Dr.
Wafa Farooq
1
Objectives:
The student will know information about the following:
1. Gas Chromatography.
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Gas Chromatography (GC)
It is well-known that many pharmacologically active components in herbal
medicines and dosage forms are volatile chemical and thermally stable
compounds. Thus, the analysis of volatile compounds by gas
chromatography is very important in the analysis of herbal medicines and
dosage forms.
✓ The advantages of GC clearly lie in its high sensitivity of detection for
almost all the volatile chemical compounds.
✓ The most serious disadvantage of GC is that it is not convenient for its
analysis of the samples of non-volatile compounds. For this, it is
necessary to use tedious sample work-up which may include
derivatization.
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Advantages of GC analysis of the volatile oils
▪ The GC of the volatile oil gives a reasonable “fingerprint” which can
be used to identify the plant. The composition and relative concentration
of the organic compounds in the volatile oil are characteristic of the
particular plant and the presence of impurities in the volatile oil can be
readily detected.
▪ The extraction of the volatile oil is relatively straightforward and can be
standardized and the components can be readily identified using GC–
MS analysis. The relative quantities of the components can be used to
monitor or assess certain characteristics of the herbal medicines.
Changes in composition of the volatile oil may also be used as
indicators of oxidation, enzymatic changes or microbial fermentation.
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Gas Chromatography
• In gas chromatography (GC), the sample is vaporized and injected onto the
head of a chromatographic column. Elution is brought about by the flow of
an inert gaseous mobile phase.
• Gas-liquid chromatography is based upon the partition or adsoption of the
analyte between a gaseous mobile phase and a liquid phase immobilized on
the surface of an inert solid.
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Block diagram of a typical gas chromatograph.
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In gas-liquid chromatography, the mobile phase is a gas, and the
stationary phase is a liquid at operating temperature that is retained on the
surface of an inert solid ,by partition or chemical bonding.
In gas-solid chromatography, the mobile phase is a gas, and the stationary
phase is a solid that retains the analytes by physical adsorption. Gas-solid
chromatography permits the separation and determination of low-
molecular-mass gases, such as air components, hydrogen sulfide, carbon
monoxide, and nitrogen oxides. Gas-solid chromatography has limited
application because of semi-permanent retention of active or polar
molecules and severe tailing of elution peaks.
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Instruments for GC
1.Carrier Gas-Supply
Carrier gases, which must be chemically inert, include helium, nitrogen, and
hydrogen. Associated with the gas supply are pressure regulators, gauges, and
flow meters. In addition, the carrier gas system often contains a molecular
sieve to remove water or other impurities.
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Properties of the carrier gas:
1- High purity (99.9%)
2- Inert so there is no interaction with the stationary phase or instrumental
component at high temp.
3- High density (larger viscosity) carrier gas is preferred.
4- Compatible with the detector because some detector require special
carrier gas.
5- Cheap and available carrier gas is an advantage.
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2. Sample Injection System
• Column efficiency requires that the sample be of suitable size and be
introduced as a “plug” of vapor; slow injection of oversized samples causes
band spreading and poor resolution.
• The most common method of sample injection involves the use of micro
syringe to inject a liquid or gaseous sample through a self-sealing, silicone-
rubber diaphragm or septum into a flash vaporizer port located at the head of
the column (the sample port is ordinarily about 50oC above the boiling
point of the least volatile component of the sample).
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Sample preparation
1. The prerequisite in GC separation is that all solutes being separated
must be:
(a) Fairly volatile, and
(b) Thermally stable.
2. Lack of volatility prevents the direct use of GC for many solute. One way
to overcome this difficulty is to derivatize the solutes into more volatile
forms.
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3. Derivatization of a solute can be used for any of the following reasons
(a) To increase the volatility of the solute.
(b) To increase the thermal stability of solute.
(c) To improve the response for the solute on certain detectors.
(d) To improve the separation of the solute from other sample components
(i.e., changing the structure of a solute will also affect its retention on the
column)
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Asst.Prof. Wafa Farooq
3. Column Configurations
• Two general types of columns are encountered in gas chromatography,
packed and open tubular, or capillary.
• Chromatographic columns vary in length from less than 2 m to 60 m or
more. They are constructed of stainless steel, glass, fused silica, or Teflon. In
order to fit into an oven for thermostating, they are usually formed as coils
having diameters of 10 to 30 cm.
Packed column, Fused-silica capillary columns. 13
Packed column
These column is fabricated from glass, stainless steel, copper or other
suitable tubes. Stainless steel is the most common tubing used with
internal diameter from 1-4 mm. The column is packed with finely divided
particles( <100-300 μm diameter),which is coated with stationary phases.
However, glass tube are also used for large-scale separation. Packed
columns contain a finely divided, inert, solid support material (commonly
based on diatomaceous earth) coated with liquid stationary phase. Most
packed columns are 1.5 – 10 m in length.
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Capillary columns
Have an internal diameter of a few tenths of a millimeter. They can be one
of two types; wall-coated open tubular (WCOT) or support-coated open
tubular (SCOT).
✓ Wall-coated columns consist of a capillary tube whose walls are coated
with liquid stationary phase < 1 μm thick liquid coating on inside of
silica tube. In support-coated columns, the inner wall of the capillary is
lined with a thin layer of liquid (30 μm) of support material such as
diatomaceous earth, onto which the stationary phase has been adsorbed.
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SCOT columns are generally less efficient than WCOT columns. Both
types of capillary column are more efficient than packed columns.
A gas encounters less resistance in the GC column which permits use of
longer column lengths.
As the column length increases the interaction time between the eluting
compounds and stationary phase increases thereby increasing column
efficiency and resolution. This results in well separated peaks. However,
the column length cannot be increased indefinitely due to practical
problems faced due to increased column head pressure and also increase in
analysis time. Dr.Wafa Farooq 17
Properties and Characteristics of Typical GC Columns. .
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Capillary columns advantages compared to packed columns
✓ Higher resolution.
✓ Shorter analysis times.
✓ Greater sensitivity.
Capillary columns disadvantage compared to packed columns
✓Smaller sample capacity.
✓Need better experience.
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4. The Stationary Phase
Desirable properties for the immobilized liquid phase in a gas-liquid
chromatographic column include:
(1) Low volatility (ideally, the boiling point of the liquid should be at
100oC higher than the maximum operating temperature for the column);
Non-volatile liquids assure minimum bleeding of the stationary phase.
(2) Thermal stability;
(3) Chemical inertness;
The retention time for a solute on a column depends upon its distribution
constant which in turn is related to the chemical nature of the stationary
phase.
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Dr.Wafa Farooq
Liquid Stationary phase:
• Should match the sample constituents(like dissolve like).
Most of them are based on polydimethylsiloxane or polyethylene
glycol(PEG) backbones.
Polydimethylsiloxane polarity is low (non-polar), can be opereted at high
temp ,while polyethylene glycol is high polarity, cannot be opereted at high
temp because it can break down.
Polydimethylsiloxane polyethylene glycol
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Some Common Liquid Stationary Phases For GLC.
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Solid Support Materials
The solid support should ideally have the following properties:
1- Large surface area.
2- Has good mechanical stability and thermal stability.
3- Inert surface in order to simplify retention behavior and prevent solute
adsorption.
4- Has a particle size in the range from 100-400 μm
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5. Column Ovens
• Column temperature is an important variable that must be controlled to a
few tenths of a degree for precise work. Thus, the column is ordinarily
housed in a thermostated oven. The optimum column temperature
depends upon the boiling point of the sample and the degree of
separation required.
• Roughly, a temperature equal to or slightly above the average boiling point
of a sample results in a reasonable elution time (2 to 30 min). For samples
with a broad boiling range, it is often desirable to employ temperature
programming, whereby the column temperature is increased either
continuously or in steps as the separation proceeds.
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Isothermal at 45°
Isothermal at 145°
Programmed 30 to 180°
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Dr.Wafa Farooq
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6. Detection Systems
Characteristics of the Ideal Detector: The ideal detector for gas
chromatography has the following characteristics:
1. Adequate sensitivity.
2. Good stability and reproducibility.
3. A linear response to solutes that extends over several orders of
magnitude.
4. A temperature range from room temperature to at least 400oC.
5. High reliability and ease of use.
6. Similarity in response toward all solutes or a highly selective response
toward one or more classes of solutes.
7. Nondestructive of sample.
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Typical Gas Chromatographic Detectors.
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Comparison of HPLC and GC.
Characteristics of both methods
▪ Efficient, highly selective, widely applicable.
▪ Only small sample required.
▪ May be non-destructive of sample.
▪ Readily adapted to quantitative analysis.
Advantages of HPLC
▪ Can accommodate nonvolatile and thermally unstable compound.
▪ Generally applicable to inorganic ions.
Advantages of GC
▪ Simple and inexpensive equipment.
▪ Rapid.
▪ Unparalleled resolution with the capillary column.
▪ Easy to interfere with mass spectrometery Dr.Wafa Farooq 29
Applications of Gas-liquid Chromatography
Qualitative Analysis
▪ Gas chromatograms are widely used as criteria of purity for organic
compounds. Contaminants, if present, are revealed by the appearance of
additional peaks; the areas under these peaks provide rough estimates of the
extent of contamination.
▪ The technique is also useful for evaluating the effectiveness of purification
procedures.
▪ Retention times should be useful for the identification of components in
mixtures. Gas chromatography provides an excellent means of confirming the
presence or absence of a suspected compound in a mixture.
Quantitative Analysis: The detector signal from a gas-liquid chromatographic
column has had wide use for quantitative analyses.
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Flash Chromatography
▪ Also called as “medium pressure chromatography” It is also known as
flash purification, due to its function as a purification method.
Differs from the conventional technique in 2 ways:
✓ Slightly smaller silica gel particles (250-400 mesh) are used, and
✓ Due to the restricted flow of solvent caused by the small gel particles,
pressurized gas (10-15 psi) is used to drive the solvent through the column
of stationary phase.
✓ The net result is rapid “over in a flash” and high-resolution
chromatography.
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Flash Chromatography
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Dr.Wafa Farooq
▪ Common solvents used dichloromethane/hexane, ether/hexane,
hexane/ethyl acetate, and dichloromethane/methanol and others.
▪ 40-63 μm silica gel (SiO2) and alumina (Al2O3) particles are used.
▪ In the modern Flash Chromatography system the glass columns are
replaced with pre-packed plastic cartridges.
▪ Faster flow rates can be achieved by using a pump or by using
compressed gas (e.g. air, nitrogen, or argon) to push the solvent
through the column.
▪ Systems may also be linked with detectors and fraction collectors. 33
Application of Flash chromatography in separation of
phytochemicals.
▪ Flash chromatography is a very valuable technique in the field of
natural compounds research because it provides a fast and economical
way to separate important phytochemical constituents of complex plant
extracts.
▪ Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves
Extract.
▪ Isolation and Purification of Ginsenosides from Red Panax Ginseng
Extract.
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Flash Chromatography of Plant Pigments
Experimental Conditions:
cartridge: 75 x 40 mm I.D. silica
sample: 0.5mL, 10 mg/mL of each
compound in dichloromethane, detection:
by the compound color.
Samples from left to right
Chlorophylline (green)
Astaxanthene (red)
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Carotene (Orange)
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Objectives:
The student will know information about the following:
1. Thin Layer Chromatography.
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Thin Layer Chromatography (TLC)
▪ It is one of the most popular and simple chromatographic technique
used of separation of compounds.
▪ TLC was the common method of choice for herbal analysis before
instrumental chromatography methods like GC and HPLC were
established.
▪ In the phytochemical evaluation of herbal drugs, TLC is being
employed extensively for the following reasons:
1. It enables rapid analysis of herbal extracts with minimum sample clean-
up requirement,
2. It provides qualitative and semi quantitative information of the resolved
compounds.
3. It enables the quantification of chemical constituents.(specially HPTLC)
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Thin Layer Chromatography (TLC)
▪ Used to separate organic compounds and to test the purity of compounds.
▪ Thin layer chromatography ( can be considered a form of liquid-solid
chromatography in which the stationary phase is a thin layer on the surface
of an appropriate plate.
▪ The mobile phase moves through the stationary phase by capillary action,
sometimes assisted by gravity or an electrical potential.
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Types of Thin Layer Chromatography
▪ Adsorption Thin Layer Chromatography.
▪ Ion Exchange Thin Layer Chromatography.
▪ Partition Thin Layer Chromatography.
▪ Reversed Phased Partition Thin Layer Chromatography.
▪ High Performance Thin Layer Chromatography.
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Principle of TLC
It is based on the principle of adsorption chromatography or
partition chromatography or combination of both, depending on
adsorbent, its treatment and nature of solvents employed.
✓ The components with more affinity towards stationary phase
travels slower.
✓ Components with less affinity towards stationary phase travels
faster.
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Preparation of Thin-Layer Plates
TLC plates are usually commercially available, with standard particle size
ranges to improve reproducibility. They are prepared by mixing the
adsorbent, such as silica gel, with a small amount of inert binder
like calcium sulfate (gypsum) and water. This mixture is spread as a thick
slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or
plastic. The resultant plate is dried and activated by heating in an oven for
thirty minutes at 110 °C. The thickness of the absorbent layer is typically
around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for
preparative TLC.
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➢ The common plate dimensions in centimeters a 5 X 20, 10 X 20, and
20 X 20.
➢ Commercial plates can be conventional and high-performance plates.
➢ Conventional Plates have thicker layers (200 to 250 μm) of
particles with particles sizes of 20 μm or greater.
➢ High-performance plates usually have film thicknesses of 100 μm
and particle diameters of 5 μm or less.
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Sample Application
➢ The sample is applied as a spot 1 to 2 cm from the edge of the plate.
Manual application of samples is performed by touching a capillary tube or
micro-syring containing the sample to the plate or by use of a syringe.
Mechanical dispensers, which increase the precision and accuracy of
sample application, are available commercially.
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capillary tube and microsyring 45
Plate Development
▪ Is the process by which a sample is carried through the stationary
phase by a mobile phase.
▪ After applying a spot and evaporating the solvent, the plate is placed
in a closed container saturated with vapors of the developing solvent.
One end of the plate is immersed in the developing solvent, with care
being taken to avoid direct contact between the sample and the
developer .
▪ After the developer has traversed one half or two thirds of the length
of the plate, the plate is removed from the container and dried. The
positions of the components are then determined in any of several
ways.
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Plate Development
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Mobile Phase
The choice of mobile phase is largely empirical but general rules can be
formulated. A mixture of an organic solvent and water with the addition of
acid, base or complexing agent to optimize the solubility of the components
of a mixture can be used. For example, good separations of polar or ionic
solutes can be achieved with a mixture of water and n-butanol. Addition of
acetic acid to the mixture allows more water to be incorporated and increases
the solubility of basic materials, whilst the addition of ammonia increases
the solubility of acidic materials. If the stationary phase is hydrophilic,
various mixtures of benzene, cyclohexane and chloroform provide
satisfactory mobile phases. It should be emphasized that a large degree of
trial and error is involved in their selection. For TLC on silica gel, a mobile
phase with as low a polarity as possible should be used consistent with
achieving a satisfactory separation. Dr.Wafa Farooq 48
The choice of the mobile phase is depends upon the following
factors:-
1. Nature of the substance to be separated.
2. Nature of the stationary phase used.
3. Mode of chromatography ( Normal phase or reverse phase).
4. Separation to be achieved- Analytical or preparative.
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Development Technique
Different development techniques are used for efficient separations. They are
1. One dimensional development (Ascending development).
2. Descending Development.
3. Horizontal development.
4. Two dimensional development.
5. Multiple development.
6. Gradient development
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1. One dimensional development (Ascending development)
• Like conventional type, the solvent flows against gravity. The spots are
kept at the bottom portion of paper and kept in a chamber with mobile
phase solvent at the bottom.
Advantages:
•Simple and quick
•Requires minimal equipment
•Good for basic separations
•Ideal for routine analysis or checking purity
Disadvantages:
•Limited resolution for complex mixtures
•Poor separation of compounds with similar Rf
values
•Can result in overlapping spots 51
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Dr.Wafa Farooq
2. Descending :
Flow of the solvent from reservoir to the plate is by means of a filter
paper strip. Solvent moves from top to bottom of the plate.
Advantages:
•Can separate compounds that don’t resolve
well with ascending
•Longer development distance possible →
better resolution
Disadvantages:
•Needs special chamber
•Setup is more complex
•Takes more time
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3. Horizontal Development
In this TLC developing method, the plate is positioned horizontally inside
the chamber, and the solvent is applied using a wick or capillary slit.
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4.Two dimensional technique
• The paper is developed in one
direction and after development, the
paper is developed in the second
direction(90ο) allowing more
compounds or complex mixtures to be
separated into individual spots. In the
second direction, either the same
solvent or different solvent system can
be used for development.
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Two dimensional technique
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Advantages:
•Excellent for complex mixtures
•Separates compounds with very similar Rf values
•Spots spread out in 2D → better distinction
Disadvantages:
•Requires precise technique and planning
•Difficult to interpret sometimes
•Time- and solvent-intensive
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5. Multiple Development
In this method, the TLC plate undergoes multiple developments with
drying between each cycle. The solvent travels repeatedly through the
layer, re-concentrating and deforming the spots, often producing
elliptical shapes or narrow bands. This significantly improves resolution
for substances with Rf values below 0.5. Multiple development can be
performed using the same solvent or different solvents of varying
polarity.
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6. Gradient Development
Description: Solvent polarity is gradually increased during development
(manually or stepwise).
Advantages:
•Fine-tunes separation for compounds with wide polarity range
•Can compress Rf values for high-polarity compounds or spread out low
ones.
•Increases the versatility of the plate
Disadvantages:
•Technically challenging to perform
•Requires careful control of solvent changes
•Not commonly used in basic labs
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Technique Advantages Disadvantages
Poor resolution for
Single Development Simple, fast, easy setup
complex mixtures
Better resolution, Time-consuming,
Double Development
versatile possible spot tailing
Excellent for complex Technically demanding,
2D-TLC
samples harder to interpret
Complex solvent control,
Gradient Fine-tuned separation
less common
Higher resolution Needs special chamber,
Descending
possible slower
Limited to simple
Radial TLC Quick, low solvent use
separations 59
Locating Analytes on the Plate
➢ Two common methods that can be applied to most organic mixtures
involve spraying with a solution of iodine or sulfuric acid. Both of these
reagents react with organic compounds to yield dark products. Several
specific reagents (such as ninhydrin) are also useful for locating
separated species.
➢ Another method of detection is based on incorporating a fluorescent
material into the stationary phase. After development, the plate is
examined under ultraviolet light. The sample components quench the
fluorescence of the material so that all of the plate fluoresces except
where the nonfluorescing sample components are located.
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Spraying with a Specific Solution
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A method of visualization is accomplished by placing the plate into iodine
vapors for a few minutes.
Most organic compounds will form a dark-colored complex with iodine.
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Locating Analytes on the Plate
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Rf values
• Rx value
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Analysing a Reaction
A + B -> Products
prepare plate
'spot' samples of both remove an aliquot from the reaction
starting materials as mixture and 'spot' on the fourth mark
shown and also on the co-spot
X X X X X X X X X X X X
A only A only reaction mixture
B only B only
co-spot co-spot
(A + B) (A + B + reaction mixture)
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Quantitative analysis
• Direct technique: Densitometer is an instrument which measures
quantitatively the density of the spots. When the optical density of the
spots for the standard and test solution are determined, the quantity of the
substance can be calculated.
• Indirect technique: In this technique, the spots are cut into portions
and eluted with solvents. This solution can be analyzed by any
conventional techniques of analysis like spectrophotometry,
electrochemical methods etc.,
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High Performance Thin Layer Chromatography (HPTLC)
▪ HPTLC technique is widely employed in pharmaceutical industry in
process development, identification and detection of adulterants in
herbal product and helps in identification of pesticide content,
mycotoxins and in quality control of herbs and health Food.
▪ It has been well reported that several samples can be run
simultaneously by use of a smaller quantity of mobile phase than in
HPLC.
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Advantages of HPTLC Over Other Chromatographic Methods:
1. In HPTLC, simultaneous processing of sample and standard – better
analytical accuracy & precision.
2. Lower analysis time & less cost per analysis.
3. HPTLC is very simple.
4. In HPTLC, the sample preparation is simple.
5. Solvent used in HPTLC needs no prior treatment like filtration &
degassing.
6. In HPTLC, the Mobile phase consumption for sample is extremely low.
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7. It promotes high separation efficiencies/ resolution of zones due to
higher number of theoretical plates.
8. Parallel separation of many samples with minimal time requirement.
9. Efficient data acquisition and processing.
10. No interference from previous analysis – fresh stationary for each
analysis -no contamination.
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In summary, the advantages of using TLC to construct the fingerprints of
herbal medicines and other compounds are its simplicity, versatility, high
velocity, specific sensitivity and simple sample preparation.
Thus, TLC is a convenient method of determining the quality and possible
adulteration of herbal products.
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Dr.Wafa Farooq
TLC Troubleshooting
A B C
D E F
TLC Troubleshooting
Appearance Cause Fix
A: Curved solvent front with Plate touching side of container Place plate in middle of
spots out of lane container
Plate not lowered level into eluent Ensure plate level when
lowering into eluent
B: Streaks, not spots Too much sample Dilute sample solution
C: Many blue spots/stripes Origin marked in pen? Only use pencil on TLC plates
D: No spots on plate Sample too dilute Remake sample/spot several
Wrong visualisation used times, Use alternative
visualisation
E: Crescent-shaped ‘spot’ Silica disturbed during spotting Be gentle when spotting
sample
F: Spot(s) smeared out Acidic/Basic groups present in Use an additive in the eluent
compound?
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References:
1- Skoog, D.A., West, D.M., Holler, F.J., Crouch, S.R., Fundamentals of
Analytical Chemistry.
2- Sanjeet Kumar, K. Jyotirmayee, Monalisa Sarangi. Thin Layer
Chromatography: A Tool of Biotechnology for Isolation of Bioactive
Compounds from Medicinal Plants. Int. J. Pharm. Sci. Rev. Res., 18(1), Jan
– Feb 2013; nᵒ 18, 126-132.
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