direct optosensing at solid surfaces
for trace-ion analysis . Trends Anal. Chem.2004 , 23 ( 1 ), 11 – 20 . [Google Scholar]] By this
way is attained a considerable reduction in the consumption of reagents in comparison with
reflectometric batch or classic FIA systems. Some examples of this are: the flow-through
optical fiber sensor for sulphide determination based on the in-line generation of the
methylene blue dye and its subsequent preconcentration in octadecyl-chemically modified
(C18) disks[39Ferrer , L.; de Armas , G.; Miró , M.; Estela , J. M.; Cerdà , V.Flow-through
optical fiber sensor for automatic sulfide determination in waters by multisyringe flow
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simultaneous multiresidues determination of nitrophenol derivatives, which is based on the
in-line anion exchange sorptive preconcentration of the target species followed by direct
detection onto the sorbent material by means of chemometric deconvolution of the

99
overlapped spectra.[40Manera , M.; Miró , M.; Estela , J. M.; Cerdà , V.; Segundo , M. A.;
Lima , J. L. F. C.Flow-through solid-phase reflectometric method for simultaneous
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Reagent Immobilization

The versatility of the MSFIA technique can be exploited for the development of analytical
methodologies using immobilized reagents. Reagents are packed in a flow-through mini-
column allowing an increased efficiency of the procedure from the point of view of GAC.

Soto et al.[41Soto , N. O.; Horstkotte , B.; March , J. G.; López de Alba , P. L.; Martínez , L.
L.; Martín , V. C.An environmental friendly method for the automatic determination of
hypochlorite in comercial products using multisyringe flow injection analysis . Anal. Chim.
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spectroscopic UV detection for the determination of hypochlorite in commercial products.
This system is based on the selective decomposition of hypochlorite by means of an
immobilized cobalt oxide catalyst. The resultant analytical methodology is almost an ideal
Green Analytical Method.

Other MSFIA approaches based on reagent immobilization are: the inclusion of a flow-
through solid-phase chemiluminescent sensor with immobilized 3-aminophtalhydrazide
(luminol), for the chemiluminometric determination of orthophosphate in waters[42Morais , I.
P. A.; Miró , M.; Manera , M.; Estela , J. M.; Cerdà , V.; Souto , M. R. S.; Rangel , A. O. S.
S.Flow-through solid-phase based optical sensor for the multisyringe flow injection trace
determination of orthophosphate in waters with chemiluminescence detection . Anal. Chim.
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of a glucose-oxidase packed-bed reactor for the conversion of the substrate followed by post-
column chemiluminescent detection of the generated oxidizing species after reaction with
luminol.[43Manera , M.; Miró , M.; Estela , J. M.; Cerdà , V.A multisyringe flow injection
system with immobilized glucose oxidase based on homogeneous chemiluminescence
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MSFIA Hyphenation with Separation Techniques Prior to UV-vis Spectrophotometric

Detection

Two of the most recent applications of MSFIA technique are based on its hyphenation with
separation techniques. Concretely, two new hyphenated flow techniques have been recently
developed, named multisyringe chromatography (MSC) and multisyringe flow injection
analysis-capillary electrophoresis (MSFIA-CE).

The MSC technique was developed by González-San Miguel et al.,[44González-San Miguel ,

H. M.; Alpízar-Lorenzo , J. M.; Cerdà-Martín , V.Development of a new high performance
low pressure chromatographic system using a multisyringe burette coupled to a
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liquid chromatographic separations, following an alternative quick and smart way. This
technique is based on the use of short monolithic columns allowing the accomplishment of
liquid chromatographic separations at lower pressures than in HPLC classic systems. With

100
the miniaturization of the chromatographic system the amount of the required reagents and
solvents is also minimized, allowing us to work in a more environmentally friendly way. The
MSC technique has been applied in the determination of β-lactamic antibiotics in
pharmaceuticals,[45González-San Miguel , H. M.; Alpízar-Lorenzo , J. M.; Cerdà ,
V.Simultaneous determination of ß-lactamic antibiotics by a new high-performance low-
pressure chromatographic system using a multisyringe burette coupled to a monolhithic
column MSC . Anal. Bioanal. Chem.2007 , 387 ( 2 ), 663 – 671 . [Google Scholar]] or in the
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M.; González , H. M.; Cerdà , V.Modulation of mobile phase composition in flow-
injection/sequential-injection chromatography exploiting multisyringe flow analysis . Anal.
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The MSC technique was also combined with in-line SPE for the determination of losartan
potassium and hydrochlorothiazide in waters.[47Obando , M. A.; Estela , J. M.; Cerdà ,
V.Multi-syringe chromatography (MSC) system for the on-line solid-phase extraction and
determination of hydrochlorothiazide and losartan potassium in superficial water,
groundwater and wastewater outlet samples . J. Pharm. Biomed. Anal.2008 , doi:
10.1016/j.jpba.2008.05.016 . [Google Scholar]] The general trend of the MSC systems, which
is shown in Table 3 for the case of the determination of B1, B6, and B12 vitamins, is the
reduction of reagent consumption in comparison to conventional HPLC systems and
concomitantly achieving higher injection throughputs. So, the MSC technique can be defined
as a low cost, quick, versatile, and greener alternative way to perform liquid chromatographic
separations.

TABLE 3 Comparison of the Analytical Features of the MSC System for the
Determination of Vitamins B1, B6, and B12 in Pharmaceuticals with Those of Earlier
HPLC Methods for the Same Purpose
CSVDisplay Table

The MSFIA-CE technique was initially characterized as a SIA-CE[48Horstkotte , B.; Elsholz ,

O.; Martín , V. C.Development of a capillary electrophoresis system coupled to sequential
injection analysis and evaluation by the analysis of nitrophenols . Int. J. Environ. An.
Ch.2007 , 87 ( 12 ), 797 – 811 .[Taylor & Francis Online], [Web of Science ®], [Google
Scholar]] technique for the direct determination of mono-substituted nitrophenolic isomers. It
was followed by the incorporation of two additional syringes, developing the first hyphenated
MSFIA-CE[49Horstkotte , B.; Elsholz , O.; Martín , V. C.Multisyringe flow injection analysis
coupled to capillary electrophoresis (MSFIA-CE) as a novel analytical tool applied to the pre-
concentration, separation and determination of nitrophenols . Talanta2008 , 76 ( 1 ), 72 –
79 . [Google Scholar]] system. This novel system took profit of the increased versatility
allowed by the two additional syringes, carrying out the efficient and miniaturized in-line
SPE of mono-substituted nitrophenolic isomers, followed by their subsequent separation and
spectrophotometric detection at a wavelength of 401 nm. The automation and miniaturization
of the required pretreatments prior to the CE separation can be accomplished effectively in an
in-line perspective by means of MSFIA technique, increasing the efficiency of the analytical
method from the point of view of GAC.

CONCLUSIONS

101
In this article has been carried out a revision of the recent literature about the newest
applications of the MSFIA technique. It has been focused to the minimization of the
consumption of reagents and other different strategies inherent to MSFIA technique and their
contribution to the Green Analytical Chemistry field.

Finally, we would like to propose the use of the MSFIA technique combined with
spectroscopic detectors as an efficient tool for the automation of sample pretreatment,
becoming analytical methodologies in a cleaner way.

ACKNOWLEDGMENTS
This work was supported from the “Ministerio de Educación y Ciencia, Gobierno de España”
through the projects CTQ2007-64331 and PROGECIC-5C. F. Maya is very grateful to the
“Conselleria d'Economia, Hisenda i Innovació, Govern de les Illes Balears,” for its support
through a Ph.D. grant.

Notes
∗Hg(II) and Fe(III) consumptions are referred to ref. [19Ruzicka , J.; Stewart , J. W. B.;
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IT—Injection throughput.

aResults obtained from references [50-55Chatzimichalakis , P. F.; Samanidou , V. F.;

Verpoorte , R.; Papadoyannis , I. N.Development of a validated HPLC method for the
determination of B-complex vitamins in pharmaceuticals and biological fluids after solid
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Marszall , M. L., Lebiedzinska , A.; Czarnowski , W.; Szefer , P.High-performance liquid
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coulometric electrochemical and ultraviolet detection . J. Chromatogr. A2005 , 1094 ( 1–2 ),
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Moreno , P.; Salvadó , V.Determination of eight water- and fat-soluble vitamins in multi-
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Heudi , O.; Kilinç , T.; Fontannaz , P.Separation of water-soluble vitamins by reversed-phase
high performance liquid chromatography with ultra-violet detection: Application to
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Li , H.-B.; Chen , F.Simultaneous determination of nine water-soluble vitamins in
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Li , L.-S.; Da , S.-L.; Feng , Y.-Q.; Liu , M.Study on the chromatographic behavior of water-
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Talanta2004 , 64 ( 2 ), 373 – 379 . ].

An invited paper submitted to a special issue on Green Spectroscopy and Analytical

Techniques, organized by Professor Miguel de la Guardia, of the Department of Chemistry,

102
University of Valencia, Spain, and Professor Arabinda Kumar Das, of the Department of
Chemistry, University of Burdwan, West Bengal, India.

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vitamin pharmaceutical formulations by high-performance liquid chromatography . J.
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 An invited paper submitted to a special issue on Green Spectroscopy and Analytical

Techniques, organized by Professor Miguel de la Guardia, of the Department of
Chemistry, University of Valencia, Spain, and Professor Arabinda Kumar Das, of the
Department of Chemistry, University of Burdwan, West Bengal, India.

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ADDITIONAL NOTES

Outlines / Summaries

Sample Preparation for Mercury Analysis

Of each dried sample, 1g was accurately weighed and transferred into a conical flask. 8ml of
a mixture of concentrated nitric acid, sulphuric and perchloric acids (HNO3:H2SO4:HClO4) in
the ratio of 3:1:1 respectively was added. These samples were put in a water bath at 50°C
for 1 hour. The digestion vessels were covered using watch glasses [Muigai et al., 1995].

114
The digestion vessels were then removed from the water bath and cooled in an ice bath.
50mlof 6% wt/vol. of potassium permanganate solution was added with constant shaking
until effervescence stopped. It was left in the oxidation medium overnight before the
second digestion step.

Second phase digestion was allowed for two hours at 50°C in a regulated water bath. The
digests were then removed from the water bath and cooled to room temperature. Then,
15ml 0f 20% wt/vol. hydroxylamine hydrochloric solution was added slowly while shaking to
avoid frothing and possible loss of flask contents. The final digest was filtered through
suitable filter paper number 40 into 100ml volumetric flask and the residue rinsed several
times with deionised water and then diluted to the mark using deionised water. The sample
extract was finally ready for cold vapour analysis by aspirating with equal volume of 25%
SnCl2 wt/vol. with 20% of conc. HCl vol. /vol [Muigai et al., 1995].

The determination was completed by measuring absorbance at 253.7nm using flameless

determination of mercury with suitable calibration standards [Muigai et al., 1995].

Hydride Generator
Metalloids and soft metals (e.g., As, Se, Sb, Sn) can be introduced into the ICP in the form of
a volatile hydride, which is formed by reacting the element of interest with sodium
borohydride (NaBH4). The gaseous hydride is then carried to the plasma for analysis.

Liquid-liquid extraction (LLE)

Liquid-liquid extraction (LLE), also known as solvent extraction and partitioning, is a
method to separate compounds based on their relative solubilities in two different
immiscible liquids, usually water and an organic solvent. It is an extraction of a
substance from one liquid into another liquid phase. It consists of transferring one (or
more) solute(s) contained in a feed solution another immiscible liquid (solvent).
Example: Caffeine can be extracted from coffee beans and tea leaves using a direct
organic extraction, by soaking in ethyl acetate which favourably dissolves the caffeine,
leaving a majority of the coffee or tea flavor remaining in the initial sample.

Headspace technology
Headspace technology is a technique developed to elucidate the odor compounds present
in the air surrounding various objects. Usually, the objects of interest are odoriferous
objects such as plants, flowers and foods. Similar techniques are also used to analyze the
interesting scents of locations and environments such as tea shops and show mills. After the
data is analyzed, the scents can then be recreated by a perfumer. Inert gases are passed into
the space containing the object or a vacuum is established such that the odor compounds
are removed from the headspace. These compounds are in turn captured using a variety of
techniques, among them cold surfaces, solvent traps, and adsorbent materials, with the

115
latter techniques capable of longer periods of collection. The samples can then be analyzed
using techniques such as Gas Chromatography, Mass Spectrometry or Carbon-13 NMR.

Flow injection analysis (FIA)

Flow injection analysis (FIA) is an approach to chemical analysis that is accomplished by
injecting a plug of sample into a flowing carrier stream. The principle is similar to that of
segmented flow analysis (SFA) but no air is injected into the sample or reagent streams. FIA
is an automated method of chemical analysis in which a simple is injected into a flowing
carrier solution that mixes with reagents before reaching a detector. Over the years, FIA
techniques have developed into a wide array of applications using spectrophotometry,
fluorescence spectroscopy, Atomic Absorption Spectroscopy, Mass Spectrometry and other
methods of instrumental analysis for detection.
Many types of detector can be used such as Colorimeter, Fluorimeter, Ion-selective
electrode and Biosensors.
- An experiment that is used in Analytical Chemistry lab courses to familiarize students
with FIA is the determination of phosphate by Flow Injection Analysis. The
experiment involves calibrating an FIA system, optimizing the system for detection of
phosphate and finding the amount of phosphate in an unknown sample.

Hydride atomization

Hydride generation techniques are specialized in solutions of specific elements. The

technique provides a means of introducing samples containing arsenic, antimony, tin,
selenium, bismuth, and lead into an atomizer in the gas phase. With these elements, hydride
atomization enhances detection limits by a factor of 10 to 100 compared to alternative
methods. Hydride generation occurs by adding an acidified aqueous solution of the sample to
a 1% aqueous solution of sodium borohydride, all of which is contained in a glass vessel. The
volatile hydride generated by the reaction that occurs is swept into the atomization chamber
by an inert gas, where it undergoes decomposition. This process forms an atomized form of
the analyte, which can then be measured by absorption or emission spectrometry.

Cold-vapor atomization

The cold-vapor technique an atomization method limited to only the determination of

mercury, due to it being the only metallic element to have a large enough vapor pressure at
ambient temperature.[citation needed] Because of this, it has an important use in determining
organic mercury compounds in samples and their distribution in the environment. The
method initiates by converting mercury into Hg2+ by oxidation from nitric and sulfuric acids,
followed by a reduction of Hg2+ with tin(II) chloride. The mercury, is then swept into a long-
pass absorption tube by bubbling a stream of inert gas through the reaction mixture. The
concentration is determined by measuring the absorbance of this gas at 253.7 nm. Detection
limits for this technique are in the parts-per-billion range making it an excellent mercury
detection atomization method.

116
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Liquid–liquid extraction
From Wikipedia, the free encyclopedia

Solvent extraction.

Liquid–liquid extraction (LLE), also known as solvent extraction and partitioning, is a

method to separate compounds based on their relative solubilities in two different immiscible
liquids, usually water and an organic solvent. It is an extraction of a substance from one
liquid into another liquid phase. It consists of transferring one (or more) solute(s) contained
in a feed solution to another immiscible liquid (solvent). The solvent that is enriched in
solute(s) is called extract. The feed solution that is depleted in solute(s) is called the raffinate.
LLE is a basic technique in chemical laboratories, where it is performed using a variety of
apparatus, from separatory funnels to countercurrent distribution equipment.[not verified in body] This
type of process is commonly performed after a chemical reaction as part of the work-up,
often including an acidic work up.

The term partitioning is commonly used to refer to the underlying chemical and physical
processes involved in liquid–liquid extraction, but on another reading may be fully
synonymous with it. The term solvent extraction can also refer to the separation of a
substance from a mixture by preferentially dissolving that substance in a suitable solvent. In
that case, a soluble compound is separated from an insoluble compound or a complex matrix.
[not verified in body]

Solvent extraction is used in nuclear reprocessing, ore processing, the production of fine
organic compounds, the processing of perfumes, the production of vegetable oils and
biodiesel, and other industries.[not verified in body] It is among the most common initial separation
techniques, though some difficulties result in extracting out closely related functional groups.

117
Liquid–liquid extraction is possible in non-aqueous systems: In a system consisting of a
molten metal in contact with molten salts, metals can be extracted from one phase to the
other. This is related to a mercury electrode where a metal can be reduced, the metal will
often then dissolve in the mercury to form an amalgam that modifies its electrochemistry
greatly. For example, it is possible for sodium cations to be reduced at a mercury cathode to
form sodium amalgam, while at an inert electrode (such as platinum) the sodium cations are
not reduced. Instead, water is reduced to hydrogen. A detergent or fine solid can be used to
stabilize an emulsion, or third phase.[not verified in body]

Contents
 1 Measures of effectiveness
o 1.1 Distribution ratio
o 1.2 Separation factors
o 1.3 Decontamination factor
o 1.4 Slopes of graphs
o 1.5 Measures of success
 2 Techniques
o 2.1 Batchwise single stage extractions
 2.1.1 Dispersive liquid–liquid microextraction (DLLME)
 2.1.2 Direct organic extraction
o 2.2 Multistage countercurrent continuous processes
 2.2.1 Mixer–settlers
 2.2.2 Centrifugal extractors
o 2.3 Extraction without chemical change
o 2.4 Solvation mechanism
o 2.5 Ion exchange mechanism
o 2.6 Ion pair extraction
 2.6.1 Types of aqueous two-phase extractions
 2.6.2 Applications
 3 Kinetics of extraction
 4 Aqueous complexing agents
 5 Industrial process design
 6 Equipment
 7 Extraction of metals
 8 See also
 9 References
 10 Further reading

Measures of effectiveness
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Distribution ratio
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118
 how and when to remove this template message)

In solvent extraction, a distribution ratio is often quoted as a measure of how well-extracted a

species is. The distribution ratio (Kd) is equal to the concentration of a solute in the organic
phase divided by its concentration in the aqueous phase. Depending on the system, the
distribution ratio can be a function of temperature, the concentration of chemical species in
the system, and a large number of other parameters. Note that D is related to the ΔG of the
extraction process.

Sometimes, the distribution ratio is referred to as the partition coefficient, which is often
expressed as the logarithm. Note that a distribution ratio for uranium and neptunium between
two inorganic solids (zirconolite and perovskite) has been reported.[1] In solvent extraction,
two immiscible liquids are shaken together. The more polar solutes dissolve preferentially in
the more polar solvent, and the less polar solutes in the less polar solvent. In this experiment,
the nonpolar halogens preferentially dissolve in the non-polar mineral oil.[2]

Although the distribution ratio and partition coefficient are often used synonymously, they
are not necessarily so. Solutes may exist in more than one form in any particular phase, which
would mean that the partition coefficient (Kd) and distribution ratio (D) will have different
values. This is an important distinction to make as whilst the partition coefficient has a fixed
value for the partitioning of a solute between two phases, the distribution ratio changes with
differing conditions in the solvent.[3]

After performing liquid–liquid extraction, a quantitative measure must be taken to determine

the ratio of the solution’s total concentration in each phase of the extraction. This quantitative
measure is known as the distribution ratio or distribution coefficient.[4]

Separation factors

The separation factor is one distribution ratio divided by another; it is a measure of the ability
of the system to separate two solutes. For instance, if the distribution ratio for nickel (DNi) is
10 and the distribution ratio for silver (DAg) is 100, then the silver/nickel separation factor
(SFAg/Ni) is equal to DAg/DNi = SFAg/Ni = 10.[5]

Decontamination factor

This is used to express the ability of a process to remove a contaminant from a product. For
instance, if a process is fed with a mixture of 1:9 cadmium to indium, and the product is a
1:99 mixture of cadmium and indium, then the decontamination factor (for the removal of
cadmium) of the process is 0.11 / 0.01 = 11.

Slopes of graphs

The easy way to work out the extraction mechanism is to draw graphs and measure the
slopes. If for an extraction system the D value is proportional to the square of the
concentration of a reagent (Z) then the slope of the graph of log10(D) against log10([[Z]]) will
be two.

119
Measures of success

Success of liquid–liquid extraction is measured through separation factors and

decontamination factors. The best way to understand the success of an extraction column is
through the liquid–liquid equilibrium (LLE) data set. The data set can then be converted into
a curve to determine the steady state partitioning behavior of the solute between the two
phases. The y-axis is the concentration of solute in the extract (solvent) phase, and the x-axis
is the concentration of the solute in the raffinate phase. From here, one can determine steps
for optimization of the process.[6]

Techniques
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Batchwise single stage extractions

This is commonly used on the small scale in chemical labs. It is normal to use a separating
funnel. Processes include DLLME and direct organic extraction.

Dispersive liquid–liquid microextraction (DLLME)

A process used to extract small amounts of organic compounds from water samples.[7] This
process is done by injecting small amounts of an appropriate extraction solvent (C2Cl4) and a
disperser solvent (acetone) into the aqueous solution. The resulting solution is then
centrifuged to separate the organic and aqueous layers. This process is useful in extraction
organic compounds such as organochloride and organophsophorus pesticides, as well as
substituted benzene compounds from water samples.[7]

Direct organic extraction

By mixing partially organic soluble samples in organic solvent (toluene, benzene, xylene),
the organic soluble compounds will dissolve into the solvent and can be separated using a
separatory funnel. This process is valuable in the extraction of proteins and specifically
phosphoprotein and phosphopeptide phosphatases.[8]

Another example of this application is extracting anisole from a mixture of water and 5%
acetic acid using ether, then the anisole will enter the organic phase. The two phases would
then be separated. The acetic acid can then be scrubbed (removed) from the organic phase by
shaking the organic extract with sodium bicarbonate. The acetic acid reacts with the sodium
bicarbonate to form sodium acetate, carbon dioxide, and water.

Caffeine can also be extracted from coffee beans and tea leaves using a direct organic
extraction. The beans or leaves can be soaked in ethyl acetate which favorably dissolves the
caffeine, leaving a majority of the coffee or tea flavor remaining in the initial sample.[9]

120
Multistage countercurrent continuous processes

Coflore continuous countercurrent extractor.

These are commonly used in industry for the processing of metals such as the lanthanides;
because the separation factors between the lanthanides are so small many extraction stages
are needed.[10] In the multistage processes, the aqueous raffinate from one extraction unit is
fed to the next unit as the aqueous feed, while the organic phase is moved in the opposite
direction. Hence, in this way, even if the separation between two metals in each stage is
small, the overall system can have a higher decontamination factor.

Multistage countercurrent arrays have been used for the separation of lanthanides. For the
design of a good process, the distribution ratio should be not too high (>100) or too low
(<0.1) in the extraction portion of the process. It is often the case that the process will have a
section for scrubbing unwanted metals from the organic phase, and finally a stripping section
to obtain the metal back from the organic phase.

Mixer–settlers

Battery of mixer-settlers counter currently interconnected. Each mixer-settler unit provides a

single stage of extraction. A mixer settler consists of a first stage that mixes the phases
together followed by a quiescent settling stage that allows the phases to separate by gravity.

In the multistage countercurrent process, multiple mixer settlers are installed with mixing and
settling chambers located at alternating ends for each stage (since the outlet of the settling
sections feed the inlets of the adjacent stage’s mixing sections). Mixer-settlers are used when
a process requires longer residence times and when the solutions are easily separated by
gravity. They require a large facility footprint, but do not require much headspace, and need
limited remote maintenance capability for occasional replacement of mixing motors. (Colven,
1956; Davidson, 1957)[11]

121
4 stage battery of mixer-settlers for counter-current extraction.

Centrifugal extractors

Centrifugal extractors mix and separate in one unit. Two liquids will be intensively mixed
between the spinning rotor and the stationary housing at speeds up to 6000 RPM. This
develops great surfaces for an ideal mass transfer from the aqueous phase into the organic
phase. At 200–2000 g, both phases will be separated again. Centrifugal extractors minimize
the solvent in the process, optimize the product load in the solvent and extract the aqueous
phase completely. Counter current and cross current extractions are easily established.[12]

Extraction without chemical change

Some solutes such as noble gases can be extracted from one phase to another without the
need for a chemical reaction (see absorption). This is the simplest type of solvent extraction.
When a solvent is extracted, two immiscible liquids are shaken together. The more polar
solutes dissolve preferentially in the more polar solvent, and the less polar solutes in the less
polar solvent. Some solutes that do not at first sight appear to undergo a reaction during the
extraction process do not have distribution ratio that is independent of concentration. A
classic example is the extraction of carboxylic acids (HA) into nonpolar media such as
benzene. Here, it is often the case that the carboxylic acid will form a dimer in the organic
layer so the distribution ratio will change as a function of the acid concentration (measured in
either phase).

For this case, the extraction constant k is described by k = [[HAorganic]]2/[[HAaqueous]]

Solvation mechanism
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Using solvent extraction it is possible to extract uranium, plutonium, or thorium from acid
solutions. One solvent used for this purpose is the organophosphate tributyl phosphate (TBP).
The PUREX process that is commonly used in nuclear reprocessing uses a mixture of tri-n-
butyl phosphate and an inert hydrocarbon (kerosene), the uranium(VI) are extracted from
strong nitric acid and are back-extracted (stripped) using weak nitric acid. An organic soluble
uranium complex [UO2(TBP)2(NO3)2] is formed, then the organic layer bearing the uranium
is brought into contact with a dilute nitric acid solution; the equilibrium is shifted away from
the organic soluble uranium complex and towards the free TBP and uranyl nitrate in dilute
nitric acid. The plutonium(IV) forms a similar complex to the uranium(VI), but it is possible
to strip the plutonium in more than one way; a reducing agent that converts the plutonium to

122
the trivalent oxidation state can be added. This oxidation state does not form a stable complex
with TBP and nitrate unless the nitrate concentration is very high (circa 10 mol/L nitrate is
required in the aqueous phase). Another method is to simply use dilute nitric acid as a
stripping agent for the plutonium. This PUREX chemistry is a classic example of a solvation
extraction.

Here in this case DU = k TBP2[[NO3]]2

Ion exchange mechanism

Another extraction mechanism is known as the ion exchange mechanism. Here, when an ion
is transferred from the aqueous phase to the organic phase, another ion is transferred in the
other direction to maintain the charge balance. This additional ion is often a hydrogen ion; for
ion exchange mechanisms, the distribution ratio is often a function of pH. An example of an
ion exchange extraction would be the extraction of americium by a combination of
terpyridine and a carboxylic acid in tert-butyl benzene. In this case

DAm = k terpyridine1carboxylic acid3H+−3

Another example is the extraction of zinc, cadmium, or lead by a dialkyl phosphinic acid
(R2PO2H) into a nonpolar diluent such as an alkane. A non-polar diluent favours the
formation of uncharged non-polar metal complexes.

Some extraction systems are able to extract metals by both the solvation and ion exchange
mechanisms; an example of such a system is the americium (and lanthanide) extraction from
nitric acid by a combination of 6,6'-bis-(5,6-dipentyl-1,2,4-triazin-3-yl)-2,2'-bipyridine and 2-
bromohexanoic acid in tert-butyl benzene. At both high- and low-nitric acid concentrations,
the metal distribution ratio is higher than it is for an intermediate nitric acid concentration.

Ion pair extraction

It is possible by careful choice of counterion to extract a metal. For instance, if the nitrate
concentration is high, it is possible to extract americium as an anionic nitrate complex if the
mixture contains a lipophilic quaternary ammonium salt.

An example that is more likely to be encountered by the 'average' chemist is the use of a
phase transfer catalyst. This is a charged species that transfers another ion to the organic
phase. The ion reacts and then forms another ion, which is then transferred back to the
aqueous phase.

For instance, the 31.1 kJ mol−1 is required to transfer an acetate anion into nitrobenzene,[13]
while the energy required to transfer a chloride anion from an aqueous phase to nitrobenzene
is 43.8 kJ mol−1.[14] Hence, if the aqueous phase in a reaction is a solution of sodium acetate
while the organic phase is a nitrobenzene solution of benzyl chloride, then, when a phase
transfer catalyst, the acetate anions can be transferred from the aqueous layer where they
react with the benzyl chloride to form benzyl acetate and a chloride anion. The chloride anion
is then transferred to the aqueous phase. The transfer energies of the anions contribute to that
given out by the reaction.

123
A 43.8 to 31.1 kJ mol−1 = 12.7 kJ mol−1 of additional energy is given out by the reaction when
compared with energy if the reaction had been done in nitrobenzene using one equivalent
weight of a tetraalkylammonium acetate.[15]

Types of aqueous two-phase extractions

Polymer–polymer systems. In a Polymer–polymer system, both phases are generated by a

dissolved polymer. The heavy phase will generally be Polyethylene glycol (PEG), and the
light phase is generally a polysaccharide. Traditionally, the polymer used is dextran.
However, dextran is relatively expensive, and research has been exploring using less
expensive polysaccharides to generate the light phase. If the target compound being separated
is a protein or enzyme, it is possible to incorporate a ligand to the target into one of the
polymer phases. This improves the target's affinity to that phase, and improves its ability to
partition from one phase into the other. This, as well as the absence of solvents or other
denaturing agents, makes polymer–polymer extractions an attractive option for purifying
proteins. The two phases of a polymer–polymer system often have very similar densities, and
very low surface tension between them. Because of this, demixing a polymer–polymer
system is often much more difficult than demixing a solvent extraction. Methods to improve
the demixing include centrifugation, and application of an electric field.

Polymer–salt systems. Aqueous two-phase systems can also be generated by introducing a

high concentration of salt to a polymer solution. The polymer phase used is generally still
PEG. Generally, a kosmotropic salt, such as Na3PO4 is used, however PEG–NaCl systems
have been documented when the salt concentration is high enough. Since polymer–salt
systems demix readily they are easier to use. However, at high salt concentrations, proteins
generally either denature, or precipitate from solution. Thus, polymer–salt systems are not as
useful for purifying proteins.

Ionic liquids systems. Ionic liquids are ionic compounds with low melting points. While they
are not technically aqueous, recent research has experimented with using them in an
extraction that does not use organic solvents.

Applications
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 DNA purification: The ability to purify DNA from a sample is important for many modern
biotechnology processes. However, samples often contain nucleases that degrade the target
DNA before it can be purified. It has been shown that DNA fragments will partition into the
light phase of a polymer–salt separation system. If ligands known to bind and deactivate
nucleases are incorporated into the polymer phase, the nucleases will then partition into the
heavy phase and be deactivated. Thus, this polymer–salt system is a useful tool for purifying
DNA from a sample while simultaneously protecting it from nucleases.
 Food industry: The PEG–NaCl system has been shown to be effective at partitioning small
molecules, such as peptides and nucleic acids. These compounds are often flavorants or
odorants. The system could then be used by the food industry to isolate or eliminate
particular flavors. Caffeine extraction used to be done using liquid–liquid extraction,
specifically direct and indirect liquid–liquid extraction (Swiss Water Method), but has since

124
 moved towards super-critical CO2 as it is cheaper and can be done on a commercial scale.[16]
[17]

 Analytical chemistry: Often there are chemical species present or necessary at one stage of
sample processing that will interfere with the analysis. For example, some air monitoring is
performed by drawing air through a small glass tube filled with sorbent particles that have
been coated with a chemical to stabilize or derivatize the analyte of interest. The coating
may be of such a concentration or characteristics that it would damage the instrumentation
or interfere with the analysis. If the sample can be extracted from the sorbent using a
nonpolar solvent (such as toluene or carbon disulfide), and the coating is polar (such as HBr
or phosphoric acid) the dissolved coating will partition into the aqueous phase. Clearly the
reverse is true as well, using polar extraction solvent and a nonpolar solvent to partition a
nonpolar interferent. A small aliquat of the organic phase (or in the latter case, polar phase)
can then be injected into the instrument for analysis.
 Purification of amines: Amines (analogously to ammonia) have a lone pair of electrons on
the nitrogen atom that can form a relatively weak bond to a hydrogen atom. It is therefore
the case that under acidic conditions amines are typically protonated, carrying a positive
charge and under basic conditions they are typically deprotonated and neutral. Amines of
sufficiently low molecular weight are rather polar and can form hydrogen bonds with water
and therefore will readily dissolve in aqueous solutions. Deprotonated amines on the other
hand, are neutral and have greasy, nonpolar organic substituents, and therefore have a
higher affinity for nonpolar inorganic solvents. As such purification steps can be carried out
where an aqueous solution of an amine is neutralized with a base such as sodium hydroxide,
then shaken in a separatory funnel with a nonpolar solvent that is immiscible with water.
The organic phase is then drained off. Subsequent processing can recover the amine by
techniques such as recrystallization, evaporation or distillation; subsequent extraction back
to a polar phase can be performed by adding HCl and shaking again in a separatory funnel
(at which point the ammonium ion could be recovered by adding an insoluble counterion),
or in either phase, reactions could be performed as part of a chemical synthesis.

Kinetics of extraction
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It is important to investigate the rate at which the solute is transferred between the two
phases, in some cases by an alteration of the contact time it is possible to alter the selectivity
of the extraction. For instance, the extraction of palladium or nickel can be very slow because
the rate of ligand exchange at these metal centers is much lower than the rates for iron or
silver complexes.

Aqueous complexing agents

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125
If a complexing agent is present in the aqueous phase then it can lower the distribution ratio.
For instance, in the case of iodine being distributed between water and an inert organic
solvent such as carbon tetrachloride then the presence of iodide in the aqueous phase can alter
the extraction chemistry.

Instead of being a constant it becomes = k[[I2.Organic]]/[I2.Aqueous] [[I−.Aqueous]]

This is because the iodine reacts with the iodide to form I3−. The I3− anion is an example of a
polyhalide anion that is quite common.

Industrial process design

This section does not cite any sources. Please help improve this section by adding citations
to reliable sources. Unsourced material may be challenged and removed. (May 2014) (Learn
how and when to remove this template message)

In a typical scenario, an industrial process will use an extraction step in which solutes are
transferred from the aqueous phase to the organic phase; this is often followed by a scrubbing
stage in which unwanted solutes are removed from the organic phase, then a stripping stage
in which the wanted solutes are removed from the organic phase. The organic phase may then
be treated to make it ready for use again.

After use, the organic phase may be subjected to a cleaning step to remove any degradation
products; for instance, in PUREX plants, the used organic phase is washed with sodium
carbonate solution to remove any dibutyl hydrogen phosphate or butyl dihydrogen phosphate
that might be present.

Equipment
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Title: Separation02.ogv
Author: Fotognome
Date: 6 June 2007

Phase separation during a laboratory scale LLE. The upper organic ether solution of MTBE is being
extracted with the lower alkaline aqueous sodium bicarbonate solution to remove benzoic acid as
the benzoate anion, leaving a non-acidic organic, benzil, (yellow in color) in the organic phase.

While solvent extraction is often done on a small scale by synthetic lab chemists using a
separatory funnel, Craig apparatus or membrane-based techniques,[18] it is normally done on
the industrial scale using machines that bring the two liquid phases into contact with each
other. Such machines include centrifugal contactors, Thin Layer Extraction, spray columns,
pulsed columns, and mixer-settlers.

Extraction of metals
The extraction methods for a range of metals include:[19]

 Cobalt – The extraction of cobalt from hydrochloric acid using alamine 336 in meta-xylene.[20]
Cobalt can be extracted also using Cyanex 272 {bis-(2,4,4-trimethylpentyl) phosphinic acid}.
 Copper – Copper can be extracted using hydroxyoximes as extractants, a recent paper
describes an extractant that has a good selectivity for copper over cobalt and nickel.[21]

127
  Neodymium – This rare earth is extracted by di(2-ethyl-hexyl)phosphoric acid into hexane by
an ion exchange mechanism.[22]
 Nickel – Nickel can be extracted using di(2-ethyl-hexyl)phosphoric acid and tributyl
phosphate in a hydrocarbon diluent (Shellsol).[23]
 Palladium and platinum – Dialkyl sulfides, tributyl phosphate and alkyl amines have been
used for extracting these metals.[24][25]
 Polonium is produced in reactors from natural 209Bi, bombarded with neutrons, creating 210Bi,
which then decays to 210Po via beta-minus decay. The final purification is done
pyrochemically followed by liquid-liquid extraction vs sodium hydroxide at 500 deg C.[26]
 Zinc and cadmium – The zinc and cadmium are both extracted by an ion exchange process,
the N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) acts as a masking agent for
the zinc and an extractant for the cadmium.[27] In the modified Zincex process, zinc is
separated from most divalent ions by solvent extraction. D2EHPA (Di (2) ethyl hexyl
phosphoric acid) is used for this. A zinc ion replaces the proton from two D2EHPA molecules.
To strip the zinc from the D2EHPA, sulfuric acid is used, at a concentration of above 170g/l
(typically 240-265g/l).

See also
 Fragrance extraction

References
1.

 (PDF) https://web.archive.org/web/20080309151803/http://www-
ssrl.slac.stanford.edu/pubs/activity_rep/ar98/2370-vance.pdf. Archived from the original (PDF) on
March 9, 2008. Retrieved January 21, 2006. Missing or empty |title= (help)
  pnjjrose. "Solvent Extraction Notes".

  "7.7: Liquid–Liquid Extractions". Chemistry LibreTexts. 2013-10-25. Retrieved 2017-03-26.

  http://courses.chem.psu.edu/chem36/Experiments/PDF's_for_techniques/Liquid_Liquid.pdf

  "Basic Technology and Tools in Chemical Engineering Field - S. Wesley - Documents".

  "Archived copy" (PDF). Archived from the original (PDF) on 2015-09-29. Retrieved 2015-09-28.

  Rezaee, Mohammad; Assadi, Yaghoub; Milani Hosseini, Mohammad-Reza; Aghaee, Elham;

Ahmadi, Fardin; Berijani, Sana (2006). "Determination of organic compounds in water using
dispersive liquid–liquid microextraction". Journal of Chromatography A. 1116 (1-2): 1–9. ISSN  0021-
9673. doi:10.1016/j.chroma.2006.03.007.

  Shacter, Emily (1984). "Organic extraction of Pi with isobutanol/toluene". Analytical

Biochemistry. 138 (2): 416–420. ISSN  0003-2697. doi:10.1016/0003-2697(84)90831-5.

128
  Senese F (20 September 2005). "How is coffee decaffeinated?". General Chemistry Online.
Retrieved 3 August 2009.

  Binnemans, Koen (2007). "Lanthanides and Actinides in Ionic Liquids". Chemical Reviews. 107
(6): 2592–2614. ISSN  0009-2665. doi:10.1021/cr050979c.

  Liquid–Liquid Extraction Equipment, Jack D. Law and Terry A. Todd, Idaho National Laboratory.

  "Riegel's Handbook of Industrial Chemistry".

  Scholz, F.; S. Komorsky-Lovric; M. Lovric (February 2000). "A new access to Gibbs energies of
transfer of ions across liquid|liquid interfaces and a new method to study electrochemical processes
at well-defined three-phase junctions". Electrochemistry Communications. Elsevier. 2 (2): 112–118.
doi:10.1016/S1388-2481(99)00156-3.

  Danil de Namor, A.F.; T. Hill (1983). Journal of the Chemical Society, Faraday Transactions: 2713.
Missing or empty |title= (help)

  zamani, Dariush. "Extraction Operation".

  Peker, Hulya; Srinivasan, M. P.; Smith, J. M.; McCoy, Ben J. (1992). "Caffeine extraction rates
from coffee beans with supercritical carbon dioxide". AIChE Journal. 38 (5): 761–770. ISSN  0001-
1541. doi:10.1002/aic.690380513.

  Emden, Lorenzo. "Decaffeination 101: Four Ways to Decaffeinate Coffee". Coffee Confidential.
Retrieved 29 October 2014.

  Adamo, Andrea; Heider, Patrick L.; Weeranoppanant, Nopphon; Jensen, Klavs F. (2013).
"Membrane-Based, Liquid–Liquid Separator with Integrated Pressure Control". Industrial &
Engineering Chemistry Research. 52 (31): 10802–10808. ISSN  0888-5885. doi:10.1021/ie401180t.

  Mackenzie, Murdoch. "The Solvent Extraction of Some Major Metals" (PDF). Cognis GmbH.
Archived from the original (PDF) on 2010-01-04. Retrieved 2008-11-18.

  Filiz, M.; Sayar, N.A.; Sayar, A.A. (2006). "Extraction of cobalt(II) from aqueous hydrochloric acid
solutions into alamine 336–m-xylene mixtures". Hydrometallurgy. 81 (3-4): 167–173. ISSN  0304-
386X. doi:10.1016/j.hydromet.2005.12.007.

  Baba, Yoshinari; Iwakuma, Minako; Nagami, Hideto (2002). "Extraction Mechanism for
Copper(II) with 2-Hydroxy-4-n-octyloxybenzophenone Oxime". Industrial & Engineering Chemistry
Research. 41 (23): 5835–5841. ISSN  0888-5885. doi:10.1021/ie0106736.

  Sanchez, J.M.; Hidalgo, M.; Salvadó, V.; Valiente, M. (1999). "EXTRACTION OF NEODYMIUM(III)
AT TRACE LEVEL WITH DI(2-ETHYL-HEXYL)PHOSPHORIC ACID IN HEXANE". Solvent Extraction and Ion
Exchange. 17 (3): 455–474. ISSN  0736-6299. doi:10.1080/07366299908934623.

  Lee W. John. "A Potential Nickel / Cobalt Recovery Process". BioMetallurgical Pty Ltd.

  "Precious Metals Refining By Solvent Extraction". Halwachs Edelmetallchemie und

Verfahrenstechnik. Retrieved 2008-11-18.

129
  Giridhar, P.; Venkatesan, K.A.; Srinivasan, T.G.; Vasudeva Rao, P.R. (2006). "Extraction of fission
palladium by Aliquat 336 and electrochemical studies on direct recovery from ionic liquid phase".
Hydrometallurgy. 81 (1): 30–39. ISSN  0304-386X. doi:10.1016/j.hydromet.2005.10.001.

  Schulz, Wallace W.; Schiefelbein, Gary F.; Bruns, Lester E. (1969). "Pyrochemical Extraction of
Polonium from Irradiated Bismuth Metal". Ind. Eng. Chem. Process Des. Dev. 8 (4): 508–515.
doi:10.1021/i260032a013.

27.  K. Takeshita; K. Watanabe; Y. Nakano; M. Watanabe (2003). "Solvent extraction

separation of Cd(II) and Zn(II) with the organophosphorus extractant D2EHPA and the
aqueous nitrogen-donor ligand TPEN". Hydrometallurgy. 70: 63–71. doi:10.1016/s0304-
386x(03)00046-x.

Further reading
 B.L. Karger, 2014, "Separation and Purification: Single-stage versus multistage processes"
and "Separation and Purification: Separations Based on Equilibrium", Encyclopædia
Britannica, see [1] and [2], accessed 12 May 2014.
 Gunt Hamburg, 2014, "Thermal Process Engineering: liquid–liquid extraction and solid-liquid
extraction", see [3], accessed 12 May 2014.
 G.W. Stevens, T.C., Lo, & M. H. I. Baird, 2007, "Extraction, liquid–liquid", in Kirk-Othmer
Encyclopedia of Chemical Technology, DOI: 10.1002/0471238961.120917211215.a01.pub2,
see [4], accessed 12 May 2014.
 Colin Poole & Michael Cooke, 2000, "Extraction", in Encyclopedia of Separation Science, 10
Vols., ISBN 9780122267703, see [5], accessed 12 May 2014.
 Sikdar, Cole, et al. Aqueous Two-Phase Extractions in Bioseparations: An Assessment.
Biotechnology 9:254. 1991
 Szlag, Giuliano. A Low-Cost Aqueous Two Phase System for Enzyme Extraction.
Biotechnology Techniques 2:4:277. 1988
 Dreyer, Kragl. Ionic Liquids for Aqueous Two-Phase Extraction and Stabilization of Enzymes.
Biotechnology and Bioengineering. 99:6:1416. 2008
 Boland. Aqueous Two-Phase Systems: Methods and Protocols. Pg 259-269
 http://ull.chemistry.uakron.edu/chemsep/extraction/

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Flow injection analysis

From Wikipedia, the free encyclopedia

Flow injection analysis (FIA) is an approach to chemical analysis that is accomplished by

injecting a plug of sample into a flowing carrier stream.[1][2][3] The principle is similar to that
of segmented flow analysis (SFA) but no air is injected into the sample or reagent streams.

Contents
 1 Overview
 2 Principles of Operation
 3 Detectors
 4 Experiments
 5 See also
 6 References
 7 Further reading
 8 External links

Overview
FIA is an automated method of chemical analysis in which a sample is injected into a flowing
carrier solution that mixes with reagents before reaching a detector. Over past 30 years, FIA
techniques developed into a wide array of applications using spectrophotometry, fluorescence
spectroscopy, atomic absorption spectroscopy, mass spectrometry, and other methods of
instrumental analysis for detection. Based on computer control, FIA evolved into Sequential
Injection and Bead Injection which are novel techniques based on flow programming. FIA
literature comprises over 22,000 scientific papers and 22 monographs.[4]

Principles of Operation

133
A sample (analyte) is injected into a carrier solution which mixes through radial and
convection diffusion with a reagent for a period of time (depending on the flow rate, coil
length, and coil diameter) before the sample passes through a detector to a waste container. A
peristaltic pump is the most commonly used pump in FIA instruments but new variants of
FIA technique use computer controlled syringe pumps that generate discontinuous flow
precisely choreographed to the needs of assay protocol. The result is dramatic decrease in
sample consumption, reagent consumption, and waste generation. The new technologies
broadened applicability of FIA techniques from laboratory serial assays to research
applications and continuous monitoring of environmental and industrial processes.[5]

Detectors
A flow-through detector is located downstream from the sample injector and records a
chemical physical parameter. Many types of detector can be used such as:[6]

 colorimeter
 fluorimeter
 ion-selective electrode
 biosensors

Experiments
An experiment that is used in analytical chemistry lab courses to familiarize students with
FIA is the determination of phosphate by flow injection analysis. The experiment involves
calibrating an FIA system, optimizing the system for detection of phosphate and finding the
amount of phosphate in an unknown sample.

See also
 AutoAnalyzer
 Colorimetric analysis

References
1.

 Xu, Weihong; Sandford, Richard; Worsfold, Paul; Carlton, Alexandra; Hanrahan, Grady (2005).
"Flow Injection Techniques in Aquatic Environmental Analysis: Recent Applications and Technological
Advances". Critical Reviews in Analytical Chemistry. 35 (3): 237. doi:10.1080/10408340500323362.
  Tyson, Julian F. (1985). "Flow injection analysis techniques for atomic-absorption spectrometry.
a review". The Analyst. 110 (5): 419–569. Bibcode:1985Ana...110..419T. PMID  4025835.
doi:10.1039/an9851000419.

  Anastos, N.; Barnett, NW; Hindson, BJ; Lenehan, CE; Lewis, SW (2004). "Comparison of soluble
manganese(IV) and acidic potassium permanganate chemiluminescence detection using flow
injection and sequential injection analysis for the determination of ascorbic acid in Vitamin C
tablets". Talanta. 64 (1): 130–4. PMID  18969577. doi:10.1016/j.talanta.2004.01.021.

134
  Ruscika, Jarda. "Flow Injection Tutorial". www.flowinjectiontutorial.com. Retrieved 2016-03-28.

  Ruzicka, Jarda. "Flow Injection Analysis". Retrieved 25 August 2013.

6.  Trojanowicz, M. (2000). Flow injection analysis  : instrumentation and applications.

World Scientific.

5. J. Ruzicka Flow Injection Analysis. Tutorial 2015 Edition

www.flowinjectiontutorial.com

Further reading
 Trojanowicz, Marek (2000). Flow injection analysis: instrumentation and applications.
Singapore: World Scientific. ISBN  981-02-2710-8.
 Hansen, Elo Harald; Růžička, Jaromír (1988). Flow injection analysis. New York: Wiley.
ISBN  0-471-81355-9.
 Martínez Calatayud, José (1996). Flow injection analysis of pharmaceuticals: automation in
the laboratory. Washington, DC: Taylor & Francis. ISBN  0-7484-0445-7.
 Pacey, Gil E.; Karlberg, Bo (1989). Flow injection analysis: a practical guide. Amsterdam:
Elsevier. ISBN  0-444-88014-3.

External links
 Technical University of Denmark
 FIAlab Instruments
 GlobalFIA

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 Analytical chemistry

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Figure 13.26 Two examples of a dual-channel manifold for flow injection analysis. In (a) the two
channels merge before the loop injector, and in (b) the two channels merge after the loop injector.

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Headspace technology
From Wikipedia, the free encyclopedia

Headspace technology is a technique developed in the 1980s to elucidate the odor

compounds present in the air surrounding various objects. Usually the objects of interest are
odoriferous objects such as plants, flowers and foods.[1] Similar techniques are also used to
analyze the interesting scents of locations and environments such as tea shops and saw mills.
After the data is analyzed, the scents can then be recreated by a perfumer.

One of the early pioneers of this technology includes Roman Kaiser who used it to measure
and characterize the scents of tropical rainforest. [2] Headspace techniques have since been
used extensively to sample in vivo floral headspace of a large variety of numerous taxa and
their aromatic compounds such as fatty acid derivatives (aldehydes, alcohols and ketones),
benzenoids and isoprenoids.[3]

Equipment
The headspace equipment involves a hollow dome or sphere-like objects which forms an
airtight seal and surrounds the objects of interest. Inert gases are passed into the space
containing the object or a vacuum is established such that the odor compounds are removed
from the headspace.[4] These compounds are in turn captured using a variety of techniques,
among them cold surfaces, solvent traps, and adsorbent materials, with the latter techniques
capable of longer periods of collection. The samples can then be analyzed using techniques
such as gas chromatography, mass spectrometry, or Carbon-13 NMR.[5]

Several companies have patented similar headspace technologies:

 Aromascope (Takasago)
 Jungle Essence (Mane)
 NaturePrint (Firmenich)
 ScentTrek (Givaudan)

References

138
 1.  Omar, Jone; Olivares, Maitane; Alonso, Ibone; Vallejo, Asier; Aizpurua-
Olaizola, Oier; Etxebarria, Nestor (2016-04-01). "Quantitative Analysis of
Bioactive Compounds from Aromatic Plants by Means of Dynamic Headspace
Extraction and Multiple Headspace Extraction-Gas Chromatography-Mass
Spectrometry". Journal of Food Science. 81 (4): C867–C873. ISSN 1750-
3841. doi:10.1111/1750-3841.13257.
2.   Kaiser, Roman (1997), "Environmental Scents at the Ligurian Coast",
Perfumer & Flavorist, 22: 7–18
3.   Knudsen, Jette T.; Tollsten, Lars; Bergström, L.Gunnar (1993),
"Floral scents—a checklist of volatile compounds isolated by head-space
techniques", Phytochemistry, 33 (2): 253–280, doi:10.1016/0031-
9422(93)85502-i
4.   Charles (Ed.), Sell; Karen Jenner (2005). "Chapter 14. The Search
for Fragrance Ingredients". The Chemistry of Fragrances (2nd ed.). Royal
Society of Chemistry Publishing. pp.  254–293. ISBN  978-0-85404-824-3.
5.  Charles (Ed.), Sell; Robin Clery (2005). "Chapter 12. Natural Product
Analysis in the Fragrance Industry". The Chemistry of Fragrances (2nd ed.).
Royal Society of Chemistry Publishing. pp.  214–228. ISBN  978-0-85404-824-
3.

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 

Hydride Generation
Atomic Absorption Spectroscopy
Introduction

Atomic absorption absorption spectroscopy (AAS) is one of the commonest instrumental

methods for analyzing for metals and some metalloids. But because of interferences, poor
reproducibility, and poor detection limits an alternative method for some elements--mostly
metalloids--has been developed. Hydride generation atomic absorption spectroscopy
(HGAAS) is available via an option for many modern AAS instruments. It "only" requires
the hydride generation module.

Metalloids like antimony,

arsenic, selenium, and
tellurium are now routinely
analyzed by HGAAS (see

www.shsu.edu/~chm_tgc/sounds/sound.html). Inductively coupled plasma (ICP) is also a

powerful analytical, instrumental method for these elements but at this point its much higher
cost limits it widespread use as compared to AAS or HGAAS.

As the animation on HGAAS here shows, many of the main parts of the HGAAS system are
identical to that of AAS: a hollow cathode lamp, air/acetylene flame, and optical system but
include (in most systems) an optical cell and the relatively complex hydride generation
system. The nebulizer required in AAS is not used in HGAAS. The system described here is
a continuous flow system, but batch flow systems have been used in the past. The job of each
component is detailed below:
 

Job of the hollow cathode lamp

141
Provide the analytical light line for the element of interest
Provide a constant yet intense beam of that analytical line

Job of the hydride generation system

Suck up (aspirate) liquid sample at a controlled rate

Mix liquid sample with sodium borohydride and HCl
Create a volatile hydride of the analyte metalloid from that reaction
Flow that gaseous hydride into the optical cell

Job of the optical cell and flame

Decompose the hydride form of the metalloid from the hydride generation module
Thereby create atoms (the elemental form) of the element of interest
Se0, Sb0, Te0, etc.

Job of the monochromator

Isolate analytical lines' photons passing through the optical cell

Remove scattered light of other wavelengths from the optical cell
In doing this, only a narrow spectral line impinges on the PMT.

Job of the photomultiplier tube (PMT)

As the detector, the PMT determines the intensity of photons of the analytical line exiting the
monochromator.

The Hollow Cathode Lamp

The hollow cathode lamp (HCL) uses a cathode made of the element of interest with a low
internal pressure of an inert gas. A low electrical current (~ 10 mA) is imposed in such a way
that the metal is excited and emits a few spectral lines characteristic of that element (for
instance, Te 214.3 nm and a couple of other lines; Se 196 nm and other lines, etc.). The light
is emitted directionally through the lamp's window, a window made of a glass transparent in
the UV and visible wavelengths.
 

142
Hydride Generation and Waste

The reaction of many metalloid oxyanions with sodium borohydride and HCl produces a
volatile hydride: H2Te, H2Se, H3As, H3Sb, etc. As with AAS, the oxidation state of the
metalloid is crucial and care must be taken to produce the specific metalloid oxidation state
before the sample is introduced into the hydride generation system.

The time from reagent mixing and when the volatile hydride is separated from the liquid and
sent to the optical cell is also important. The timing of that process is controlled by flowing
reagents together using a peristaltic pump and tubing of specific lengths. After being mixed
together the liquid mixture flows through a tube of a specific length (read this as a controlled
reaction time) and is ultimately flowed into a gas/liquid separator where the hydride and
some gaseous hydrogen (produced by the NaBH4 + H2 reaction) bubble out and are purged
(via a high purity inert gas) into the optical cell via a gas transfer line.

Most of the reagents introduced into the system flow to a waste container, and since the acid
content is very high, often approaching 50%, as with AAS, the waste container is glass and
must be handled carefully and labeled well.
 

The Optical Cell and Flame

The optical cell is fused silica glass tube (transparent in the visible and UV wavelengths and
thermally stable at high temperatures) through which the HCL's beam passes on the way to
the monochromator and PMT. In some instruments it sits on top of the normal AAS
air/acetylene flame. The gaseous, metalloidal hydride flows into the optical cell from the

143
hydride generation module pushes by an inert purge gas. In the optical cell it decomposes into
the elemental form
which can absorb the
HCL's beam.

The Monochromator
and PMT

Tuned to a specific
wavelength and with a
specified slit width
chosen, the
monochromator isolates
the hollow cathode
lamp's analytical line.
Since the basis for the
HGAAS process, like
AAS, is atomic
ABSORPTION, the
monochromator seeks to only allow the light not absorbed by the analyte atoms in the optical
cell to reach the PMT. That is, before an analyte is aspirated, a measured signal is generated
by the PMT as light from the HCL passes through the optical cell. When analyte atoms are
present in the cell from hydride decomposition--while the sample is aspirated--some of that
light is absorbed by those atoms (remember only volatile hydride gets to the optical cell and
then only decomposed hydride produces the elemental form). This causes a decrease in PMT
signal that is proportional to the amount of analyte. This last is true inside the linear range for
that element using that slit and that analytical line. The signal is therefore a decrease in
measure light: atomic absorption spectroscopy.
 

Acidic Content and Oxidation State of Samples and Standards

144
The samples and standards are often prepared with duplicate acid concentrations to replicate
the
analyte's
chemical
matrix as
closely
as
possible.
In
HGAAS,
acid
contents
of
samples
and
standards
of 10%
to 50%
are
common;
this is
much
much
higher
than in normal AAS.

The oxidation state of the analyte metalloid is important in HGAAS. For instance, HGAAS
analysis of selenium requires the Se(IV) oxidation state (selenite). Se(VI), the more highly
oxidized state of the element (selenate), responds erratically and non reproducibly in the
system. Therefore, all selenium in Se calibration standards and Se containing samples must
be in the Se(IV) form for analysis. This can be accomplished by oxidizing all Se in the
sample to selenate using a strong oxidizer such as nitric acid or hydrogen peroxide
(decomposing the excess oxidant) and then reducing the contained selenate to selenite with
boiling HCl. After that reduction step, the final acid content is made up to the required
content before the sample is introduced into the hydride generation module. The literature
also suggests that the time from reduction to introduction into the hydride module is
important: Shorter is best.

Also important is the concentration of sodium borohydride and hydrochloric acid reagents
feed into the hydride generation reaction vessel: optimization of this is important and may be
different for different elements. Example concentrations are 0.35% NaBH4 and 50% HCl.
Note that this acid content is not necessarily identical with the acid content of the samples
and standards themselves. The reagent acid's content is aimed at producing a reproducible
amount of hydride in the module.
 

Double Beam Instruments

The light from the HCL is split into two paths using a rotating mirror: one pathway passes
through the optical cell and another around. Both beams are recombined in space so they both

145
hit the PMT but separated in time. The beams alternate quickly back and forth along the two
paths: one instant the PMT beam is split by the rotating mirror and the sample beam passes
through the cell and hits the PMT. The next instance, the HCL beam passes through a hole in
the mirror and passes directly to the PMT without passing through the optical cell. The
difference between these beams is the amount of light absorbed by atoms in the optical cell.

The purpose of a double beam instrument is to help compensate for drift of the output of the
hollow cathode lamp or PMT. If the HCL output drifts slowly the subtraction process
described immediately above will correct for this because both beams will drift equally on the
time scale of the analysis. Likewise if the PMT response changes the double beam
arrangement take this into account.
 

Ignition, Flame conditions, and Shut Down

The process of lighting the AAS flame involves first putting the optical cell in place and
connecting the hydride gas transfer line. Next the fuel and the oxidant are turned on and then
the flame is lit with the instrument's auto ignition system (a small flame or red-hot glow
plug). After only a few minutes the flame is stable. Deionized water or a dilute acid solution
can be aspirated between samples (but experimentation is required to ascertain what produces
the best reproducibility). An aqueous solution with the correct amount of acid and no analyte
is often used as the blank. To stabilize the HGAAS system the acidic blank is often flowed
through the sample inlet tube for 5 or 10 minutes; although the longer this goes on, the more
acidic waste is produced.

Careful control of the fuel/air mixture is important because each element's response depends
on successful decomposition of the volatile hydride in the heated optical cell. Remember that
the flame's heat must break down the hydride and reproducibly create the elemental form of
the analyte atom. Optimization is accomplished by aspirating a solution containing the
element (with analyte content about that of the middle of the linear response range) and then
adjusting the fuel/oxidant mix until the maximum light absorbance is achieved. Also the
position of the burned head, optical cell, and sample uptake rate are similarly "tuned." Most
computer controlled systems can save variable settings so that methods for different elements
can be easily saved and reloaded.

Shut down involves aspirating deionized water through all three inlet tubes (borohydride,
acid, and sample inlets) for a short period and then closing the fuel off first. Most modern
instruments control the ignition and shutdown procedures automatically. The plastic tubing
that is stretched around the peristaltic pump head is loosened to length its lifetime. Finally the
purge gas is turned off.
 

These notes were written by Dr. Thomas G. Chasteen; Department of

Chemistry, Sam Houston State University, Huntsville, Texas 77341. © 2000,
2009.

146
Introduction

Contamination of industrial and municipal water

supplies with arsenic (As), selenium (Se), and mercury

(Hg) can occur from natural deposits, industrial

discharge, runoff from mining, landfill and agricultural

operations. Consumption of contaminated water can

cause skin damage (As), kidney and nervous system

damage (Hg) and numbness in the fingers and toes

(Se).
1

The U.S. Environmental Protection Agency

(EPA) and the Canadian Council of Ministers of the

Environment (CCME) have guidelines on the concen

tration of As, Se and Hg for the protection of marine

and freshwater aquatic life and the protection of agriculture.

1,2

Due to the low levels of

these guidelines for As, Se, and Hg, it is important to have analytical measurements that
are

precise and accurate with low amounts of noise.

Hydride generation (HG) is a very effective analytical technique developed to separate

hydride forming metals, such as Se and As, from a range of matrices and varying acid

concentrations. The heated quartz tube atomizer is particularly useful for the
determination

147
of arsenic and selenium because the absorption wavelengths for these elements are
below

200 nm in an area subject to intense interference from flame radicals that can
significantly

affect detection limits. Mercury can be easily reduced in solution to generate elemental

mercury, otherwise known as cold vapor (CV). This technique is also effective at
separating

mercury from a range of matrices. These analytical techniques can improve detection
limits

by a factor of approximately 3000 times that of flame detection limits and typically have
less

interference than graphite furnace techniques.

Atomic Absorption
APPLICATION NOTE
Author

Aaron Hineman

PerkinElmer, Inc.

Ontario, Canada

Determination of As,
Se and Hg in Waters by
Hydride Generation/
Cold Vapor Atomic
Absorption Spectroscopy
2

148
The generated hydrides were decomposed and atomized in

a heated quartz cell (Part No. B0507486) placed in a heating

mantle (Figure 2) with adaptor (Part No. N3160162 for

PinAAcle 900T, 900H, 900F models and N3160161 for the

PinAAcle 900Z model) in the place of the burner assembly.

The quartz cell was heated to 900 ̊C for the hydrides and

100 ̊C for mercury vapor analysis to avoid any condensation

in the cell. The quartz cell used for hydride analysis was

cleaned prior to use with 30% hydrofluoric acid.

High-energy electrodeless discharge lamps (EDLs) were

used for all the elements (Part Nos. As: N3050605; Se:

N3050672; Hg: N3050634). EDLs typically provide higher

energy than the corresponding HCLs and improve sensitivity

and detection limits, especially for arsenic and selenium. The

sample loop size was 500 μL for all analytes and could have

been increased or decreased to improve detection limits or

throughput, respectively.

The carrier gas stream has a large influence on sensitivity.

The objective is to have a fast enough carrier flow to obtain

a sharp peak, therefore greater sensitivity. If the flow is too

high, the atom or hydride cloud is dispersed too rapidly and

Separating the analyte from the matrix can improve the

sensitivity of the atomic absorption technique and avoids

physical, matrix and spectral interferences. The separation

of the hydride from the matrix allows for high efficiency

of analyte introduction into the atomic absorption spectro

photometer (AAS). The concentration effect and added

149
sensitivity of these techniques ultimately enables laboratories

to meet the lower detection limits required for environmental

regulations.

Experimental Conditions

Instrumentation

The measurements were performed using a PerkinElmer

®

PinAAcle
™

900T atomic absorption spectrophotometer

(Shelton, CT, USA) equipped with the intuitive WinLab32

™

for AA software, which features all the tools to analyze

samples, report and archive data and ensure regulatory

compliance. The PinAAcle spectrometer was coupled to a

FIAS 400 flow injection analysis system that incorporates

two peristaltic pumps, a 5-port flow injection valve and a

regulated gas supply. Default parameters found in the

software were used for all three elements: As, Se, and Hg.

Using a FIAS-AAS system, a sample loop on the flow injection

valve is filled with the acidified sample, blank, or standard.

The valve is automatically switched to the inject position and

the sample is mixed with a pumped stream of reductant,

sodium borohydride for hydrides or stannous chloride for

mercury to produce the gaseous vapors. At the point of

reaction with the reductant, arsenic or selenium hydrides or

elemental mercury vapor are produced, along with hydrogen

from the sodium borohydride, resulting in a two-phase mixture:

150
vapor with the analyte in it and the used-up reductant.

A flow of argon is added to this mixture and the vapors

are carried through a gas/liquid separator. This allows the

gaseous phase which contains the analyte vapor to enter

the quartz cell on the AAS for analysis while the remaining

liquids are pumped to a waste container. Refer to Table 1

for the FIAS parameters and Figure 1 for a schematic of the

FIAS system.

Table 1.

FIAS pump and valve timing.

Pump 1

Pump 2

Valve

Read

Step #

Time

(rpm)

(rpm)

Position

Trigger

Fill Inject

Prefill 15

100

120

10 100

151
120

15

120

100 120

Figure 2.

PerkinElmer heating mantle for the PinAAcle 900T spectrometer.

Figure 1.

Schematic diagram of FIAS 400 system for automated hydride

generation.

the sample was ready to run. The final dilution factor for the

SRMs was 10.

Selenium –

The samples and standards for selenium analysis

were pre-reduced (Se

6+

to Se
4+

) prior to analysis by diluting

1:1 with concentrated HCl followed by heating at 90 ̊C

152
for 30 minutes. The Se solutions were prepared using the

PerkinElmer SPB 50-24 Block Digestion System (Part No.

N9308019) with the SBP Touch controller (Part No. N9308007).

The graphite blocks are PTFE coated to resist the aggressive

corrosive attack that is common when pre-reducing Se

solutions for hydride analysis. Following the pre-reduction,

there is no risk of back oxidation (Se

4+

to Se
6+

). Therefore,

the solutions were diluted to the appropriate volume and

concentration for analysis. The final dilution factor for the

SRMs was 10.

Mercury –

No sample preparation was used for mercury

analysis.

Results and Discussion

System Performance

The system performed to the specifications as described in

Recommended Conditions and General Information for Flow

Injection Mercury/Hydride Analysis using the PerkinElmer

FIAS 100/400

technical note and in the WinLab32

™

for AA

software recommended conditions.

The sensitivity and detection limits of the system were estab

153
-

lished for each element and the information is presented in

Tables 2 and 3 (Page 4), respectively. The method detection

limits (MDLs) were determined from 10 analyses (3 replicates

each) of the blank solution that was taken through the prepa

ration procedures described above (Table 3, Figure 3 – Page 4).

Table 2.

System sensitivity.

Concentration

Analyte

(μg/L)

Peak Height (Abs)

As

10

0.44

Se

10

0.18

Hg

10

0.10

The method detection limits obtained were sufficient to

meet the U.S. EPA maximum contaminant levels (MCLs) for

drinking water.
1

Arsenic and selenium detection limits were

154
sufficient to meet the lowest Canadian CCME limit.

The mercury MDL obtained was sufficient for the analysis

of soils as per Canadian regulations.

2

However, either FIMS

(flow injection mercury system) or a larger sample loop is

recommended for meeting the CCME mercury guideline for

the protection of marine life.

could be pushed out with the waste to the drain. If the flow

is too low, the hydrides will come out slowly leading to a

broader peak, therefore the resulting signal and sensitivity

are lower. As a result, approximate flows of 50, 80 and

100 mL/min were used for the arsenic, selenium and mercury

determinations, respectively. To obtain the highest sensitivity,

the carrier gas flow was slightly optimized for each element

prior to calibration.

Standards, Chemicals and Standard Reference Materials

PerkinElmer Single-Element Calibration Standards for Atomic

Spectroscopy were used as the stock standards for preparing

the working standards (Part Nos. As: N9300180; Se: N9300182;

Hg: N9300174). Working standards were prepared by serial

volume/volume (v/v) dilution in polypropylene vials (Part No.

B0193234). All working standard solutions were freshly

prepared the day of analysis from the stock standards.

ASTM
®

Type I water (from an ELGA

®

155
filtration system –

ELGA LLC, Woodridge, Illinois, USA) acidified to 1% nitric

acid was used to make the calibration blank and standards.

Micropipettes (Eppendorf
®

, Germany) with disposable tips

were used for pipetting solutions.

The carrier solution for As and Se determination was a

10% (v/v) hydrochloric acid (HCl) solution. The carrier solution

for mercury was 3% (v/v) HCl acid. The selenium hydride

generation reducing agent was an aqueous solution of 0.2%

weight/volume (w/v) NaBH

4

in a 0.05% (w/v) NaOH solution,

which should be freshly prepared each day. For As, the NaBH
4

concentration was increased to 0.5% in order to improve

sensitivity. Mercury was determined using SnCl

2

as the

reducing agent, the reducing solution consisted of 1.1% (w/v)

SnCl
2

(from SnCl
2

2H
2

O) in 3.0% (v/v) hydrochloric acid.

Standard reference material (SRM) NIST

156
®

1640 Trace Elements

in Natural Water and SRM NIST

®

1643e Trace Elements in

Water were used to validate the method developed. The

NIST
®

reference materials were not certified for mercury,

therefore a second source standard from SPEX CertiPrep

®

(Metuchen, NJ, USA) was used for validation purposes.

Sample and Standard Reference Material Preparation

Arsenic –

The samples and standards for arsenic analysis

were pre-reduced (As

+5

to As
+3

) prior to analysis. This was

achieved by adding a reducing solution containing 5% (w/v)

KI and 5% (w/v) ascorbic acid. An appropriate volume of

standard or sample (up to 10 mL) was placed in a 50 mL

polypropylene autosampler tube. To this, 1 mL of the reducing

solution and 5 mL of concentrated HCl was added. The

treated samples or standards were set to stand at room

temperature for 30-60 minutes prior to analysis. The tube

was brought to the 50 mL mark with deionized water and

Conclusions

157
An accurate and reliable sample pretreatment and analysis

procedure for the determination of arsenic, selenium and

mercury in waters using FIAS-AAS is described. Hydride

generation is widely used in the determination of low levels

of elements whose salts readily form hydrides with sodium

borohydride. The advantage of hydride generation is that it

provides a concentration step and the analyte is transported

to the atomizer much more efficiently compared to that of

solution nebulization systems. This enhanced transportation

efficiency, combined with the heated quartz tube atomizer,

results in a large increase in sensitivity over flame (as well

as furnace) atomization and is valuable for very low-level

determinations.

The results presented here demonstrate that the PinAAcle

900T spectrometer coupled with a FIAS 400 flow injection

system can provide accurate and precise data for the analysis

of arsenic, selenium and mercury in waters. The unique

design of the PinAAcle 900T system allows for simple

installation and optimization of the quartz cell heating

mantle. This permits the user to easily switch between

flame, furnace and mercury/hydride techniques. This

application can be used on all of the PinAAcle spectro-

meter models with the appropriate adaptor kit.

References

1. U.S. EPA: http://water.epa.gov/drink/contaminants/#List

2. CCME: http://st-ts.ccme.ca/?chems=9,132,197&chapte

rs=1,2,3,4

Table 3.

158
System detection limits with U.S. EPA and CCME

limits.

MDL

U.S. EPA

Lowest CCME

Analyte

(μg/L)

MCL (μg/L)

Limits (μg/L)

As

0.020

10

Se

0.027

50

Hg

0.035

0.016

Water Samples

The two NIST

®

SRMs and the second source standard of

mercury were used to establish the accuracy and precision

of the methods. The accuracy and precision determined

159
for the validation samples for all elements was very good

(Table 4). The SRM samples were diluted 10x prior to analysis.

Figure 3.

Three replicates of 10 μg/L As.

Table 4.

NIST
®

SRM hydride results and mercury second source solution results.

Reference Material

Analyte

Target (μg/L)

Average Found (μg/L)

% Recovery

Std Dev

NIST
®

1640

As

26.71

27.76

104%

0.75

NIST
®

1640

Se

160
21.99

23.61

107%

11

0.19

NIST
®

1643e

As

60.45

61.30

101%

0.67

NIST
®

1643e

Se

11.97

12.38

103%

11

0.22

Second Source

Hg

1.00

1.024

161
102%

10

0.007
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Copyright ©2011-2012, PerkinElmer, Inc. All rights reserved. PerkinElmer

is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.

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Acid Digestions of Organic Samples

Trace Analysis Guide: Part 12 By Paul Gaines, Ph.D.

 Overview
 Nitric and Perchloric Acid Digestions
 Sample Preparation Procedure
 Microwave Digestion References
 Further Reading
 View as one page

Overview
The lack of negative side effects is unfortunately limited to the inorganic side of the table.
The ability of nitric acid to react with alcohols and aromatic rings forming explosive
compounds (nitro glycerine and TNT, to name two) calls for caution when using nitric acid
alone or in combination with other reagents in the decomposition of organic matrices. If your
sample contains -OH functionality it is best to pre-treat the sample with concentrated sulfuric
acid. When concentrated, the sulfuric will act as a dehydrating agent:

165
R-CH2-CH(OH)-R' + H2SO4     R-CH = C(OH)-R' + H2O

I do not recommend the use of nitric acid for the digestion of highly aromatic samples.

Nitric and Perchloric Acid Digestions

Nitric acid is rarely used alone. It is best used in combination with sulfuric and/or perchloric
acids for organic sample digestion. For samples that are not highly aromatic and/or contain a
high -OH functionality, I prefer to use nitric acid followed by perchloric acid. The only
element that may be lost from a nitric/perchloric digestion is Hg. Care should be exercised
and the literature consulted before attempting to use nitric acid in combination with other
acids for organic sample digestions.

CAUTION: The use of perchloric acid should only be attempted by those individuals well-
versed in the safe use of this reagent. Consult "Perchloric Acid and Perchlorates"1 for safety
guidelines.

The following are some key rules that I recommend when using nitric/perchloric acid
digestions:

1. Organic matrices should always be pre-treated with nitric acid (see exceptions above).
2. Perchloric acid should never be used alone.
3. Perchloric acid digestions should never be allowed to go to dryness.
4. Hot perchloric acid should never be added to an organic matrix.
5. Sample sizes should never exceed 1 gram (dry weight for biologicals).
6. Perchloric acid fumes should be not be allowed to 'go free' unless a perchloric acid hood is
used.
7. Unknown organic matrices should be analyzed by molecular spectroscopy to determine
primary structure before attempting the use of either nitric or perchloric acid.

Sample Preparation Procedure

The following preparation procedure was taken from our procedures manual for the acid
digestion of biological samples. Only excerpts pertaining to the sample handling and
digestion are included. It should be noted that this procedure takes advantage of the sliding
oxidation / reduction potential scale of perchloric acid and does not require a "high"
temperature finish to perchloric acid fumes.

Introduction and Scope

Determination of Trace Metals in Biological Samples

This procedure is intended for the determination trace metals (excluding Hg) in biological
tissues and biological liquids involved in biologically related tests (such as skin absorption
studies). This method is applicable for the determination of trace metals down to the ppb
concentration level. The detection limit is often determined by the blank due to the sensitivity
of current ICP-OES instrumentation. A mixture of nitric and perchloric acids is used to
decompose the tissue and serum sample types. Yttrium is added to the sample prior to
digestion and is used as an internal standard. All measurements are made using ICP-OES.

166
Apparatus and Chemicals

1. 20 by 150 mm borosilicate test tubes with Teflon-lined screw caps, cardboard holder for
weighing, and stainless steel racks for holding.
2. Borosilicate glass digestion beads.
3. Aluminum heating blocks with bore size of 20 mm equipped with thermometer and
aluminum heating plate.
4. An all plastic metal-free acid digestion hood.
5. 4-place and 2-place analytical balances.
6. ICP spectrometer capable of making simultaneous internal standard measurements and
storing spectra on computer disk.
7. Trace Metals grade 70% nitric acid.
8. Trace Metals grade 72% perchloric acid.
9. 5 and 50 mL class "A" pipets.
10. 500 mL class "A" volumetric flasks.
11. NIST/SRM 1577b bovine liver.
12. High purity 18 megaohm water.
13. 3 mL graduated LDPE dropping pipettes.
14. 1 mL Eppendorf pipette and plastic tips made form natural PE.
15. 0.1 mL Eppendorf pipette and plastic tips made form natural PE.
16. Plastic disposable weighing dishes.

Glassware Cleaning

1. The digestions' tubes and boiling beads are cleaned by heating in boiling 1:1 nitric acid / 18
meg-ohm water for at least 1 hour. After drying at 110 °C, the vessels and beads are allowed
to come to room temperature before use. Two boiling beads are added to each vessel and it
is capped with the Teflon lined screw on caps.
2. The digestion vessel caps and the volumetric glassware is cleaned by first sitting in 5 % v/v
nitric acid for 24 hours and then rinsed with 18 meg-ohm water a minimum of six times. No
heating of caps or volumetric glassware is allowed -- only air drying is acceptable.

Sample Handling, Identification, and Storage

1. Immediately upon arrival, all samples are to be inspected and compared with the chain of
custody information to confirm the proper shipping procedure, labeling procedure, and
number of samples (i.e. - it must be confirmed that the samples shipped were exactly those
received with respect to type, quantity, labeling, and that they were shipped properly and
received in good condition).
2. After signing off on the chain of custody document(s) it is required that each sample be
assigned an ID number. The number assigned is to be written using permanent magic marker
on a plastic bag into which the sample is placed and entered into the laboratory notebook
along with the submitter ID (The sample and the plastic bag are to accompany one another
throughout their existence at the lab). The assigned numbers are always to be in sequence
starting with 1 so it will be obvious to the operator if a sample is missing, was not prepared
for analysis, etc.. The assigned number is to never appear in any laboratory notebooks,
reports, or other documents without the accompaniment of the identification given by the
submitter. This guarantees that no mix-ups will occur.
3. The tissue and serum samples are stored in a freezer until 2-3 hours before they are
scheduled for analysis, at which time they are allowed to thaw at room temperature. Any
remaining sample is stored in the freezer. Plastic forceps and plastic spatulas are used to

167
 handle the tissue samples. Plastic pipettes are used to withdraw the serum samples. If the
whole tissue is not to be taken for analysis, it is transferred from the sample container to a
disposable PE dish where it is divided with plastic forceps or spatulas.

Sample Preparation of Tissues and Serum

1. Place a digestion vessel holder on a 4-place analytical balance and tare. Uncap a cleaned
digestion vessel and add two boiling beads.
2. Label the digestion vessel using a graphite pencil with the assigned ID. Obtain the vial weight
using a 4-place analytical balance. Record the digestion vessel weight in the analytical
notebook in the space provided across from the submitter and the assigned IDs written in
the notebook as described in step 2 under "Sample Handling, Identification, and Storage"
(see above). At no time is the vessel cap to be included in the vessel weight.
3. Use only plastic forceps, spatula, or dropping pipettes to handle the tissue and serum
samples. Tissue samples are removed from their shipping container to a plastic HDPE dish
for tearing (if necessary) just prior to weighing.
4. After recording the digestion vessel weight, tare the balance. It should read 0.0000 grams to
± 0.0001 grams.
5. Add 100 µL of 1000 µg/mL Yttrium internal standard to the digestion vessel. Record the
weight in the analytical notebook and tare the balance.
6. Add between 0.5 and 1.5 grams of tissue sample or 0.1 to 0.15 grams of NIST bovine liver QC
sample or 2.5 to 3.0 grams of serum sample to the digestion vessel and record the sample
weight to the nearest 0.0001 gram.
7. In a acid fume hood, add 3 mL of 70% nitric acid using a disposable LDPE dropping pipette.
8. Each group of digestions is to be accompanied with a blank and a NIST/SRM 1577b bovine
liver QC sample. A group of samples consists of a full digestion block of samples. One group
is equal to 24 vessels (22 samples and 2 controls). Since the NIST liver is dried, the sample
weight should not exceed 0.15 grams.
9. Place the digestion vessel in the digestion block which should be maintained at 110 °C
throughout the digestion.
10. Brown nitrogen dioxide fumes should be observed within 5 minutes. Do not leave the
digestion for the first 15 minutes. The sample should be completely dissolved within 15
minutes. Swirl the digestate to render homogeneous. With the sample weights
recommended, foaming should not be a problem. If foaming does occur, remove the sample
from the digestion block periodically to cool until dissolved.
11. Continue digesting the sample with nitric acid until the brown NOX fumes are barely visible.
Place the explosion shield in front of the digestion block, put on a face shield and heavy
rubber gloves. Carefully add 2 mL of 72% perchloric acid using a graduated 3 mL LDPE
dropping pipette.
12. Continue the digestion at 110 °C for 16 hours. The digestate should appear a very pale
yellow to water white.
13. Allow the digestate to cool to room temperature. Weigh the digestion vessel + digestate and
record this weight in the analytical notebook.
14. Calculate the weight of the digestate and record this value in the notebook. The density of
the digestate has been determined to be 1.49 ± 0.02 g/mL. Calculate the volume of the
digestate by dividing the digestate weight by the density. Enter this value in the notebook.
The final volume of the digestate is brought to 10 mL using 18 meg-ohm water. Calculate the
mL of water to add by subtracting the digestate volume from 10 mL and enter this value in
the notebook. Calculate the weight of water to add by multiplying the calculated mL by
0.997 g/mL and record this value in the notebook. Tare the analytical balance and add the

168
 calculated weight of water to the nearest ± 0.02 grams. A 2-place analytical balance can be
used.
15. Mix the final sample solution after capping by hand-shaking. The sample is ready for
analysis.

The above procedure has been used at our laboratory for processing large numbers of
biological tissues from animal feeding studies. For smaller sample numbers, a Kjeldahl
digestion rack with a glass hood and caustic scrubber is more convenient and is very effective
in removing any perchloric acid fumes. For samples that are harder to digest, higher
temperatures reaching fumes of perchloric acid may be required.

Microwave Digestion References

Dr. Skip Kingston has devoted a significant portion of his career to microwave chemistry. I
recommend several books by Dr. Kingston for researchers with interest in acid chemistry,
digestion, and safety.

 Microwave-Enhanced Chemistry Fundamentals, Sample Preparation, and Applications;

Kingston, H. M., Haswell, S. J., Eds.; American Chemical Society,: Washington, D.C., 1997.
 Introduction to Microwave Sample Preparation: Theory and Practice; Kingston, H. M., Jassie,
L. B., Eds.; American Chemical Society,: Washington, D.C., 1998.

1. G. Frederick, Perchloric Acid and Perchlorates Smith Chemical Co.: Columbus, OH

(available on request from GF Smith Co.)

Further Reading
 Part 13: Sample Preparation by Fusion
 Trace Analysis Guide: Table of Contents
 More Guides and Papers

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169
ADDITIONAL NOTES FOR SCH 2358 - Sample preparation Methods

ADDITIONAL AND SUMMARY NOTES

Methods
Method A (nitric-hydrochloric acid digestion 1:3) Metal ores, alloys and Soils – (AQUA
REGIA METHOD)

To about 1.0 g of the sample, 9 mL of freshly prepared acid mixture (1:3, v/v,) of 65 %
HNO3 and 37 % HCl is added. Then, the mixture is boiled gently over a water bath (95 °C)
for 4–5 h (or until the sample has completely dissolved) (Ang and Lee 2005).

During the digestion procedures, the inner walls of the beakers are washed with 2 mL of
deionized water to prevent the loss of the sample, and at the last part of the digestion
processes, the samples are filtered with Whatman 42 (2.5-μm particle retention) filter paper.
Then, a sufficient amount of deionized water is added to make the final volume up to 50 mL
ready for AAS analysis. The Heavy metals are determined using a suitable Atomic
Absorption Spectrometer, using a suitable AAS atomization technique, e.g. Flame AAS and
Graphite Furnace AAS, with the correct calibration, reagents, etc.

Method B (Nitric-Perchloric acid digestion 5:1) – Plant and animal tissues (AOAC
method) e.g. for Cd, Pb, Zn, Fe and Cu

To about 1.0 g of the sample, 5 mL of 65 % HNO3 is added, and then the mixture is boiled
gently for 30–45 min. After cooling, 1.0 mL of 70 % HClO4 is added, and the mixture is gently
boiled until dense white fumes appear. Later, the mixture is allowed to cool, and 10 mL of
deionized water is added followed by further boiling until the fumes are totally released.

The Heavy metals are determined using a suitable Atomic Absorption Spectrometer, using a
suitable AAS atomization technique, e.g. Flame AAS and Graphite Furnace AAS, with the
correct calibration, reagents, etc.

Analytical procedure for Graphite Furnace AAS

Heavy metals are determined, e.g. using a Perkin Elmer atomic absorption spectrometer (AAnalyst
800). The instrument is designed to analyze the sample using three different atomization techniques:
FAAS for Zn and Fe; GFAAS for Cd, Pb, and Ni; and HGAAS for As. In FAAS, the aqueous sample is
aspirated in the flame atomizer by the nebulizer to measure the analyte concentration at a parts per
million (ppm) concentration level with good precision. Pb, Cd, and Ni could be analyzed by GFAAS. It
is an efficient measurement system for a number of elements at relatively low levels of
concentration with the use of several matrix modifiers (Shah et al. 2009). In GFAAS, the instrument is
equipped with a transverse heated graphite atomizer (THGA) which provides uniform temperature
distribution across the entire length of the graphite tube atomizer to overcome the potential

170
chemical interference effects; also, the instrument offers an auto-sampler system and provides an
accurate background correction (Zeeman correction). Arsenic is detected by the HGAAS method
which is based on the reaction of NaBH4 with acidified sample results in total separation of the
analyte as hydride from the matrix before measurement which reduces the matrix interferences. In
this technique, standards and samples are pre-reduced from an arsenate pentavalent (V) to an
arsenite trivalent (III) state. This is achieved by adding a reducing solution containing 5 % (w/v) KI,
5 % (w/v) ascorbic acid, and 10 % HCl. The treated samples and standards are allowed to stand at
room temperature for approximately 40 min prior to analysis.

Hydride Generator (Outline – Summary)

Metalloids and soft metals (e.g., As, Se, Sb, Sn) can be introduced into the ICP in the form of
a volatile hydride, which is formed by reacting the element of interest with sodium
borohydride (NaBH4). The gaseous hydride is then carried to the plasma for analysis.

Hydride atomization

Hydride generation techniques are specialized in solutions of specific elements. The

technique provides a means of introducing samples containing arsenic, antimony, tin,
selenium, bismuth, and lead into an atomizer in the gas phase. With these elements, hydride
atomization enhances detection limits by a factor of 10 to 100 compared to alternative
methods. Hydride generation occurs by adding an acidified aqueous solution of the sample to
a 1% aqueous solution of sodium borohydride, all of which is contained in a glass vessel. The
volatile hydride generated by the reaction that occurs is swept into the atomization chamber
by an inert gas, where it undergoes decomposition. This process forms an atomized form of
the analyte, which can then be measured by absorption or emission spectrometry. (Refer to
other details later).

Cold-Vapor Atomization

The cold-vapor technique an atomization method limited to only the determination of

mercury, due to it being the only metallic element to have a large enough vapor pressure at
ambient temperature.[citation needed] Because of this, it has an important use in determining
organic mercury compounds in samples and their distribution in the environment. The
method initiates by converting mercury into Hg2+ by oxidation from nitric and sulfuric acids,
followed by a reduction of Hg2+ with tin(II) chloride. The mercury, is then swept into a long-
pass absorption tube by bubbling a stream of inert gas through the reaction mixture. The
concentration is determined by measuring the absorbance of this gas at 253.7 nm. Detection
limits for this technique are in the parts-per-billion range making it an excellent mercury
detection atomization method.

171
Sample Preparation for Mercury Analysis

Of each dried sample, 1g was accurately weighed and transferred into a conical flask. 8ml of
a mixture of concentrated nitric acid, sulphuric and perchloric acids (HNO3:H2SO4:HClO4) in
the ratio of 3:1:1 respectively was added. These samples were put in a water bath at 50°C
for 1 hour. The digestion vessels were covered using watch glasses [Muigaiet al., 1995].

The digestion vessels were then removed from the water bath and cooled in an ice bath.
50mlof 6% wt/vol. of potassium permanganate solution was added with constant shaking
until effervescence stopped. It was left in the oxidation medium overnight before the
second digestion step.

Second phase digestion was allowed for two hours at 50°C in a regulated water bath. The
digests were then removed from the water bath and cooled to room temperature. Then,
15ml 0f 20% wt/vol. hydroxylamine hydrochloric solution was added slowly while shaking to
avoid frothing and possible loss of flask contents. The final digest was filtered through
suitable filter paper number 40 into 100ml volumetric flask and the residue rinsed several
times with deionised water and then diluted to the mark using deionised water. The sample
extract was finally ready for cold vapour analysis by aspirating with equal volume of 25%
SnCl2 wt/vol. with 20% of conc. HCl vol. /vol [Muigaiet al., 1995].

The determination was completed by measuring absorbance at 253.7nm using flameless

determination of mercury with suitable calibration standards [Muigaiet al., 1995].

Liquid-liquid extraction (LLE)

Liquid-liquid extraction (LLE), also known as solvent extraction and partitioning, is a
method to separate compounds based on their relative solubilities in two different
immiscible liquids, usually water and an organic solvent. It is an extraction of a
substance from one liquid into another liquid phase. It consists of transferring one (or
more) solute(s) contained in a feed solution another immiscible liquid (solvent).
Example: Caffeine can be extracted from coffee beans and tea leaves using a direct
organic extraction, by soaking in ethyl acetate which favourably dissolves the caffeine,
leaving a majority of the coffee or tea flavor remaining in the initial sample.

Headspace technology
Headspace technology is a technique developed to elucidate the odor compounds present
in the air surrounding various objects. Usually, the objects of interest are odoriferous
objects such as plants, flowers and foods. Similar techniques are also used to analyze the
interesting scents of locations and environments such as tea shops and show mills. After the
data is analyzed, the scents can then be recreated by a perfumer. Inert gases are passed into
the space containing the object or a vacuum is established such that the odor compounds
are removed from the headspace. These compounds are in turn captured using a variety of
techniques, among them cold surfaces, solvent traps, and adsorbent materials, with the
latter techniques capable of longer periods of collection. The samples can then be analyzed
using techniques such as Gas Chromatography, Mass Spectrometry or Carbon-13 NMR.

172
 Flow injection analysis (FIA)
Flow injection analysis (FIA) is an approach to chemical analysis that is accomplished by
injecting a plug of sample into a flowing carrier stream. The principle is similar to that of
segmented flow analysis (SFA) but no air is injected into the sample or reagent streams. FIA
is an automated method of chemical analysis in which a simple is injected into a flowing
carrier solution that mixes with reagents before reaching a detector. Over the years, FIA
techniques have developed into a wide array of applications using spectrophotometry,
fluorescence spectroscopy, Atomic Absorption Spectroscopy, Mass Spectrometry and other
methods of instrumental analysis for detection.
Many types of detector can be used such as Colorimeter, Fluorimeter, Ion-selective
electrode and Biosensors.
- An experiment that is used in Analytical Chemistry lab courses to familiarize students
with FIA is the determination of phosphate by Flow Injection Analysis. The
experiment involves calibrating an FIA system, optimizing the system for detection of
phosphate and finding the amount of phosphate in an unknown sample.

Liquid–liquid extraction
From Wikipedia, the free encyclopedia

Solvent extraction.

Liquid–liquid extraction (LLE), also known as solvent extraction and partitioning, is a

method to separate compounds based on their relative solubilities in two different immiscible
liquids, usually water and an organic solvent. It is an extraction of a substance from one
liquid into another liquid phase. It consists of transferring one (or more) solute(s) contained
in a feed solution to another immiscible liquid (solvent). The solvent that is enriched in
solute(s) is called extract. The feed solution that is depleted in solute(s) is called the raffinate.
LLE is a basic technique in chemical laboratories, where it is performed using a variety of
apparatus, from separatory funnels to countercurrent distribution equipment.[not verified in body]This
type of process is commonly performed after a chemical reaction as part of the work-up,
often including an acidic work up.

The term partitioning is commonly used to refer to the underlying chemical and physical
processes involved in liquid–liquid extraction, but on another reading may be fully
synonymous with it. The term solvent extraction can also refer to the separation of a
substance from a mixture by preferentially dissolving that substance in a suitable solvent. In
that case, a soluble compound is separated from an insoluble compound or a complex matrix.
[not verified in body]

173
Solvent extraction is used in nuclear reprocessing, ore processing, the production of fine
organic compounds, the processing of perfumes, the production of vegetable oils and
biodiesel, and other industries.[not verified in body] It is among the most common initial separation
techniques, though some difficulties result in extracting out closely related functional groups.

Liquid–liquid extraction is possible in non-aqueous systems: In a system consisting of a

molten metal in contact with molten salts, metals can be extracted from one phase to the
other. This is related to a mercuryelectrode where a metal can be reduced, the metal will often
then dissolve in the mercury to form an amalgam that modifies its electrochemistry greatly.
For example, it is possible for sodiumcations to be reduced at a mercury cathode to form
sodium amalgam, while at an inert electrode (such as platinum) the sodium cations are not
reduced. Instead, water is reduced to hydrogen. A detergent or fine solid can be used to
stabilize an emulsion, or third phase.[not verified in body]

Contents
 1Measures of effectiveness
o 1.1Distribution ratio
o 1.2Separation factors
o 1.3Decontamination factor
o 1.4Slopes of graphs
o 1.5Measures of success
 2Techniques
o 2.1Batchwise single stage extractions
 2.1.1Dispersive liquid–liquid microextraction (DLLME)
 2.1.2Direct organic extraction
o 2.2Multistage countercurrent continuous processes
 2.2.1Mixer–settlers
 2.2.2Centrifugal extractors
o 2.3Extraction without chemical change
o 2.4Solvation mechanism
o 2.5Ion exchange mechanism
o 2.6Ion pair extraction
 2.6.1Types of aqueous two-phase extractions
 2.6.2Applications
 3Kinetics of extraction
 4Aqueous complexing agents
 5Industrial process design
 6Equipment
 7Extraction of metals
 8See also
 9References
 10Further reading

174
Measures of effectiveness
Distribution ratio

In solvent extraction, a distribution ratio is often quoted as a measure of how well-extracted a

species is. The distribution ratio (Kd) is equal to the concentration of a solute in the organic
phase divided by its concentration in the aqueous phase. Depending on the system, the
distribution ratio can be a function of temperature, the concentration of chemical species in
the system, and a large number of other parameters. Note that D is related to the ΔG of the
extraction process.

Sometimes, the distribution ratio is referred to as the partition coefficient, which is often
expressed as the logarithm. Note that a distribution ratio for uranium and neptunium between
two inorganic solids (zirconolite and perovskite) has been reported.[1] In solvent extraction,
two immiscible liquids are shaken together. The more polar solutes dissolve preferentially in
the more polar solvent, and the less polar solutes in the less polar solvent. In this experiment,
the nonpolar halogens preferentially dissolve in the non-polar mineral oil.[2]

Although the distribution ratio and partition coefficient are often used synonymously, they
are not necessarily so. Solutes may exist in more than one form in any particular phase, which
would mean that the partition coefficient (Kd) and distribution ratio (D) will have different
values. This is an important distinction to make as whilst the partition coefficient has a fixed
value for the partitioning of a solute between two phases, the distribution ratio changes with
differing conditions in the solvent.[3]

After performing liquid–liquid extraction, a quantitative measure must be taken to determine

the ratio of the solution’s total concentration in each phase of the extraction. This quantitative
measure is known as the distribution ratio or distribution coefficient.[4]

Separation factors

The separation factor is one distribution ratio divided by another; it is a measure of the ability
of the system to separate two solutes. For instance, if the distribution ratio for nickel (DNi) is
10 and the distribution ratio for silver (DAg) is 100, then the silver/nickel separation factor
(SFAg/Ni) is equal to DAg/DNi = SFAg/Ni = 10.[5]

Decontamination factor

This is used to express the ability of a process to remove a contaminant from a product. For
instance, if a process is fed with a mixture of 1:9 cadmium to indium, and the product is a
1:99 mixture of cadmium and indium, then the decontamination factor (for the removal of
cadmium) of the process is 0.11 / 0.01 = 11.

175
Slopes of graphs

The easy way to work out the extraction mechanism is to draw graphs and measure the
slopes. If for an extraction system the D value is proportional to the square of the
concentration of a reagent (Z) then the slope of the graph of log10(D) against log10([[Z]]) will
be two.

Measures of success

Success of liquid–liquid extraction is measured through separation factors and

decontamination factors. The best way to understand the success of an extraction column is
through the liquid–liquid equilibrium (LLE) data set. The data set can then be converted into
a curve to determine the steady state partitioning behavior of the solute between the two
phases. The y-axis is the concentration of solute in the extract (solvent) phase, and the x-axis
is the concentration of the solute in the raffinate phase. From here, one can determine steps
for optimization of the process.[6]

Techniques
Batchwise single stage extractions

This is commonly used on the small scale in chemical labs. It is normal to use a separating
funnel. Processes include DLLME and direct organic extraction.

Dispersive liquid–liquid microextraction (DLLME)

A process used to extract small amounts of organic compounds from water samples.[7] This
process is done by injecting small amounts of an appropriate extraction solvent (C2Cl4) and a
disperser solvent (acetone) into the aqueous solution. The resulting solution is then
centrifuged to separate the organic and aqueous layers. This process is useful in extraction
organic compounds such as organochloride and organophsophorus pesticides, as well as
substituted benzene compounds from water samples.[7]

Direct organic extraction

By mixing partially organic soluble samples in organic solvent (toluene, benzene, xylene),
the organic soluble compounds will dissolve into the solvent and can be separated using a
separatory funnel. This process is valuable in the extraction of proteins and specifically
phosphoprotein and phosphopeptide phosphatases.[8]

Another example of this application is extracting anisole from a mixture of water and 5%
acetic acid using ether, then the anisole will enter the organic phase. The two phases would
then be separated. The acetic acid can then be scrubbed (removed) from the organic phase by
shaking the organic extract with sodium bicarbonate. The acetic acid reacts with the sodium
bicarbonate to form sodium acetate, carbon dioxide, and water.

Caffeine can also be extracted from coffee beans and tea leaves using a direct organic
extraction. The beans or leaves can be soaked in ethyl acetate which favorably dissolves the
caffeine, leaving a majority of the coffee or tea flavor remaining in the initial sample.[9]

176
Multistage countercurrent continuous processes

Coflore continuous countercurrent extractor.

These are commonly used in industry for the processing of metals such as the lanthanides;
because the separation factors between the lanthanides are so small many extraction stages
are needed.[10] In the multistage processes, the aqueous raffinate from one extraction unit is
fed to the next unit as the aqueous feed, while the organic phase is moved in the opposite
direction. Hence, in this way, even if the separation between two metals in each stage is
small, the overall system can have a higher decontamination factor.

Multistage countercurrent arrays have been used for the separation of lanthanides. For the
design of a good process, the distribution ratio should be not too high (>100) or too low
(<0.1) in the extraction portion of the process. It is often the case that the process will have a
section for scrubbing unwanted metals from the organic phase, and finally a stripping section
to obtain the metal back from the organic phase.

Mixer–settlers

Battery of mixer-settlers counter currently interconnected. Each mixer-settler unit provides a

single stage of extraction. A mixer settler consists of a first stage that mixes the phases
together followed by a quiescent settling stage that allows the phases to separate by gravity.

In the multistage countercurrent process, multiple mixer settlers are installed with mixing and
settling chambers located at alternating ends for each stage (since the outlet of the settling
sections feed the inlets of the adjacent stage’s mixing sections). Mixer-settlers are used when
a process requires longer residence times and when the solutions are easily separated by
gravity. They require a large facility footprint, but do not require much headspace, and need
limited remote maintenance capability for occasional replacement of mixing motors. (Colven,
1956; Davidson, 1957)[11]

177
4 stage battery of mixer-settlers for counter-current extraction.

Centrifugal extractors

Centrifugal extractors mix and separate in one unit. Two liquids will be intensively mixed
between the spinning rotor and the stationary housing at speeds up to 6000 RPM. This
develops great surfaces for an ideal mass transfer from the aqueous phase into the organic
phase. At 200–2000 g, both phases will be separated again. Centrifugal extractors minimize
the solvent in the process, optimize the product load in the solvent and extract the aqueous
phase completely. Counter current and cross current extractions are easily established.[12]

Extraction without chemical change

Some solutes such as noble gases can be extracted from one phase to another without the
need for a chemical reaction (see absorption). This is the simplest type of solvent extraction.
When a solvent is extracted, two immiscible liquids are shaken together. The more polar
solutes dissolve preferentially in the more polar solvent, and the less polar solutes in the less
polar solvent. Some solutes that do not at first sight appear to undergo a reaction during the
extraction process do not have distribution ratio that is independent of concentration. A
classic example is the extraction of carboxylic acids (HA) into nonpolar media such as
benzene. Here, it is often the case that the carboxylic acid will form a dimer in the organic
layer so the distribution ratio will change as a function of the acid concentration (measured in
either phase).

For this case, the extraction constant k is described by k = [[HAorganic]]2/[[HAaqueous]]

Solvation mechanism

Using solvent extraction it is possible to extract uranium, plutonium, or thorium from acid
solutions. One solvent used for this purpose is the organophosphatetributyl phosphate (TBP).
The PUREX process that is commonly used in nuclear reprocessing uses a mixture of tri-n-
butyl phosphate and an inerthydrocarbon (kerosene), the uranium(VI) are extracted from
strong nitric acid and are back-extracted (stripped) using weak nitric acid. An organic soluble
uranium complex [UO2(TBP)2(NO3)2] is formed, then the organic layer bearing the uranium
is brought into contact with a dilute nitric acid solution; the equilibrium is shifted away from
the organic soluble uranium complex and towards the free TBP and uranyl nitrate in dilute
nitric acid. The plutonium(IV) forms a similar complex to the uranium(VI), but it is possible
to strip the plutonium in more than one way; a reducing agent that converts the plutonium to
the trivalent oxidation state can be added. This oxidation state does not form a stable complex
with TBP and nitrate unless the nitrate concentration is very high (circa 10 mol/L nitrate is
required in the aqueous phase). Another method is to simply use dilute nitric acid as a

178
stripping agent for the plutonium. This PUREX chemistry is a classic example of a
solvationextraction.

Here in this case DU = k TBP2[[NO3]]2

Ion exchange mechanism

Another extraction mechanism is known as the ion exchange mechanism. Here, when an ion
is transferred from the aqueous phase to the organic phase, another ion is transferred in the
other direction to maintain the charge balance. This additional ion is often a hydrogen ion; for
ion exchange mechanisms, the distribution ratio is often a function of pH. An example of an
ion exchange extraction would be the extraction of americium by a combination of
terpyridine and a carboxylic acid in tert-butylbenzene. In this case

DAm = kterpyridine1carboxylic acid3H+−3

Another example is the extraction of zinc, cadmium, or lead by a dialkyl phosphinic acid
(R2PO2H) into a nonpolar diluent such as an alkane. A non-polar diluent favours the
formation of uncharged non-polar metal complexes.

Some extraction systems are able to extract metals by both the solvation and ion exchange
mechanisms; an example of such a system is the americium (and lanthanide) extraction from
nitric acid by a combination of 6,6'-bis-(5,6-dipentyl-1,2,4-triazin-3-yl)-2,2'-bipyridine and 2-
bromohexanoic acid in tert-butylbenzene. At both high- and low-nitric acid concentrations,
the metal distribution ratio is higher than it is for an intermediate nitric acid concentration.

Ion pair extraction

It is possible by careful choice of counterion to extract a metal. For instance, if the nitrate
concentration is high, it is possible to extract americium as an anionic nitrate complex if the
mixture contains a lipophilicquaternary ammonium salt.

An example that is more likely to be encountered by the 'average' chemist is the use of a
phase transfer catalyst. This is a charged species that transfers another ion to the organic
phase. The ion reacts and then forms another ion, which is then transferred back to the
aqueous phase.

For instance, the 31.1 kJmol−1 is required to transfer an acetate anion into nitrobenzene,[13]
while the energy required to transfer a chloride anion from an aqueous phase to nitrobenzene
is 43.8 kJ mol−1.[14] Hence, if the aqueous phase in a reaction is a solution of sodium acetate
while the organic phase is a nitrobenzene solution of benzyl chloride, then, when a phase
transfer catalyst, the acetate anions can be transferred from the aqueous layer where they
react with the benzylchloride to form benzyl acetate and a chloride anion. The chloride anion
is then transferred to the aqueous phase. The transfer energies of the anions contribute to that
given out by the reaction.

A 43.8 to 31.1 kJ mol−1 = 12.7 kJ mol−1 of additional energy is given out by the reaction when
compared with energy if the reaction had been done in nitrobenzene using one equivalent
weight of a tetraalkylammonium acetate.[15]

179
Types of aqueous two-phase extractions

Polymer–polymer systems. In a Polymer–polymer system, both phases are generated by a

dissolved polymer. The heavy phase will generally be Polyethylene glycol (PEG), and the
light phase is generally a polysaccharide. Traditionally, the polymer used is dextran.
However, dextran is relatively expensive, and research has been exploring using less
expensive polysaccharides to generate the light phase. If the target compound being separated
is a protein or enzyme, it is possible to incorporate a ligand to the target into one of the
polymer phases. This improves the target's affinity to that phase, and improves its ability to
partition from one phase into the other. This, as well as the absence of solvents or other
denaturing agents, makes polymer–polymer extractions an attractive option for purifying
proteins. The two phases of a polymer–polymer system often have very similar densities, and
very low surface tension between them. Because of this, demixing a polymer–polymer
system is often much more difficult than demixing a solvent extraction. Methods to improve
the demixing include centrifugation, and application of an electric field.

Polymer–salt systems. Aqueous two-phase systems can also be generated by introducing a

high concentration of salt to a polymer solution. The polymer phase used is generally still
PEG. Generally, a kosmotropic salt, such as Na3PO4 is used, however PEG–NaCl systems
have been documented when the salt concentration is high enough. Since polymer–salt
systems demix readily they are easier to use. However, at high salt concentrations, proteins
generally either denature, or precipitate from solution. Thus, polymer–salt systems are not as
useful for purifying proteins.

Ionic liquids systems. Ionic liquids are ionic compounds with low melting points. While they
are not technically aqueous, recent research has experimented with using them in an
extraction that does not use organic solvents.

Applications

 DNA purification: The ability to purify DNA from a sample is important for many modern
biotechnology processes. However, samples often contain nucleases that degrade the target
DNA before it can be purified. It has been shown that DNA fragments will partition into the
light phase of a polymer–salt separation system. If ligands known to bind and deactivate
nucleases are incorporated into the polymer phase, the nucleases will then partition into the
heavy phase and be deactivated. Thus, this polymer–salt system is a useful tool for purifying
DNA from a sample while simultaneously protecting it from nucleases.
 Food industry: The PEG–NaCl system has been shown to be effective at partitioning small
molecules, such as peptides and nucleic acids. These compounds are often flavorants or
odorants. The system could then be used by the food industry to isolate or eliminate
particular flavors. Caffeine extraction used to be done using liquid–liquid extraction,
specifically direct and indirect liquid–liquid extraction (Swiss Water Method), but has since
moved towards super-critical CO2 as it is cheaper and can be done on a commercial scale.[16]
[17]

 Analytical chemistry: Often there are chemical species present or necessary at one stage of
sample processing that will interfere with the analysis. For example, some air monitoring is
performed by drawing air through a small glass tube filled with sorbent particles that have
been coated with a chemical to stabilize or derivatize the analyte of interest. The coating
may be of such a concentration or characteristics that it would damage the instrumentation
or interfere with the analysis. If the sample can be extracted from the sorbent using a

180
 nonpolar solvent (such as toluene or carbon disulfide), and the coating is polar (such as HBr
or phosphoric acid) the dissolved coating will partition into the aqueous phase. Clearly the
reverse is true as well, using polar extraction solvent and a nonpolar solvent to partition a
nonpolar interferent. A small aliquat of the organic phase (or in the latter case, polar phase)
can then be injected into the instrument for analysis.
 Purification of amines: Amines (analogously to ammonia) have a lone pair of electrons on
the nitrogen atom that can form a relatively weak bond to a hydrogen atom. It is therefore
the case that under acidic conditions amines are typically protonated, carrying a positive
charge and under basic conditions they are typically deprotonated and neutral. Amines of
sufficiently low molecular weight are rather polar and can form hydrogen bonds with water
and therefore will readily dissolve in aqueous solutions. Deprotonated amines on the other
hand, are neutral and have greasy, nonpolar organic substituents, and therefore have a
higher affinity for nonpolar inorganic solvents. As such purification steps can be carried out
where an aqueous solution of an amine is neutralized with a base such as sodium hydroxide,
then shaken in a separatory funnel with a nonpolar solvent that is immiscible with water.
The organic phase is then drained off. Subsequent processing can recover the amine by
techniques such as recrystallization, evaporation or distillation; subsequent extraction back
to a polar phase can be performed by adding HCl and shaking again in a separatory funnel
(at which point the ammonium ion could be recovered by adding an insoluble counterion),
or in either phase, reactions could be performed as part of a chemical synthesis.

Kinetics of extraction

It is important to investigate the rate at which the solute is transferred between the two
phases, in some cases by an alteration of the contact time it is possible to alter the selectivity
of the extraction. For instance, the extraction of palladium or nickel can be very slow because
the rate of ligand exchange at these metal centers is much lower than the rates for iron or
silver complexes.

Aqueous complexing agents

If a complexing agent is present in the aqueous phase then it can lower the distribution ratio.
For instance, in the case of iodine being distributed between water and an inert organic
solvent such as carbon tetrachloride then the presence of iodide in the aqueous phase can alter
the extraction chemistry.

Instead of being a constant it becomes = k[[I2.Organic]]/[I2.Aqueous] [[I−.Aqueous]]

This is because the iodine reacts with the iodide to form I3−. The I3− anion is an example of a
polyhalideanion that is quite common.

181
Industrial process design

In a typical scenario, an industrial process will use an extraction step in which solutes are
transferred from the aqueous phase to the organic phase; this is often followed by a scrubbing
stage in which unwanted solutes are removed from the organic phase, then a stripping stage
in which the wanted solutes are removed from the organic phase. The organic phase may then
be treated to make it ready for use again.

After use, the organic phase may be subjected to a cleaning step to remove any degradation
products; for instance, in PUREX plants, the used organic phase is washed with sodium
carbonate solution to remove any dibutyl hydrogen phosphate or butyl dihydrogen phosphate
that might be present.

Equipment

Close

Credits

Title: Separation02.ogv
Author: Fotognome
Date: 6 June 2007

Phase separation during a laboratory scale

LLE. The upper organic ether solution of
MTBE is being extracted with the lower
alkaline aqueous sodium bicarbonate

182
solution to remove benzoic acid as the benzoate anion, leaving a non-acidic organic, benzil, (yellow
in color) in the organic phase.

While solvent extraction is often done on a small scale by synthetic lab chemists using a
separatory funnel, Craig apparatus or membrane-based techniques,[18] it is normally done on
the industrial scale using machines that bring the two liquid phases into contact with each
other. Such machines include centrifugal contactors, Thin Layer Extraction, spray columns,
pulsed columns, and mixer-settlers.

Extraction of metals
The extraction methods for a range of metals include:[19]

 Cobalt – The extraction of cobalt from hydrochloric acid using alamine 336 in meta-xylene.[20]
Cobalt can be extracted also using Cyanex 272 {bis-(2,4,4-trimethylpentyl) phosphinic acid}.
 Copper – Copper can be extracted using hydroxyoximes as extractants, a recent paper
describes an extractant that has a good selectivity for copper over cobalt and nickel.[21]

 Neodymium – This rare earth is extracted by di(2-ethyl-hexyl)phosphoric acid into hexane by

an ion exchange mechanism.[22]
 Nickel – Nickel can be extracted using di(2-ethyl-hexyl)phosphoric acid and tributyl
phosphate in a hydrocarbon diluent (Shellsol).[23]
 Palladium and platinum – Dialkyl sulfides, tributyl phosphate and alkyl amines have been
used for extracting these metals.[24][25]
 Polonium is produced in reactors from natural 209Bi, bombarded with neutrons, creating 210Bi,
which then decays to 210Po via beta-minus decay. The final purification is done
pyrochemically followed by liquid-liquid extraction vs sodium hydroxide at 500 deg C.[26]
 Zinc and cadmium – The zinc and cadmium are both extracted by an ion exchange process,
the N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) acts as a masking agent for
the zinc and an extractant for the cadmium.[27] In the modified Zincex process, zinc is
separated from most divalent ions by solvent extraction. D2EHPA (Di (2) ethyl hexyl
phosphoric acid) is used for this. A zinc ion replaces the proton from two D2EHPA molecules.
To strip the zinc from the D2EHPA, sulfuric acid is used, at a concentration of above 170g/l
(typically 240-265g/l).

See also
 Fragrance extraction

References
2.

183
(PDF)https://web.archive.org/web/20080309151803/http://www-
ssrl.slac.stanford.edu/pubs/activity_rep/ar98/2370-vance.pdf. Archived from the original(PDF) on
March 9, 2008. Retrieved January 21, 2006.Missing or empty |title= (help)
pnjjrose. "Solvent Extraction Notes".

"7.7: Liquid–Liquid Extractions". Chemistry LibreTexts. 2013-10-25. Retrieved 2017-03-26.

http://courses.chem.psu.edu/chem36/Experiments/PDF's_for_techniques/Liquid_Liquid.pdf

"Basic Technology and Tools in Chemical Engineering Field - S. Wesley - Documents".

"Archived copy"(PDF). Archived from the original(PDF) on 2015-09-29. Retrieved 2015-09-28.

Rezaee, Mohammad; Assadi, Yaghoub; Milani Hosseini, Mohammad-Reza; Aghaee, Elham;

Ahmadi, Fardin; Berijani, Sana (2006). "Determination of organic compounds in water using
dispersive liquid–liquid microextraction". Journal of Chromatography A. 1116 (1-2): 1–9. ISSN  0021-
9673. doi:10.1016/j.chroma.2006.03.007.

Shacter, Emily (1984). "Organic extraction of Pi with isobutanol/toluene". Analytical Biochemistry.

138 (2): 416–420. ISSN  0003-2697. doi:10.1016/0003-2697(84)90831-5.

Senese F (20 September 2005). "How is coffee decaffeinated?". General Chemistry Online.
Retrieved 3 August 2009.

Binnemans, Koen (2007). "Lanthanides and Actinides in Ionic Liquids". Chemical Reviews. 107 (6):
2592–2614. ISSN  0009-2665. doi:10.1021/cr050979c.

Liquid–Liquid Extraction Equipment, Jack D. Law and Terry A. Todd, Idaho National Laboratory.

"Riegel's Handbook of Industrial Chemistry".

Scholz, F.; S. Komorsky-Lovric; M. Lovric (February 2000). "A new access to Gibbs energies of
transfer of ions across liquid|liquid interfaces and a new method to study electrochemical processes
at well-defined three-phase junctions". Electrochemistry Communications. Elsevier. 2 (2): 112–118.
doi:10.1016/S1388-2481(99)00156-3.

Danil de Namor, A.F.; T. Hill (1983). Journal of the Chemical Society, Faraday Transactions:
2713.Missing or empty |title= (help)

zamani, Dariush. "Extraction Operation".

Peker, Hulya; Srinivasan, M. P.; Smith, J. M.; McCoy, Ben J. (1992). "Caffeine extraction rates from
coffee beans with supercritical carbon dioxide". AIChE Journal. 38 (5): 761–770. ISSN  0001-1541.
doi:10.1002/aic.690380513.

Emden, Lorenzo. "Decaffeination 101: Four Ways to Decaffeinate Coffee". Coffee Confidential.
Retrieved 29 October 2014.

184
Adamo, Andrea; Heider, Patrick L.; Weeranoppanant, Nopphon; Jensen, Klavs F. (2013).
"Membrane-Based, Liquid–Liquid Separator with Integrated Pressure Control". Industrial &
Engineering Chemistry Research. 52 (31): 10802–10808. ISSN  0888-5885. doi:10.1021/ie401180t.

Mackenzie, Murdoch. "The Solvent Extraction of Some Major Metals" (PDF). Cognis GmbH.
Archived from the original(PDF) on 2010-01-04. Retrieved 2008-11-18.

Filiz, M.; Sayar, N.A.; Sayar, A.A. (2006). "Extraction of cobalt(II) from aqueous hydrochloric acid
solutions into alamine 336–m-xylene mixtures". Hydrometallurgy. 81 (3-4): 167–173. ISSN  0304-
386X. doi:10.1016/j.hydromet.2005.12.007.

Baba, Yoshinari; Iwakuma, Minako; Nagami, Hideto (2002). "Extraction Mechanism for Copper(II)
with 2-Hydroxy-4-n-octyloxybenzophenone Oxime". Industrial & Engineering Chemistry Research. 41
(23): 5835–5841. ISSN  0888-5885. doi:10.1021/ie0106736.

Sanchez, J.M.; Hidalgo, M.; Salvadó, V.; Valiente, M. (1999). "EXTRACTION OF NEODYMIUM(III) AT
TRACE LEVEL WITH DI(2-ETHYL-HEXYL)PHOSPHORIC ACID IN HEXANE". Solvent Extraction and Ion
Exchange. 17 (3): 455–474. ISSN  0736-6299. doi:10.1080/07366299908934623.

Lee W. John. "A Potential Nickel / Cobalt Recovery Process". BioMetallurgical Pty Ltd.

"Precious Metals Refining By Solvent Extraction". Halwachs Edelmetallchemie und

Verfahrenstechnik. Retrieved 2008-11-18.

Giridhar, P.; Venkatesan, K.A.; Srinivasan, T.G.; Vasudeva Rao, P.R. (2006). "Extraction of fission
palladium by Aliquat 336 and electrochemical studies on direct recovery from ionic liquid phase".
Hydrometallurgy. 81 (1): 30–39. ISSN  0304-386X. doi:10.1016/j.hydromet.2005.10.001.

Schulz, Wallace W.; Schiefelbein, Gary F.; Bruns, Lester E. (1969). "Pyrochemical Extraction of
Polonium from Irradiated Bismuth Metal". Ind. Eng. Chem. Process Des. Dev. 8 (4): 508–515.
doi:10.1021/i260032a013.

28. K. Takeshita; K. Watanabe; Y. Nakano; M. Watanabe (2003). "Solvent extraction

separation of Cd(II) and Zn(II) with the organophosphorus extractant D2EHPA and the
aqueous nitrogen-donor ligand TPEN". Hydrometallurgy. 70: 63–71. doi:10.1016/s0304-
386x(03)00046-x.

Further reading
 B.L. Karger, 2014, "Separation and Purification: Single-stage versus multistage processes"
and "Separation and Purification: Separations Based on Equilibrium", Encyclopædia
Britannica, see [1] and [2], accessed 12 May 2014.
 Gunt Hamburg, 2014, "Thermal Process Engineering: liquid–liquid extraction and solid-liquid
extraction", see [3], accessed 12 May 2014.
 G.W. Stevens, T.C., Lo, & M. H. I. Baird, 2007, "Extraction, liquid–liquid", in Kirk-Othmer
Encyclopedia of Chemical Technology, DOI: 10.1002/0471238961.120917211215.a01.pub2,
see [4], accessed 12 May 2014.
 Colin Poole & Michael Cooke, 2000, "Extraction", in Encyclopedia of Separation Science, 10
Vols., ISBN 9780122267703, see [5], accessed 12 May 2014.

185
  Sikdar, Cole, et al. Aqueous Two-Phase Extractions in Bioseparations: An Assessment.
Biotechnology 9:254. 1991
 Szlag, Giuliano. A Low-Cost Aqueous Two Phase System for Enzyme Extraction.
Biotechnology Techniques 2:4:277. 1988
 Dreyer, Kragl. Ionic Liquids for Aqueous Two-Phase Extraction and Stabilization of Enzymes.
Biotechnology and Bioengineering. 99:6:1416. 2008
 Boland. Aqueous Two-Phase Systems: Methods and Protocols. Pg 259-269
 http://ull.chemistry.uakron.edu/chemsep/extraction/

Edit links

 This page was last edited on 23 August 2017, at 00:37.

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terms may apply. By using this site, you agree to the Terms of Use and Privacy Policy.
Wikipedia® is a registered trademark of the Wikimedia Foundation, Inc., a non-profit
organization.

Flow injection analysis

From Wikipedia, the free encyclopedia

Flow injection analysis (FIA) is an approach to chemical analysis that is accomplished by

injecting a plug of sample into a flowing carrier stream.[1][2][3] The principle is similar to that
of segmented flow analysis (SFA) but no air is injected into the sample or reagent streams.

Contents
 1Overview
 2Principles of Operation
 3Detectors
 4Experiments
 5See also
 6References
 7Further reading
 8External links

Overview
FIA is an automated method of chemical analysis in which a sample is injected into a flowing
carrier solution that mixes with reagents before reaching a detector. Over past 30 years, FIA
techniques developed into a wide array of applications using spectrophotometry, fluorescence
spectroscopy, atomic absorption spectroscopy, mass spectrometry, and other methods of

186
instrumental analysis for detection. Based on computer control, FIA evolved into Sequential
Injection and Bead Injection which are novel techniques based on flow programming. FIA
literature comprises over 22,000 scientific papers and 22 monographs.[4]

Principles of Operation
A sample (analyte) is injected into a carrier solution which mixes through radial and
convectiondiffusion with a reagent for a period of time (depending on the flow rate, coil
length, and coil diameter) before the sample passes through a detector to a waste container. A
peristaltic pump is the most commonly used pump in FIA instruments but new variants of
FIA technique use computer controlled syringe pumps that generate discontinuous flow
precisely choreographed to the needs of assay protocol. The result is dramatic decrease in
sample consumption, reagent consumption, and waste generation. The new technologies
broadened applicability of FIA techniques from laboratory serial assays to research
applications and continuous monitoring of environmental and industrial processes.[5]

Detectors
A flow-through detector is located downstream from the sample injector and records a
chemical physical parameter. Many types of detector can be used such as:[6]

 colorimeter
 fluorimeter
 ion-selective electrode
 biosensors

Experiments
An experiment that is used in analytical chemistry lab courses to familiarize students with
FIA is the determination of phosphate by flow injection analysis. The experiment involves
calibrating an FIA system, optimizing the system for detection of phosphate and finding the
amount of phosphate in an unknown sample.

References
2.

Xu, Weihong; Sandford, Richard; Worsfold, Paul; Carlton, Alexandra; Hanrahan, Grady (2005).
"Flow Injection Techniques in Aquatic Environmental Analysis: Recent Applications and Technological
Advances". Critical Reviews in Analytical Chemistry. 35 (3): 237. doi:10.1080/10408340500323362.
Tyson, Julian F. (1985). "Flow injection analysis techniques for atomic-absorption spectrometry. a
review". The Analyst. 110 (5): 419–569. Bibcode:1985Ana...110..419T. PMID  4025835.
doi:10.1039/an9851000419.

Anastos, N.; Barnett, NW; Hindson, BJ; Lenehan, CE; Lewis, SW (2004). "Comparison of soluble
manganese(IV) and acidic potassium permanganate chemiluminescence detection using flow

187
injection and sequential injection analysis for the determination of ascorbic acid in Vitamin C
tablets". Talanta. 64 (1): 130–4. PMID  18969577. doi:10.1016/j.talanta.2004.01.021.

Ruscika, Jarda. "Flow Injection Tutorial". www.flowinjectiontutorial.com. Retrieved 2016-03-28.

Ruzicka, Jarda. "Flow Injection Analysis". Retrieved 25 August 2013.

7. Trojanowicz, M. (2000). Flow injection analysis  : instrumentation and applications.

World Scientific.

5. J. Ruzicka Flow Injection Analysis. Tutorial 2015 Edition

www.flowinjectiontutorial.com

Further reading
 Trojanowicz, Marek (2000). Flow injection analysis: instrumentation and applications.
Singapore: World Scientific. ISBN  981-02-2710-8.
 Hansen, Elo Harald; Růžička, Jaromír (1988). Flow injection analysis. New York: Wiley.
ISBN  0-471-81355-9.
 Martínez Calatayud, José (1996). Flow injection analysis of pharmaceuticals: automation in
the laboratory. Washington, DC: Taylor & Francis. ISBN  0-7484-0445-7.
 Pacey, Gil E.; Karlberg, Bo (1989). Flow injection analysis: a practical guide. Amsterdam:
Elsevier. ISBN  0-444-88014-3.

Figure 13.26 Two examples of a dual-channel manifold for flow injection analysis. In (a) the two
channels merge before the loop injector, and in (b) the two channels merge after the loop injector.

188
Headspace technology
From Wikipedia, the free encyclopedia

Headspace technology is a technique developed in the 1980s to elucidate the odor

compounds present in the air surrounding various objects. Usually the objects of interest are
odoriferous objects such as plants, flowers and foods.[1] Similar techniques are also used to
analyze the interesting scents of locations and environments such as tea shops and saw mills.
After the data is analyzed, the scents can then be recreated by a perfumer.

One of the early pioneers of this technology includes Roman Kaiser who used it to measure
and characterize the scents of tropical rainforest. [2] Headspace techniques have since been
used extensively to sample in vivo floral headspace of a large variety of numerous taxa and
their aromatic compounds such as fatty acid derivatives (aldehydes, alcohols and ketones),
benzenoids and isoprenoids.[3]

Equipment
The headspace equipment involves a hollow dome or sphere-like objects which forms an
airtight seal and surrounds the objects of interest. Inert gases are passed into the space
containing the object or a vacuum is established such that the odor compounds are removed
from the headspace.[4] These compounds are in turn captured using a variety of techniques,
among them cold surfaces, solvent traps, and adsorbent materials, with the latter techniques
capable of longer periods of collection. The samples can then be analyzed using techniques
such as gas chromatography, mass spectrometry, or Carbon-13 NMR.[5]

Several companies have patented similar headspace technologies:

 Aromascope (Takasago)
 Jungle Essence (Mane)
 NaturePrint (Firmenich)
 ScentTrek (Givaudan)

References
1. Omar, Jone; Olivares, Maitane; Alonso, Ibone; Vallejo, Asier; Aizpurua-
Olaizola, Oier; Etxebarria, Nestor (2016-04-01). "Quantitative Analysis of
Bioactive Compounds from Aromatic Plants by Means of Dynamic Headspace
Extraction and Multiple Headspace Extraction-Gas Chromatography-Mass
Spectrometry". Journal of Food Science. 81 (4): C867–C873. ISSN 1750-
3841. doi:10.1111/1750-3841.13257.
2. Kaiser, Roman (1997), "Environmental Scents at the Ligurian Coast",
Perfumer & Flavorist, 22: 7–18
3. Knudsen, Jette T.; Tollsten, Lars; Bergström, L.Gunnar (1993), "Floral
scents—a checklist of volatile compounds isolated by head-space techniques",
Phytochemistry, 33 (2): 253–280, doi:10.1016/0031-9422(93)85502-i

189
 4. Charles (Ed.), Sell; Karen Jenner (2005). "Chapter 14. The Search for
Fragrance Ingredients". The Chemistry of Fragrances (2nd ed.). Royal
Society of Chemistry Publishing. pp.  254–293. ISBN  978-0-85404-824-3.
5. Charles (Ed.), Sell; Robin Clery (2005). "Chapter 12. Natural Product
Analysis in the Fragrance Industry". The Chemistry of Fragrances (2nd ed.).
Royal Society of Chemistry Publishing. pp.  214–228. ISBN  978-0-85404-824-
3.

Hydride Generation
Atomic Absorption Spectroscopy
Introduction

Atomic absorption absorption spectroscopy (AAS) is one of the commonest instrumental

methods for analyzing for metals and some metalloids. But because of interferences, poor
reproducibility, and poor detection limits an alternative method for some elements--mostly
metalloids--has been developed. Hydride generation atomic absorption spectroscopy
(HGAAS) is available via an option for many modern AAS instruments. It "only" requires
the hydride generation module.

Metalloids like antimony,

arsenic, selenium, and
tellurium are now routinely
analyzed by HGAAS (see

www.shsu.edu/~chm_tgc/sounds/sound.html). Inductively coupled plasma (ICP) is also a

powerful analytical, instrumental method for these elements but at this point its much higher
cost limits it widespread use as compared to AAS or HGAAS.

As the animation on HGAAS here shows, many of the main parts of the HGAAS system are
identical to that of AAS: a hollow cathode lamp, air/acetylene flame, and optical system but
include (in most systems) an optical cell and the relatively complex hydride generation
system. The nebulizer required in AAS is not used in HGAAS. The system described here is
a continuous flow system, but batch flow systems have been used in the past. The job of each
component is detailed below:

190
 

Job of the hollow cathode lamp

Provide the analytical light line for the element of interest

Provide a constant yet intense beam of that analytical line

Job of the hydride generation system

Suck up (aspirate) liquid sample at a controlled rate

Mix liquid sample with sodium borohydride and HCl
Create a volatile hydride of the analyte metalloid from that reaction
Flow that gaseous hydride into the optical cell

Job of the optical cell and flame

Decompose the hydride form of the metalloid from the hydride generation module
Thereby create atoms (the elemental form) of the element of interest
Se0, Sb0, Te0, etc.

Job of the monochromator

Isolate analytical lines' photons passing through the optical cell

Remove scattered light of other wavelengths from the optical cell
In doing this, only a narrow spectral line impinges on the PMT.

Job of the photomultiplier tube (PMT)

As the detector, the PMT determines the intensity of photons of the analytical line exiting the
monochromator.

The Hollow Cathode Lamp

The hollow cathode lamp (HCL) uses a cathode made of the element of interest with a low
internal pressure of an inert gas. A low electrical current (~ 10 mA) is imposed in such a way
that the metal is excited and emits a few spectral lines characteristic of that element (for
instance, Te 214.3 nm and a couple of other lines; Se 196 nm and other lines, etc.). The light
is emitted directionally through the lamp's window, a window made of a glass transparent in
the UV and visible wavelengths.
 

191
Hydride Generation and Waste

The reaction of many metalloid oxyanions with sodium borohydride and HCl produces a
volatile hydride: H2Te, H2Se, H3As, H3Sb, etc. As with AAS, the oxidation state of the
metalloid is crucial and care must be taken to produce the specific metalloid oxidation state
before the sample is introduced into the hydride generation system.

The time from reagent mixing and when the volatile hydride is separated from the liquid and
sent to the optical cell is also important. The timing of that process is controlled by flowing
reagents together using a peristaltic pump and tubing of specific lengths. After being mixed
together the liquid mixture flows through a tube of a specific length (read this as a controlled
reaction time) and is ultimately flowed into a gas/liquid separator where the hydride and
some gaseous hydrogen (produced by the NaBH4 + H2 reaction) bubble out and are purged
(via a high purity inert gas) into the optical cell via a gas transfer line.

Most of the reagents introduced into the system flow to a waste container, and since the acid
content is very high, often approaching 50%, as with AAS, the waste container is glass and
must be handled carefully and labeled well.

The Optical Cell and Flame

The optical cell is fused silica glass tube (transparent in the visible and UV wavelengths and
thermally stable at high temperatures) through which the HCL's beam passes on the way to

192
the monochromator and PMT. In some instruments it sits on top of the normal AAS
air/acetylene flame. The gaseous, metalloidal hydride flows into the optical cell from the
hydride generation module pushes by an inert purge gas. In the optical cell it decomposes into
the elemental form
which can absorb the
HCL's beam.

The Monochromator
and PMT

Tuned to a specific
wavelength and with a
specified slit width
chosen, the
monochromator isolates
the hollow cathode
lamp's analytical line.
Since the basis for the
HGAAS process, like
AAS, is atomic
ABSORPTION, the
monochromator seeks to only allow the light not absorbed by the analyte atoms in the optical
cell to reach the PMT. That is, before an analyte is aspirated, a measured signal is generated
by the PMT as light from the HCL passes through the optical cell. When analyte atoms are
present in the cell from hydride decomposition--while the sample is aspirated--some of that
light is absorbed by those atoms (remember only volatile hydride gets to the optical cell and
then only decomposed hydride produces the elemental form). This causes a decrease in PMT
signal that is proportional to the amount of analyte. This last is true inside the linear range for
that element using that slit and that analytical line. The signal is therefore a decrease in
measure light: atomic absorption spectroscopy.

193
 

Acidic Content and Oxidation State of Samples and Standards

The samples and standards are often prepared with duplicate acid concentrations to replicate
the
analyte's
chemical
matrix as
closely
as
possible.
In
HGAAS,
acid
contents
of
samples
and
standards
of 10%
to 50%
are
common;
this is
much
much
higher
than in
normal AAS.

The oxidation state of the analyte metalloid is important in HGAAS. For instance, HGAAS
analysis of selenium requires the Se(IV) oxidation state (selenite). Se(VI), the more highly
oxidized state of the element (selenate), responds erratically and non reproducibly in the
system. Therefore, all selenium in Se calibration standards and Se containing samples must
be in the Se(IV) form for analysis. This can be accomplished by oxidizing all Se in the
sample to selenate using a strong oxidizer such as nitric acid or hydrogen peroxide
(decomposing the excess oxidant) and then reducing the contained selenate to selenite with
boiling HCl. After that reduction step, the final acid content is made up to the required
content before the sample is introduced into the hydride generation module. The literature
also suggests that the time from reduction to introduction into the hydride module is
important: Shorter is best.

Also important is the concentration of sodium borohydride and hydrochloric acid reagents
feed into the hydride generation reaction vessel: optimization of this is important and may be
different for different elements. Example concentrations are 0.35% NaBH4 and 50% HCl.
Note that this acid content is not necessarily identical with the acid content of the samples
and standards themselves. The reagent acid's content is aimed at producing a reproducible

194
amount of hydride in the module.

Double Beam Instruments

The light from the HCL is split into two paths using a rotating mirror: one pathway passes
through the optical cell and another around. Both beams are recombined in space so they both
hit the PMT but separated in time. The beams alternate quickly back and forth along the two
paths: one instant the PMT beam is split by the rotating mirror and the sample beam passes
through the cell and hits the PMT. The next instance, the HCL beam passes through a hole in
the mirror and passes directly to the PMT without passing through the optical cell. The
difference between these beams is the amount of light absorbed by atoms in the optical cell.

The purpose of a double beam instrument is to help compensate for drift of the output of the
hollow cathode lamp or PMT. If the HCL output drifts slowly the subtraction process
described immediately above will correct for this because both beams will drift equally on the
time scale of the analysis. Likewise if the PMT response changes the double beam
arrangement take this into account.
 

Ignition, Flame conditions, and Shut Down

The process of lighting the AAS flame involves first putting the optical cell in place and
connecting the hydride gas transfer line. Next the fuel and the oxidant are turned on and then
the flame is lit with the instrument's auto ignition system (a small flame or red-hot glow
plug). After only a few minutes the flame is stable. Deionized water or a dilute acid solution
can be aspirated between samples (but experimentation is required to ascertain what produces
the best reproducibility). An aqueous solution with the correct amount of acid and no analyte
is often used as the blank. To stabilize the HGAAS system the acidic blank is often flowed
through the sample inlet tube for 5 or 10 minutes; although the longer this goes on, the more
acidic waste is produced.

Careful control of the fuel/air mixture is important because each element's response depends
on successful decomposition of the volatile hydride in the heated optical cell. Remember that
the flame's heat must break down the hydride and reproducibly create the elemental form of
the analyte atom. Optimization is accomplished by aspirating a solution containing the
element (with analyte content about that of the middle of the linear response range) and then
adjusting the fuel/oxidant mix until the maximum light absorbance is achieved. Also the
position of the burned head, optical cell, and sample uptake rate are similarly "tuned." Most
computer controlled systems can save variable settings so that methods for different elements
can be easily saved and reloaded.

Shut down involves aspirating deionized water through all three inlet tubes (borohydride,
acid, and sample inlets) for a short period and then closing the fuel off first. Most modern
instruments control the ignition and shutdown procedures automatically. The plastic tubing
that is stretched around the peristaltic pump head is loosened to length its lifetime. Finally the
purge gas is turned off.
 

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