RAJALAKSHMI ENGINEERING COLLEGE, THANDALAM

DEPARTMENT OF BIOTECHNOLOGY

FINALYEAR-VII SEMESTER SEC A&B

BT-2401 DOWNSTREAM PROCESSING

Bachelor of Biotechnology
DEPARTMENT OF BIOTECHNOLOGY

PREPARED BY

KAVITHA VIJAYARAGHAVAN(BT102)

LECTURER

DEPARTMENT OF BIOTECHNOLOGY
 UNIT-1 DOWNSTREAM PROCESSING

Downstream processing refers to the recovery and purification of biosynthetic products,

particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation
broth, including the recycling of salvageable components and the proper treatment and disposal
of waste. It is an essential step in the manufacture of pharmaceuticals such as antibiotics,
hormones (e.g. insulin and human growth hormone), antibodies (e.g. infliximab and abciximab)
and vaccines; antibodies and enzymes used in diagnostics; industrial enzymes; and natural
fragrance and flavor compounds. Downstream processing is usually considered a specialized
field in biochemical engineering, itself a specialization within chemical engineering, though
many of the key technologies were developed by chemists and biologists for laboratory-scale
separation of biological products.

Downstream processing and analytical bioseparation both refer to the separation or purification
of biological products, but at different scales of operation and for different purposes.
Downstream processing implies manufacture of a purified product fit for a specific use, generally
in marketable quantities, while analytical bioseparation refers to purification for the sole purpose
of measuring a component or components of a mixture, and may deal with sample sizes as small
as a single cell.

Stages in Downstream Processing

A widely recognized heuristic for categorizing downstream processing operations divides them
into four groups which are applied in order to bring a product from its natural state as a
component of a tissue, cell or fermentation broth through progressive improvements in purity
and concentration.

Removal of insolubles is the first step and involves the capture of the product as a solute in a
particulate-free liquid, for example the separation of cells, cell debris or other particulate matter
from fermentation broth containing an antibiotic. Typical operations to achieve this are filtration,
centrifugation, sedimentation, flocculation, electro-precipitation, and gravity settling. Additional
operations such as grinding, homogenization, or leaching, required to recover products from
solid sources such as plant and animal tissues, are usually included in this group.

Product Isolation is the removal of those components whose properties vary markedly from that
of the desired product. For most products, water is the chief impurity and isolation steps are
designed to remove most of it, reducing the volume of material to be handled and concentrating
the product. Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit
operations involved.
Product Purification is done to separate those contaminants that resemble the product very
closely in physical and chemical properties. Consequently steps in this stage are expensive to
carry out and require sensitive and sophisticated equipment. This stage contributes a significant
fraction of the entire downstream processing expenditure. Examples of operations include
affinity, size exclusion, reversed phase chromatography, crystallization and fractional
precipitation.

Product Polishing describes the final processing steps which end with packaging of the product
in a form that is stable, easily transportable and convenient. Crystallization, desiccation,
lyophilization and spray drying are typical unit operations. Depending on the product and its
intended use, polishing may also include operations to sterilize the product and remove or
deactivate trace contaminants which might compromise product safety. Such operations might
include the removal of viruses or depyrogenation.

A few product recovery methods may be considered to combine two or more stages. For
example, expanded bed adsorption accomplishes removal of insolubles and product isolation in a
single step. Affinity chromatography often isolates and purifies in a single step.

Cell disruption is a method or process for releasing biological molecules from inside a cell.

Choice of disruption method

The production of biologically-interesting molecules using cloning and culturing methods allows
the study and manufacture of relevant molecules.Except for excreted molecules, cells producing
molecules of interest must be disrupted. This page discusses various methods.

Major factors
Several factors must be considered.

Volume or sample size of cells to be disrupted

If only a few microliters of sample are available, care must be taken to minimize loss and to
avoid cross-contamination.

Disruption of cells, when hundreds or even thousands of liters of material are being processed in
a production environment, presents a different challenge. Throughput, efficiency, and
reproducibility are key factors.

How many different samples need to be disrupted at one time?

Frequently when sample sizes are small, there are many samples. As sample sizes increase, fewer
samples are usually processed. Issues are sample cross contamination, speed of processing, and
equipment cleaning .
How easily are the cells disrupted?

As the difficulty of disruption increases (e.g. E. coli), more force is required to efficiently disrupt
the cells. For even more difficult samples (e.g. yeast), there is a parallel increase in the processor
power and cost. The most difficult samples (e.g. spores) require mechanical forces combined
with chemical or enzymatic efforts, often with limited disruption efficiency.

What efficiency of disruption is required?

Over-disruption may impact the desired product. For example, if subcellular fractionation studies
are undertaken, it is often more important to have intact subcellular components, while
sacrificing disruption efficiency.

For production scale processes, the time to disrupt the cells and the reproducibility of the method
become more important factors.

How stable is the molecule(s) or component that needs to be isolated?

In general, the cell disruption method is closely matched with the material that is desired from
the cell studies. It is usually necessary to establish the minimum force of the disruption method
that will yield the best product. Additionally, once the cells are disrupted, it is often essential to
protect the desired product from normal biological processes (e.g. proteases) and from oxidation
or other chemical events.

What purification methods will be used following cell disruption?

It is rare that a cell disruption process produces a directly usable material; in almost all cases,
subsequent purification events are necessary. Thus, when the cells are disrupted, it is important to
consider what components are present in the disruption media so that efficient purification is not
impeded.

Is the sample being subjected to the method biohazardous?

Preparation of cell-free extracts of pathogens presents unique difficulties. Mechanical disruption

techniques are not always applicable owing to potential biohazard problems associated with
contamination of equipment and generation of aerosols.

Lysis
For easily disrupted cells such as insect and mammalian cells grown in culture media, a mild
osmosis-based method for cell disruption (lysis) is commonly used. Quite frequently, simply
lowering the ionic strength of the media will cause the cells to swell and burst. In some cases it is
also desirable to add a mild surfactant and some mild mechanical agitation or sonication to
completely disassociate the cellular components. Due to the cost and relative effort to grow these
cells, there is often only a small quantity of cells to be processed, and preferred methods for cell
disruption tend to be a manual mechanical homogenizer, nitrogen burst methods, or ultrasound
with a small probe. Because these methods are performed under very mild conditions, they are
often used for subcellular fractionation studies.

For cells that are more difficult to disrupt, such as bacteria, yeast, and algae, hypotonic shock
alone generally is insufficient to open the cell and stronger methods must be used, due to the
presence of cell walls that must be broken to allow access to intracellular components. These
stronger methods are discussed below.

Laboratory-scale methods
Enzymatic method

The use of enzymatic methods to remove cell walls is well-established for preparing cells for
disruption, or for preparation of protoplasts (cells without cell walls) for other uses such as
introducing cloned DNA or subcellular organelle isolation. The enzymes are generally
commercially available and, in most cases, were originally isolated from biological sources (e.g.
snail gut for yeast or lysozyme from hen egg white). The enzymes commonly used include
lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase etc.

Disadvantages include:

 Not always reproducible.

In addition to potential problems with the enzyme stability, the susceptibility of the cells to the
enzyme can be dependent on the state of the cells. For example, yeast cells grown to maximum
density (stationary phase) possess cell walls that are notoriously difficult to remove whereas
midlog growth phase cells are much more susceptible to enzymatic removal of the cell wall.

 Not usually applicable to large scale.

Large scale applications of enzymatic methods tend to be costly and irreproducible.

The enzyme must be removed (or inactivated) to allow cell growth or permit isolation of the
desired material.

Bead method

Another common laboratory-scale mechanical method for cell disruption uses small glass,
ceramic, or steel beads and a high level of agitation by stirring or shaking of the mix. The
method, often referred to as "beadbeating", works well for all types of cellular material - from
spores to animal and plant tissues.

At the lowest levels of the technology, beads are added to the cell or tissue suspension in a
testtube and the sample is mixed on a common laboratory vortex mixer. While processing time is
3-10 times longer than that in specially machines (see below), it works for easily disrupted cells
and is inexpensive.

At the more sophisticated level, beadbeating is done in closed vials. The sample and the beads
are vigorously agitated at about 2000 oscillation per minute in a specially designed shaker driven
by a high energy electric motor. In some machines hundreds of samples can be processed
simultaneously. When samples larger that 2 ml are processed, some form of cooling is required
because samples heat due to collisions of the beads. Another configuration suitable for larger
sample volumes uses a rotor inside a sealed 15, 50 or 200 ml chamber to agitate the beads. The
chamber can be surrounded by a cooling jacket. Using this same configuation, commercial
machines capable of processing many liters of cell suspension are available.

Disadvantages include:

 Occasional problems with foaming and sample heating, especially for larger samples.
 Tough tissue samples such as skin or seeds are difficult to disrupt unless the sample is
very small or has been pre-chopped into small pieces.

Sonication

Main article: Sonication

Another common laboratory-scale method for cell disruption applies ultrasound (typically 20-50
kHz) to the sample (sonication). In principle, the high-frequency is generated electronically and
the mechanical energy is transmitted to the sample via a metal probe that oscillates with high
frequency. The probe is placed into the cell-containing sample and the high-frequency oscillation
causes a localized low pressure region resulting in cavitation and impaction, ultimately breaking
open the cells. Although the basic technology was developed over 50 years ago, newer systems
permit cell disruption in smaller samples (including multiple samples under 200 µL in microplate
wells) and with an increased ability to control ultrasonication parameters.

Disadvantages include:

 Heat generated by the ultrasound process must be dissipated.

 High noise levels (most systems require hearing protection and sonic enclosures)
 Yield variability
 Free radicals are generated that can react with other molecules.

Detergent methods

Detergent-based cell lysis is an alternative to physical disruption of cell membranes, although it

is sometimes used in conjunction with homogenization and mechanical grinding. Detergents
disrupt the lipid barrier surrounding cells by disrupting lipid:lipid, lipid:protein and
protein:protein interactions. The ideal detergent for cell lysis depends on cell type and source and
on the downstream applications following cell lysis. Animal cells, bacteria and yeast all have
differing requirements for optimal lysis due to the presence or absence of a cell wall. Because of
the dense and complex nature of animal tissues, they require both detergent and mechanical lysis
to effectively lyse cells.

In general, nonionic and zwitterionic detergents are milder, resulting in less protein denaturation
upon cell lysis, than ionic detergents and are used to disrupt cells when it is critical to maintain
protein function or interactions. CHAPS, a zwitterionic detergent, and the Triton X series of
nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are
strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and
function. SDS, an ionic detergent that binds to and denatures proteins, is used extensively for
studies assessing protein levels by gel electrophoresis and western blotting.

In addition to the choice of detergent, other important considerations for optimal cell lysis
include the buffer, pH, ionic strength and temperature.

Solvent Use

A method was developed for the extraction of proteins from both pathogenic and nonpathogenic
bacteria. The method involves the treatment of cells with sodium dodecyl sulfate followed by
extraction of cellular proteins with acetone. This method is simple, rapid and particularly well
suited when the material is biohazardous.[1]

Simple and rapid method for disruption of bacteria for protein studies. S Bhaduri and P H
Demchick Disadvantages include:

 Proteins are denatured

The 'cell bomb'

Another laboratory-scale system for cell disruption is rapid decompression or the "cell bomb"
method. In this process, cells in question are placed under high pressure (usually nitrogen or
other inert gas up to about 25,000 psi) and the pressure is rapidly released. The rapid pressure
drop causes the dissolved gas to be released as bubbles that ultimately lyse the cell.

Disadvantages include:

 Only easily disrupted cells can be effectively disrupted (stationary phase E. coli, yeast,
fungi, and spores do not disrupt well by this method).
 Large scale processing is not practical.
 High gas pressures have a small risk of personal hazard if not handled carefully.

High-shear mechanical methods.

High-shear mechanical methods for cell disruption fall into three major classes: rotor-stator
disruptors, valve-type processors, and fixed-geometry processors. (These fluid processing
systems also are used extensively for homogenization and deaggregation of a wide range of
materials – uses that will not be discussed here.) These processors all work by placing the bulk
aqueous media under shear forces that literally pull the cells apart. These systems are especially
useful for larger scale laboratory experiments (over 20 mL) and offer the option for large-scale
production.

Rotor-stator Processors

Most commonly used as tissue disruptors.

Disadvantages include:

 Do not work well with difficult-to-lyse cells like yeast and fungi
 Often variable in product yield.
 Poorly suited for culture use.

Valve-type processors

Valve-type processors disrupt cells by forcing the media with the cells through a narrow valve
under high pressure (20,000–30,000 psi or 140–210 MPa). As the fluid flows past the valve, high
shear forces in the fluid pull the cells apart. By controlling the pressure and valve tension, the
shear force can be regulated to optimize cell disruption. Due to the high energies involved,
sample cooling is generally required, especially for samples requiring multiple passes through
the system. Two major implementations of the technology exist: the French pressure cell press
and pumped-fluid processors.

French press technology uses an external hydraulic pump to drive a piston within a larger
cylinder that contains the sample. The pressurized solution is then squeezed past a needle valve.
Once past the valve, the pressure drops to atmospheric pressure and generates shear forces that
disrupt the cells. Disadvantages include:

 Not well suited to larger volume processing.

 Awkward to manipulate and clean due to the weight of the assembly (about 30 lb or 14
kg).

Mechanically pumped-fluid processors function by forcing the sample at a constant volume flow
past a spring-loaded valve.

Disadvantages include:

 Requires 10 mL or more of media.

 Prone to valve-clogging events.
 Due to variations in the valve setting and seating, less reproducible than fixed-geometry
fluid processors.

Fixed-geometry fluid processors

Fixed-geometry fluid processors are marketed under the name of Microfluidizer processors. The
processors disrupt cells by forcing the media with the cells at high pressure (typically 20,000–
30,000 psi or 140–210 MPa) through an interaction chamber containing a narrow channel. The
ultra-high shear rates allow for:

 Processing of more difficult samples

 Fewer repeat passes to ensure optimum sample processing

The systems permit controlled cell breakage without the need to add detergent or to alter the
ionic strength of the media. The fixed geometry of the interaction chamber ensures
reproducibility. Especially when samples are processed multiple times, the processors require
sample cooling.

UNIT II-PHYSICAL METHODS OF SEPERATION

Unit operations

In chemical engineering and related fields, a unit operation is a basic step in a process. For
example in milk processing, homogenization, pasteurization, chilling, and packaging are each
unit operations which are connected to create the overall process. A process may have many unit
operations to obtain the desired product.
Historically, the different chemical industries were regarded as different industrial processes and
with different principles. In 1923 William H. Walker, Warren K. Lewis and William H.
McAdams wrote the book The Principles of Chemical Engineering and explained the variety of
chemical industries have processes which follow the same physical laws. They summed-up these
similar processes into unit operations. Each unit operation follows the same physical laws and
may be used in all chemical industries. The unit operations form the fundamental principles of
chemical engineering.

Chemical engineering unit operations consist of five classes:

1. Fluid flow processes, including fluids transportation, filtration, solids fluidization

2. Heat transfer processes, including evaporation, condensation
3. Mass transfer processes, including gas absorption, distillation, extraction, adsorption,
drying
4. Thermodynamic processes, including gas liquefaction, refrigeration
5. Mechanical processes, including solids transportation, crushing and pulverization,
screening and sieving

Chemical engineering unit operations also fall in the following categories:

 Combination (mixing)
 Separation (distillation)
 Reaction (chemical reaction)

Chemical engineering unit operations and chemical engineering unit processing form the main
principles of all kinds of chemical industries and are the foundation of designs of chemical
plants, factories, and equipment useIn chemistry and chemical engineering, a separation process
is used to transform a mixture of substances into two or more distinct products. The separated
products could differ in chemical properties or some physical property, such as size, or crystal
modification or other.

Barring a few exceptions, almost every element or compound is found naturally in an impure
state such as a mixture of two or more substances. Many times the need to separate it into its
individual components arises. Separation applications in the field of chemical engineering are
very important. A good example is that of crude oil. Crude oil is a mixture of various
hydrocarbons and is valuable in this natural form. Demand is greater, however, for the purified
various hydrocarbons such as natural gases, gasoline, diesel, jet fuel, lubricating oils, asphalt,etc.

Seperation process

Separation processes can essentially be termed as mass transfer processes. The classification can
be based on the means of separation, mechanical or chemical. The choice of separation depends
on the pros and cons of each. Mechanical separations are usually favored if possible due to the
lower cost of the operations as compared to chemical separations. Systems that can not be
separated by purely mechanical means (e.g. crude oil), chemical separation is the remaining
solution. The mixture at hand could exist as a combination of any two or more states: solid-solid,
solid-liquid, solid-gas, liquid-liquid, liquid-gas, gas-gas, solid-liquid-gas mixture, etc.

Depending on the raw mixture, various processes can be employed to separate the mixtures.
Many times two or more of these processes have to be used in combination to obtain the desired
separation. In addition to chemical processes, mechanical processes can also be applied where
possible. In the example of crude oil, one upstream distillation operation will feed its two or
more product streams into multiple downstream distillation operations to further separate the
crude, and so on until final products are purified.

Various types of separation processes

 Adsorption
 Centrifugation and Cyclones - density differences
 Chromatography involves the separation of different dissolved substances as they travel
through a material. The dissolved substances are separated based on their interaction with
the stationary phase.
 Crystallization
 Decantation
 Demister (Vapor) - removing liquid droplets from gas streams
 Dissolved air flotation - suspended solids are encouraged to float to the top of a fluid by
rising air bubbles.
 Distillation - used for mixtures of liquids with different boiling points, or for a solid
dissolved in a liquid.
 Drying - removing liquid from a solid by vaporising it
 Electrophoresis Organic molecules, such as protein are placed in a gel. A voltage is
applied and the molecules move through the gel because they are charged. The gel
restricts the motion so that different proteins will make different amounts of progress in
any given time.
 Elutriation
 Evaporation
 Extraction
o Leaching
o Liquid-liquid extraction
o Solid phase extraction
 Flocculation - density differences utilization a flocculant such as soap or detergent
 Fractional freezing
 Filtration. Mesh, bag and paper filters are used to remove large particulates suspended in
fluids, eg. fly ash, while membrane processes including microfiltration, ultrafiltration,
nanofiltration, reverse osmosis, dialysis (biochemistry) utilising synthetic membranes can
separate micrometre-sized or smaller species.
 Oil-water separation - gravimetric separator used to remove suspended oil droplets from
wastewaters in oil refineries, petrochemical and chemical plants, natural gas processing
plants and similar industries.
 Precipitation
 Recrystallization
  Sedimentation - density differences
o Gravity separation
 Sieving
 Stripping
 Sublimation
 Vapor-liquid separation - designed by using the Souders-Brown equation.
 Winnowing
 Zone refining


Filtration

Filtration is a mechanical or physical operation which is used for the separation of solids from
fluids (liquids or gases) by interposing a medium to fluid flow through which the fluid can pass,
but the solids (or at least part of the solids) in the fluid are retained. It has to be emphasized that
the separation is NOT complete, and it will depend on the pore size and the thickness of the
medium as well as the mechanisms that occur during filtration.

 Filtration is used for the purification of fluids: for instance separating dust from the
atmosphere to clean ambient air.

 Filtration, as a physical operation is very important in chemistry for the separation of

materials of different chemical composition in solution (or solids which can be dissolved)
by first using a reagent to precipitate one of the materials and then use a filter to separate
the solid from the other material(s).

 Filtration is also important and widely used as one of the unit operations of chemical
engineering.

It is important not to confuse filtration with sieving. In sieving there is only a single layer of
medium where size separation occurs purely by the fact that the fraction of the particulate solid
matter which is too large to be able to pass through the holes of the sieve, scientifically called
oversize (See particle size distribution) are retained. In filtration a multilayer medium is
involved, where other mechanisms are included as well, for instance direct interception, diffusion
and centrifugal action, where in this latter those particles, which are unable to follow the tortuous
channels of the filter will also adhere to the structure of the medium and are retained.[1]

Depending on the application, either one or both of the components may be isolated. Examples
of filtration include A) a coffee filter to keep the coffee separate from the grounds and B) the use
of HEPA filters in air conditioning to remove particles from air.

The filtration process separates particles and fluid from a suspension, and the fluid can be either
a liquid or a gas (or a supercritical fluid). To separate a mixture of chemical compounds, a
solvent is chosen which dissolves one component, while not dissolving the other. By dissolving
the mixture in the chosen solvent, one component will go into the solution and pass through the
filter, while the other will be retained. This is one of the most important techniques used by
chemists to purify compounds.

Filtration also cleans up air streams or other gas streams. Furnaces use filtration to prevent the
furnace elements from fouling with particulates. Pneumatic conveying systems often employ
filtration to stop or slow the flow of material that is transported, through the use of a baghouse.

The remainder of this article focuses primarily on liquid filtration.

Contents
[
 1 Methods
 2 Flowing
 3 Filter media
 4 Filter aid
 5 Alternatives
 6 Filter types
 7 In kidney
 8 See also
 9 Footnotes
 10 References

 11 Further reading

Methods
There are many different methods of filtration; all aim to attain the separation of substances. This
is achieved by some form of interaction between the substance or objects to be removed and the
filter. In addition the substance that is to pass through the filter must be a fluid, i.e. a liquid or
gas.

The simplest method of filtration is to pass a solution of a solid and fluid through a porous
interface so that the solid is trapped, while the fluid passes through. This principle relies upon the
size difference between the particles making up the fluid, and the particles making up the solid.
In the laboratory, a Büchner funnel is often used, with a filter paper serving as the porous barrier.

For example an experiment to prove the existence of microscopic organisms involves the
comparison of water passed through unglazed porcelain and unfiltered water. When left in sealed
containers the filtered water takes longer to go foul, showing that very small items (such as
bacteria) can be removed from fluids by filtration.[citation needed] Alternate methods often take the
form of electrostatic attractions. These form of filters again have the problem of either becoming
clogged, or the active sites on the filter all become used by the undesirable. However, most
chemical filters are designed so that the filter can be flushed with a chemical that will remove the
undesirables and allow the filter to be re-used.

Flowing
Liquids usually flow through the filter by gravity. This is the simplest method, and can be seen in
the coffeemaker example. For chemical plants, this is usually the most economical method as
well. In the laboratory, pressure in the form of compressed air may be applied to make the
filtration process faster, though this may lead to clogging or the passage of fine particles.
Alternatively, the liquid may flow through the filter by the force exerted by a pump. In this case,
the filter need not be mounted vertically.

Filter media
There are two main types of filter media — a solid sieve which traps the solid particles, with or
without the aid of filter paper, and a bed of granular material which retains the solid particles as
it passes. The first type allows the solid particles, i.e. the residue, to be collected intact; the
second type does not permit this. However, the second type is less prone to clogging due to the
greater surface area where the particles can be trapped. Also, when the solid particles are very
fine, it is often cheaper and easier to discard the contaminated granules than to clean the solid
sieve.

Filter media can be cleaned by rinsing with solvents or detergents. Alternatively, in engineering
applications, such as swimming pool water treatment plants, they may be cleaned by
backwashing.

Examples of the first type include filter paper used with a Buchner, Hirsch, filter funnel or other
similar funnel. A sintered-glass funnel is often used in chemistry laboratories because it is able
to trap very fine particles, while permitting the particles to be removed by a spatula.

Examples of the second type include filters at municipal and swimming pool water treatment
plants, where the granular material is sand. In the laboratory, Celite or diatomaceous earth is
packed in a Pasteur pipette (microscale) or loaded on top of a sintered-glass funnel to serve as
the filter bed.

The following points should be considered while selecting the filter media:

 ability to build the solid.

 minimum resistance to flow the filtrate.
 resistance to chemical attack.
 minimum cost.
 long life.

Filter aid
Certain filter aids may be used to aid filtration. These are often incompressible diatomaceous
earth or kieselguhr, which is composed primarily of silica. Also used are wood cellulose and
other inert porous solids.

These filter aids can be used in two different ways. They can be used as a precoat before the
slurry is filtered. This will prevent gelatinous-type solids from plugging the filter medium and
also give a clearer filtrate. They can also be added to the slurry before filtration. This increases
the porosity of the cake and reduces resistance of the cake during filtration. In a rotary filter, the
filter aid may be applied as a precoat; subsequently, thin slices of this layer are sliced off with the
cake.

The use of filter aids is usually limited to cases where the cake is discarded or where the
precipitate can be separated chemically from the filter.

Alternatives

v•d•e
Separation processes

Acid-base http://en.wikipedia.org/wiki/Image:ChemSepProcDi
agram.svg
extraction ·
Chromatography ·
Crystallization ·
Dissolved air
flotation ·
Distillation ·
Drying ·
Electrochromatograp
Processes
hy · Filtration ·
Flocculation · Froth
flotation · Liquid-
liquid extraction ·
Recrystallization ·
Sedimentation ·
Solid Phase
Extraction ·
Sublimation
 API oil-water
separator ·
Centrifuge · Depth
filter · Mixer-settler ·
Devices
Protein skimmer ·
Spinning cone ·
Still · Sublimation
apparatus

Aqueous two phase

Multiphase
systems system · Azeotrope ·
Eutectic

Filtration is a more efficient method for the separation of mixtures than decantation, but is much
more time consuming. If very small amounts of solution are involved, most of the solution may
be soaked up by the filter medium.

An alternative to filtration is centrifugation — instead of filtering the mixture of solid and liquid
particles, the mixture is centrifuged to force the (usually) denser solid to the bottom, where it
often forms a firm cake. The liquid above can then be decanted. This method is especially useful
for separating solids which do not filter well, such as gelatinous or fine particles. These solids
can clog or pass through the filter, respectively.

Filter types
 Gravity filter (open system that operates with water column pressure only)
 Pressure filter (closed system that operates under pressure from a pump)
 Side stream filter (filter in a closed loop, that filters part of the media per cycle only)
 Depth filter
 Continuous rotary filters

Centrifugation

Centrifugation is a process that involves the use of the centrifugal force for the separation of
mixtures, used in industry and in laboratory settings. More-dense components of the mixture
migrate away from the axis of the centrifuge, while less-dense components of the mixture
migrate towards the axis. In chemistry and biology, increasing the effective gravitational force on
a test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the
bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant
liquid".
Since "supernatant" is an adjective, its usage alone is technically incorrect, although many
examples can be found in scientific literature. The supernatant liquid is then either quickly
decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.
The rate of centrifugation is specified by the acceleration applied to the sample, typically
measured in revolutions per minute (RPM) or g. The particles' settling velocity in centrifugation
is a function of their size and shape, centrifugal acceleration, the volume fraction of solids
present, the density difference between the particle and the liquid, and the viscosity.

A simple example of a centrifuge is a household washing machine which separates liquids

(water) from solids (fabric/clothing) during the spin cycle.

In the chemical and food industries, special centrifuges can process a continous stream of
particle-laden liquid.

It is worth noting that centrifugation is the most common method used for uranium enrichment,
relying on the slight mass difference between atoms of U238 and U235 in uranium hexafluoride
gas.

Types
There are various types of centrifugation:

 Differential centrifugation
 Isopycnic centrifugation
 Sucrose gradient centrifugation

Other applications
 Separating textile.
 Removing water from lettuce after washing it.
 Separating particles from an air-flow using cyclonic separation.

Differential centrifugation is a common procedure in microbiology and cytology used to

separate certain organelles from whole cells for further analysis of specific parts of cells. In the
process, a tissue sample is first homogenised to break the cell membranes and mix up the cell
contents. The homogenate is then subjected to repeated centrifugations, each time removing the
pellet and increasing the centrifugal force. Finally, purification may be done through equilibrium
sedimentation, and the desired layer is extracted for further analysis.

Separation is based on size and density, with larger and denser particles pelleting at lower
centrifugal forces. As an example, unbroken whole cells will pellet at low speeds and short
intervals such as 1,000g for 5 minutes. Whereas smaller cell fragments and organelles remain in
the supernatant and require more force and greater times to pellet. In general, one can enrich for
the following cell components, in the separating order in actual application:
  Whole cells and nuclei;
 Mitochondria, lysosomes and peroxisomes;
 Microsomes (vesicles of disrupted endoplasmic reticulum); and
 Ribosomes and cytosol.

Sample preparation

Before differential centrifugation can be carried out to separate different portions of a cell from
one another, the tissue sample must first be homogenised. In this process, a blender, usually a
piece of porous porcelain of the same shape and dimension as the container, is used. The
container is, in most cases, a glass boiling tube.

The tissue sample is first crushed and a buffer solution is added to it, forming a liquid suspension
of crushed tissue sample. The buffer solution is a dense, inert, aqueous solution which is
designed to suspend the sample in a liquid medium without damaging it through chemical
reactions or osmosis. In most cases, the solution used is sucrose solution; in certain cases brine
will be used. Then, the blender, connected to a high-speed rotor, is inserted into the container
holding the sample, pressing the crushed sample against the wall of the container.

With the rotator turned on, the tissue sample is ground by the porcelain pores and the container
wall into tiny fragments. This grinding process will break the cell membranes of the sample's
cells, leaving individual organelles suspended in the solution. This process is called
homogenization. A portion cells will remain intact after grinding and some organelles will be
damaged, and these will be catered for in the later stages of centrifugation.

Ultracentrifugation
The homogenised sample is now ready for centrifugation in an ultracentrifuge. An
ultracentrifuge consists of a refrigerated, evacuated chamber containing a rotor which is driven
by an electrical motor capable of high speed rotation. Samples are placed in tubes within or
attached to the rotor. Rotational speed may reach around 70,000 rpm, creating centrifugal speed
forces of 500,000g. This force causes sedimentation of macromolecules, and can even cause non-
uniform distributions of small molecules.

Since different fragments of a cell have different sizes and densities, each fragment will settle
into a pellet with different minimum centrifugal forces. Thus, separation of the sample into
different layers can be done by first centrifuging the original homogenate under weak forces,
removing the pellet, then exposing the subsequent supernatants to sequentially greater centrifugal
fields. Each time a portion of different density is sedimented to the bottom of the container and
extracted, and repeated application produces a rank of layers which includes different parts of the
original sample. Additional steps can be taken to further refine each of the obtained pellets.
Sedimentation depends on mass, shape, and partial specific volume of a macromolecule, as well
as solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored
during the experiment to calculate molecular weight. Values of sedimentation coefficient (S) can
be calculated. Large values of S (faster sedimentation rate) correspond to larger molecular
weight. Dense particle sediments more rapidly. Elongated proteins have larger frictional
coefficients, and sediment more slowly.

Equilibrium (isopycnic) sedimentation

Equilibrium sedimentation uses a gradient of a solution such as Caesium Chloride or Sucrose

to separate particles based on their individual densities (mass/volume). It is used as a purifying
process for differential centrifugation. A solution is prepared with the densest portion of the
gradient at the bottom. Particles to be separated are then added to the gradient and centrifuged.
Each particle proceeds (either up or down) until it reaches an environment of comparable
density. Such a density gradient may be continuous or prepared in a stepped manner. For
instance, when using sucrose to prepare density gradients, one can carefully float a solution of
40% sucrose onto a layer of 45% sucrose and add further less dense layers above. The
homogenate, prepared in a dilute buffer and centrifuged briefly to remove tissue and unbroken
cells, is then layered on top. After centrifugation typically for an hour at about 100,000 x g, one
can observe disks of cellular components residing at the change in density from one layer to the
next. By carefully adjusting the layer densities to match the cell type, one can enrich for specific
cellular components. Caesium chloride allows for greater precision in separating particles of
similar density. In fact, with a caesium chloride gradient, DNA particles that have incorporated
heavy isotopes (13C or 15N for example) can be separated from DNA particles without heavy
isotopes

Isopycnic centrifugation or equilibrium centrifugation is a process used to isolate nucleic acids

such as DNA. To begin the analysis a mixture of caesium chloride and DNA is placed in a
centrifuge for several hours at high speed to generate a force of about 10^5 x g (earth's gravity).
Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mol it is similar to the density
of DNA. After some time a gradient of the caesium ions is formed, caused by two opposing
forces: diffusion and centrifugal force. The diffusive force arises due to the density gradient of
solvated caesium chloride and it always directed towards the center of the rotor. The sedimenting
particles will sediment away from the rotor until their density is equivalent to the local density of
the caesium gradient, at which point the diffusive force is equivalent to the centrifugal force.

The DNA molecules will then be separated based on the relative proportions of AT (adenine and
thymine base pairs) to GC (guanine and cytosine base pairs). An AT base pair has a lower
molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the
one with the greater proportion of AT base pairs will have a lower density, all other factors being
equal. Different types of nucleic acids will also be separated into bands, e.g. RNA is denser than
supercoiled plasmid DNA, which is denser than linear chromosomal DNA.
UNIT111-ISOLATION OF PRODUCTS
Liquid-liquid extraction, 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 phase into
another liquid phase. Liquid-liquid extraction is a basic technique in chemical laboratories,
where it is performed using a separatory funnel. This type of process is commonly performed
after a chemical reaction as part of the work-up.

Solvent extraction is used in nuclear reprocessing, ore processing, the production of fine organic
compounds, the processing of perfumes and other industries. In an industrial application, this
process is done continuously by pumping an organic and aqueous stream into a mixer. This
mixes the organic component with the aqueous component and allows ion transfer between them.
The mixing continues until equilibrium is reached. Once the ion transfer is complete (equilibrium
is reached), the mixture flows into a vessel, where the organic and aqueous are allowed to
separate, similar to the way oil and water would separate after mixing them. Fresh material is
continuously fed into the mixer, and a two continuous streams is removed from the settler (one
organic, and one aqueous). The process is commonly used to process copper and uranium, but
has recently been adapted for zinc, at Skorpion Zinc mine in Namibia.

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

metal in contact with molten salt, 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 which 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.

If a detergent or fine solid can stabilise an emulsion which in the solvent extraction community is
known as a third phase.

Contents

 1 Distribution ratio
 2 Separation factors
 3 Decontamination factor
 4 Slopes of graphs
 5 Batchwise single stage extractions
 6 Multistage countercurrent continuous processes
 7 Extraction without chemical change
 8 Extraction with chemical change
o 8.1 Solvation mechanism
o 8.2 Ion exchange mechanism
o 8.3 Ion pair extraction
 9 Kinetics of extraction
 10 Aqueous complexing agents
 11 Industrial process design
  12 Equipment
 13 Extraction of metals
o 13.1 Palladium and platinum
o 13.2 Neodymium
o 13.3 Cobalt
o 13.4 Nickel
o 13.5 Copper
o 13.6 Zinc and cadmium
 14 Terms
 15 See also

 16 References

Distribution ratio
In solvent extraction, a distribution ratio is often quoted as a measure of how well-extracted a
species is. The distribution ratio (D) 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. See partition coefficient for more details. Note that a distribution
ratio for uranium and neptunium between two inorganic solids (zirconolite and perovskite) has
been reported.[1]

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.

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 0.1 / 0.01 = 10.

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.

Batchwise single stage extractions

This is commonly used on the small scale in chemical labs, it is normal to use a separating funnel

For instance if a chemist was to extract 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.

Multistage countercurrent continuous processes

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. In the multistage processes the aqueous raffinate from one extraction unit is feed as 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 win back
the metal from the organic phase.

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 (chemistry)). This is the most simple type of solvent
extraction. Some solutes which do not at first sight appear to undergo a reaction during the
extraction process do not have distribution ratio which is independent of concentration, a classic
example is the extraction of carboxylic acids (HA) into non polar 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]]

Extraction with chemical change

A small review on the subject of the main classes of extraction agents (extractants) can be found
at [2].

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 organophosphate tri-n-butyl phosphate. The
PUREX process 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 can be added which converts the plutonium to
the trivalent oxidation state. 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 would be the extraction of zinc, cadmium or lead by a dialkyl phosphinic acid
(R2PO2H) into a non polar 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 intermidate 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 which is more likely to be encountered by the 'average' chemist is the use of a phase
transfer catalyst, these are charged species which transfer 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 according to F. Scholz, S. Komorsky-Lovric, M. Lovric, Electrochem. Comm.,

2000, 2, 112-118 the 31.1 kJ mol-1 is required to transfer an acetate anion into nitrobenzene,
while according to A.F.Danil de Namor and T.Hill, J.Chem. Soc Fraraday Trans., 1983, 2713 the
energy required to transfer a chloride anion from an aqueous phase to nitrobenzene is 43.8 kJ
mol-1.

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 the 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.

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 due to the fact that
the rate of ligand exchange at these metal centres 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
polyhalide anion which is quite common.

Industrial process design

Typically 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 which might
be present.

Equipment
Two layers separating during a liquid-liquid extraction

An organic MTBE solution is extracted with aqueous sodium bicarbonate solution. This
base removes benzoic acid as benzoate but leaves non-acidic benzil (yellow) behind in
the upper organic phase.
details and copyright info
In case of problems, see media help.

While solvent extraction is often done on a small scale by synthetic lab chemists using a
separating funnel, it is normally done on the industrial scale using machines which bring the two
liquid phases into contact with each other. Such machines include centrifugal contactors, spray
columns, pulsed columns and mixer-settlers.

Extraction of metals
A review of the extraction methods for a range of metals is to be found here [3].

Palladium and platinum

Dialkyl sulfides, tributyl phosphate and alkyl amines have been used for extracting these metals.
[4][1]

Neodymium

This rare earth is extracted by di(2-ethyl-hexyl)phosphoric acid into hexane by an ion exchange
mechanism.[2]

Cobalt

The extraction of cobalt from hydrochloric acid using alamine 336 in meta-xylene.[3]

Cobalt can be extracted also using Cyanex 272 {bis-(2,4,4-trimethylpentyl) phosphinic acid}.

Nickel

Nickel can be extracted using di(2-ethyl-hexyl)phosphoric acid and tributyl phosphate in a

hydrocarbon diluent (Shellsol).[4]

Copper

Copper can be extracted using hydroxyoximes as extractants, a recent paper describes an

extractant which has a good selectivity for copper over cobalt and nickel.[5]

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.[6]

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 at a high pH (around pH 4-5 Zinc is selective). To strip the Zinc
from the D2EHPA, sulfuric acid is used, at a strength of about 170g/l.

Terms
 Solvent is the term for the organic layer
 Diluent is the term for an inert liquid used to dissolve an extractant, and to dilute the
system.
 Extractant is the term for a metal extraction agent
 Raffinate is the term for the aqueous layer after a solute has been extracted from it
 Scrubbing is the term for the back extraction of an unwanted solute from the organic
phase
 Stripping is the term for the back extraction from the organic phase
Aqueous two phase extraction

Aqueous biphasic systems (ABS) or aqueous two phase systems are clean alternatives for
traditional organic-water solvent extraction systems.

ABS are formed when 2 polymers, one polymer and one kosmotropic salt, or two salts (one
chaotropic salt and the other a kosmotropic salt) are mixed together at appropriate concentrations
or at a particular temperature. The two phases are mostly composed of water and non volatile
components, thus eliminating volatile organic compounds. They have been used for many years
in biotechnological applications as denaturing and benign separation media. Recently, they have
been used for metal ion separations, environmental remediation, metallurgical applications and
as a reaction media.

Contents

 1 Introduction
 2 The two phases
o 2.1 PEG–dextran system
 3 Advantages

 4 External links

Introduction

In 1896, Beijerinck first noted an 'incompatibility' in solutions of agar, a water-soluble polymer,

with soluble starch or gelatine. Upon mixing, they separated into two immiscible phases.
Subsequent investigation has led to the determination of many other aqueous biphasic systems,
of which the polyethylene glycol (PEG) - dextran system is the most extensively studied. Other
systems that form aqueous biphases are: PEG - sodium carbonate or PEG and phosphates,
citrates or sulfates. Aqueous biphasic systems are used during downstream processing mainly in
biotechnological and chemical industries.

The two phases

It is a common observation that when oil and water are poured into the same container, they
separate into two phases or layers, because they are immiscible. In general, aqueous (or water-
based) solutions, being polar, are immiscible with non-polar organic solvents (chloroform,
toluene, hexane etc) and form a two-phase system. However, in an ABS, both immiscible
components are water-based.

The formation of the distinct phases is affected by the pH, temperature and ionic strength of the
two components, and separation occurs when the amount of a polymer present exceeds a certain
limiting concentration (which is determined by the above factors).
PEG–dextran system

The "upper phase" is formed by the more hydrophobic polyethylene glycol (PEG), which is of
lower density than the "lower phase," consisting of the more hydrophilic and denser dextran
solution.

Although PEG is inherently denser than water, it occupies the upper layer. This is believed to be
due to its solvent 'ordering' properties, which excludes excess water, creating a low density water
environment.[1] The degree of polymerisation of PEG also affects the phase separation and the
partitioning of molecules during extraction.

Advantages

ABS is an excellent method to employ for the extraction of proteins/enzymes and other labile
biomolecules from crude cell extracts or other mixtures. Most often, this technique is employed
in enzyme technology during industrial or laboratory production of enzymes.

 They provide mild conditions that do not harm or denature unstable/labile biomolecules
 The interfacial stress (at the interface between the two layers) is far lower (400-fold less)
than water-organic solvent systems used for solvent extraction, causing less damage to
the molecule to be extracted
 The polymer layer stabilizes the extracted protein molecules, favouring a higher
concentration of the desired protein in one of the layers, resulting in an effective
extraction
 Specialised systems may be developed (by varying factors such as temperature, degree of
polymerisation, presence of certain ions etc ) to favour the enrichment of a specific
compound, or class of compounds, into one of the two phases. They are sometimes used
simultaneously with ion-exchange resins for better extraction
 Separation of the phases and the partitioning of the compounds occurs rapidly. This
allows the extraction of the desired molecule before endogenous proteases can degrade
them.
 These systems are amenable to scale-ups, from laboratory-sized setups to those that can
handle the requirements of industrial production. They may be employed in continuous
protein-extraction proceses.

Specificity may be further increased by tagging ligands specific to the desired enzyme, onto the
polymer. This results in a preferential binding of the enzyme to the polymer, increasing the
effectiveness of the extraction.

One major disadvantage, however, is the cost of materials involved, namely high-purity dextrans
employed for the purpose. However, other low-cost alternatives such as less refined Dextrans,
hydroxypropyl starch derivatives and high-salt solutions.

Precipitation
Precipitation is widely used in downstream processing of biological products, such as proteins.
[1] This unit operation serves to concentrate and fractionate the target product from various
contaminants. For example, in the biotechnology industry protein precipitation is used to
eliminate contaminants commonly contained in blood. [2] Academic research on protein
precipitation explores new protein precipitation methods. [3] The underlying mechanism of
precipitation is to alter the solvation potential of the solvent and thus lower the solubility of the
solute by addition of a reagent.

Contents

 1 Protein solubility
 2 Repulsive electrostatic force
 3 Attractive electrostatic force
 4 Precipitate formation
 5 Salting out
o 5.1 Energetics involved in salting out
o 5.2 Hofmeister series
o 5.3 Salting out in practice
 6 Isoelectric point precipitation
 7 Precipitation with organic solvents
 8 Non-ionic hydrophilic polymers
 9 Flocculation by polyelectrolytes
 10 Polyvalent metallic ions
 11 Precipitation reactors
o 11.1 Batch reactors
o 11.2 Tubular reactors
o 11.3 Continuous stirred tank reactors (CSTR)

 12 References

Protein solubility
The solubility of proteins in aqueous buffers depends on the distribution of hydrophilic and
hydrophobic amino acid residues on the protein’s surface. Hydrophobic residues predominantly
occur in the globular protein core, but some exist in patches on the surface. Proteins that have
high hydrophobic amino acid content on the surface have low solubility in an aqueous solvent.
Charged and polar surface residues interact with ionic groups in the solvent and increase
solubility. Knowledge of amino acid composition of a protein will aid in determining an ideal
precipitation solvent and method.

Repulsive electrostatic force

Repulsive electrostatic forces form when proteins are suspended in an electrolyte solution. These
repulsive forces between proteins prevent aggregation and facilitate dissolution. Solvent
counterions migrate towards charged surface residues on the protein, forming a rigid matrix of
counterions attached to the protein surface. The adjacent solvation layer, which is less rigid,
consists of a decreasing concentration profile of the counterions and an increasing concentration
profile of the co-ions. In effect, the protein’s potential to engage in ionic interactions with each
other will decrease. Proteins will be less likely to form aggregates. Water molecules can have a
similar effect. Water forms a solvation layer around hydrophilic surface residues of a protein.
Water establishes a concentration gradient around the protein, with the highest concentration at
the protein surface. This water network has a damping effect on the attractive forces between
proteins.

Ionic Solvation LayerHydration Layer

Attractive electrostatic force

Dispersive or attractive forces exist between proteins through permanent and induced dipoles.
For example, basic residues on a protein can have electrostatic interactions with acidic residues
on another protein. However, solvation by ions in an electrolytic solution or water will decrease
protein-protein attractive forces. Protein accumulation and precipitation can be enhanced by
decreasing the hydration layer around the protein. The purpose of the added reagents in protein
precipitation is to reduce the hydration layer.

Hydration Layer

Precipitate formation
Protein precipitate formation occurs in a stepwise process. The addition of a precipitating agent
and steady mixing destabilizes the protein solution. Mixing causes the precipitant and the target
product to collide. Enough mixing time is required for molecules to diffuse across the fluid
eddies. During the following nucleation phase, submicroscopic sized particles are generated.
Growth of these particles is under Brownian diffusion control. Once the growing particles reach
a critical size (0.1 µm to 10 µm for high and low shear fields, respectively), by diffusive addition
of individual protein molecules, they continue to grow by colliding into each other and sticking
or flocculating. This phase occurs at a slower rate. During the final step, aging in a shear filed,
the precipitate particles repeatedly collide and stick, then break apart, until a stable mean particle
size is reached, which is dependent upon individual proteins. The mechanical strength of the
protein particles correlates with the product of the mean shear rate and the aging time, which is
known as the Camp number. Aging helps particles withstand the fluid shear forces encountered
in pumps and centrifuge feed zones without reducing in size.

Salting out
Salting out is the most common method used to precipitate a target protein. Addition of a neutral
salt, such as ammonium sulfate, compresses the solvation layer and increases protein-protein
interactions. As the salt concentration of a solution is increased, more of the bulk water becomes
associated with the ions. As a result, less water is available to partake in the solvation layer
around the protein, which exposes hydrophobic patches on the protein surface. Proteins may then
exhibit hydrophobic interactions, aggregate and precipitate from solution.

Energetics involved in salting out

Salting out is a spontaneous process when the right concentration of the salt is reached in
solution. The hydrophobic patches on the protein surface generate highly ordered water shells.
This results in a small decrease in enthalpy, ΔH, and a larger decrease in entropy, ΔS, of the
ordered water molecules relative to the molecules in the bulk solution. The overall free energy
change, ΔG, of the process is given by the Gibbs free energy equation:

ΔG = ΔH − TΔS.

ΔG = Free energy change, ΔH = Enthalpy change upon precipitation, ΔS = Entropy change upon
precipitation, T = Absolute temperature When water molecules in the rigid solvation layer are
brought back into the bulk phase through interactions with the added salt, their greater freedom
of movement causes a significant increase in their entropy. Thus, ΔG becomes negative and
precipitation occurs spontaneously.

Hofmeister series

Kosmotropes or "water structure makers" are salts which promote the dissipation of water from
the solvation layer around a protein. Hydrophobic patches are then exposed on the protein’s
surface, and they interact with hydrophobic patches on other proteins. These salts enhance
protein aggregation and precipitation. Chaotropes or “water structure breakers,” have the
opposite effect of Kosmotropes. These salts promote an increase in the solvation layer around a
protein. The effectiveness of the kosmotropic salts in precipitating proteins follows the order of
the Hofmeister series:

Most precipitation least precipitation

Most precipitation least precipitation

Salting out in practice

The decrease in protein solubility follows a normalized solubility curve of the type shown. The
relationship between the solubility of a protein and increasing ionic strength of the solution can
be represented by the Cohn equation:

S = solubility of the protein, B is idealized solubility, K is a salt-specific constant and I is the

ionic strength of the solution, which is attributed to the added salt.

zi is the ion charge of the salt and ci is the salt concentration. The ideal salt for protein
precipitation is most effective for a particular amino acid composition, inexpensive, non-
buffering, and non-polluting. The most commonly used salt is ammonium sulfate. There is a low
variation in salting out over temperatures 0 °C to 30 °C. Protein precipitates left in the salt
solution can remain stable for years-protected from proteolysis and bacterial contamination by
the high salt concentrations. Ammonium sulfate salt cannot be used in solutions that have pH > 8
because the ammonium ion has a buffering effect on the solution. Sodium citrate is a good
alternative for solutions above pH 8.

Solubility Curve

Isoelectric point precipitation

The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein
becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly
negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise,
at a solution pH that is below the pI, the surface of the protein is predominantly positively
charged and repulsion between proteins occurs. However, at the pI the negative and positive
charges cancel, repulsive electrostatic forces are reduced and the dispersive forces predominate.
The dispersive forces will cause aggregation and precipitation. The pI of most proteins is in the
pH range of 4-6. Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants.
The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused
by the mineral acids. For this reason isoelectric point precipitation is most often used to
precipitate contaminant proteins, rather than the target protein. The precipitation of casein during
cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation. tkr

Precipitation with organic solvents

Addition of miscible solvents such as ethanol or methanol to a solution may cause proteins in the
solution to precipitate. The solvation layer around the protein will decrease as the organic solvent
progressively displaces water from the protein surface and binds it in hydration layers around the
organic solvent molecules. With smaller hydration layers, the proteins can aggregate by attractive
electrostatic and dipole forces. Important parameters to consider are temperature, which should
be less than 0 °C to avoid denaturation, pH and protein concentration in solution. Miscible
organic solvents decrease the dielectric constant of water, which in effect allows two proteins to
come close together. At the isoelectric point the relationship between the dielectric constant and
protein solubility is given by:

S0 is an extrapolated value of S, e is the dielectric constant of the mixture and k is a constant that
relates to the dielectric constant of water. The Cohn process for plasma protein fractionation
relies on solvent precipitation with ethanol to isolate individual plasma proteins.

Non-ionic hydrophilic polymers

Polymers, such as dextrans and polyethylene glycols, are frequently used to precipitate proteins
because they have low flammability and are less likely to denature biomaterials than isoelectric
precipitation. These polymers in solution attract water molecules away from the solvation layer
around the protein. This increases the protein-protein interactions and enhances precipitation. For
the specific case of polyethylene glycol, precipitation can be modeled by the equation:

C is the polymer concentration, P is a protein-protein interaction coefficient, a is a protein-

polymer interaction coefficient and

μ is the chemical potential of component I, R is the universal gas constant and T is the absolute
temperature.

Flocculation by polyelectrolytes
Alginate, carboxymethycellulose, polyacrylic acid, tannic acid and polyphosphates can form
extended networks between protein molecules in solution. The effectiveness of these
polyelectrolytes depend on the pH of the solution. Anionic polyelectrolytes are used at pH values
less than the isoelectric point. Cationic polyelectrolytes are at pH values above the pI. It is
important to note that an excess of polyelectrolytes will cause the precipitate to dissolve back
into the solution. An example of polyelectrolyte flocculation is the removal of protein cloud from
beer wort using Irish moss.

Polyvalent metallic ions

Metal salts can be used at low concentrations to precipitate enzymes and nucleic acids from
solutions. Polyvalent metal ions frequently used are Ca+, Mg2+, Mn2+ or Fe+.

Precipitation reactors
There are numerous industrial scaled reactors than can be used to precipitate large amounts of
proteins, such as recombinant DNA polymerases from a solution.[4]

Batch reactors
Batch reactors are the simplest type of precipitation reactor. The precipitating agent is slowly
added to the protein solution under mixing. The aggregating protein particles tend to be compact
and regular in shape. Since the particles are exposed to a wide range of shear stresses for a long
period of time, they tend to be compact, dense and mechanically stable.

Tubular reactors

In tubular reactors, feed protein solution and the precipitating reagent are contacted in a zone of
efficient mixing then fed into long tubes where precipitation takes place. The fluid in volume
elements approach plug flow as they move though the tubes of the reactor. Turbulent flow is
promoted through wire mesh inserts in the tube. The tubular reactor does not require moving
mechanical parts and is inexpensive to build. However, the reactor can become impractically
long if the particles aggregate slowly.

Continuous stirred tank reactors (CSTR)

CSTR reactors run at steady state with a continuous flow of reactants and products in a well-
mixed tank. Fresh protein feed contacts slurry that already contains precipitate particles and the
precipitation reagents.
 UNIT-IV PRODUCT PURIFICATION

Chromatography (from Greek χρώμα:chroma, color and γραφειν:"graphein" to write) is the

collective term for a family of laboratory techniques for the separation of mixtures. It involves
passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the
analyte to be measured from other molecules in the mixture and allows it to be isolated.

Chromatography may be preparative or analytical. Preparative chromatography seeks to separate

the components of a mixture for further use (and is thus a form of purification). Analytical
chromatography normally operates with smaller amounts of material and seeks to measure the
relative proportions of analytes in a mixture. The two are not mutually exclusive.

Contents

 1 Explanation
 2 History
 3 Chromatography terms
 4 Techniques by chromatographic bed shape
o 4.1 Column chromatography
o 4.2 Planar Chromatography
 4.2.1 Paper Chromatography
 4.2.2 Thin layer chromatography
 5 Techniques by physical state of mobile phase
o 5.1 Gas chromatography
o 5.2 Liquid chromatography
 6 Affinity chromatography
o 6.1 Supercritical fluid chromatography
 7 Techniques by separation mechanism
o 7.1 Ion exchange chromatography
o 7.2 Size exclusion chromatography
  8 Special techniques
o 8.1 Reversed-phase chromatography
o 8.2 Two-dimensional chromatography
o 8.3 Simulated Moving-Bed Chromatography
o 8.4 Pyrolysis gas chromatography
o 8.5 Fast protein liquid chromatography
o 8.6 Countercurrent chromatography
o 8.7 Chiral chromatography
 9 See also
 10 References

 11 External links

Explanation
http://en.wikipedia.org/wiki/Image:Wiktionary-logo-en.svg
.

An analogy which is sometimes useful is to suppose a mixture of bees and wasps passing over a
flower bed. The bees would be more attracted to the flowers than the wasps, and would become
separated from them. If one were to observe at a point past the flower bed, the wasps would pass
first, followed by the bees. In this analogy, the bees and wasps represent the analytes to be
separated, the flowers represent the stationary phase, and the mobile phase could be thought of as
the air. The key to the separation is the differing affinities among analyte, stationary phase, and
mobile phase. The observer could represent the detector used in some forms of analytical
chromatography. A key point is that the detector need not be capable of discriminating between
the analytes, since they have become separated before passing the detector.

History
It was the Russian botanist Mikhail Semyonovich Tsvet who invented the first chromatography
technique in 1900 during his research on chlorophyll. He used a liquid-adsorption column
containing calcium carbonate to separate plant pigments. The method was described on
December 30, 1901 at the 11th Congress of Naturalists and Doctors (XI съезд
естествоиспытателей и врачей) in Saint Petersburg. The first printed description was in 1903,
in the Proceedings of the Warsaw Society of Naturalists, section of biology. He first used the
term chromatography in print in 1906 in his two papers about chlorophyll in the German
botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his
chromatograph for the German Botanical Society. Interestingly, Mikhail's surname "Цвет" means
"color" in Russian, so there is the possibility that his naming the procedure chromatography
(literally "color writing") was a way that he could make sure that he, a commoner in Tsarist
Russia, could be immortalized.

In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the
Chemistry Nobel Prize for their invention of partition chromatography.[1] Since then, the
technology has advanced rapidly. Researchers found that the principles underlying Tsvet's
chromatography could be applied in many different ways, giving rise to the different varieties of
chromatography described below. Simultaneously, advances continually improved the technical
performance of chromatography, allowing the separation of increasingly similar molecules. In
1987 Pedro Cuatrecasas and Meir Wilchek were awarded the Wolf Prize in Medicine for the
invention and development of affinity chromatography and its applications to biomedical
sciences.

Chromatography terms
 The analyte is the substance that is to be separated during chromatography.
 Analytical chromatography is used to determine the existence and possibly also the
concentration of analyte(s) in a sample.
 A bonded phase is a stationary phase that is covalently bonded to the support particles or
to the inside wall of the column tubing.
 A chromatogram is the visual output of the chromatograph. In the case of an optimal
separation, different peaks or patterns on the chromatogram correspond to different
components of the separated mixture.

http://en.wikipedia.org/wiki/Image:Rt_5_9.pnghttp://en.wikipedia.org/wiki/Image:Rt_5_
12.png
Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example
obtained by a spectrophotometer, mass spectrometer or a variety of other detectors)
corresponding to the response created by the analytes exiting the system. In the case of an
optimal system the signal is proportional to the concentration of the specific analyte
separated.
 A chromatograph is equipment that enables a sophisticated separation e.g. gas
chromatographic or liquid chromatographic separation.
 Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary (stationary
phase) while the other (the mobile phase) moves in a definite direction.
 The effluent is the mobile phase leaving the column.
 An immobilized phase is a stationary phase which is immobilized on the support
particles, or on the inner wall of the column tubing.
 The mobile phase is the phase which moves in a definite direction. It may be a liquid
(LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography,
SFC). A better definition: The mobile phase consists of the sample being
separated/analyzed and the solvent that moves the sample through the column. In one
case of HPLC the solvent consists of a carbonate/bicarbonate solution and the sample is
the anions being separated. The mobile phase moves through the chromatography column
(the stationary phase) where the sample interacts with the stationary phase and is
separated.
 Preparative chromatography is used to purify sufficient quantities of a substance for
further use, rather than analysis.
  The retention time is the characteristic time it takes for a particular analyte to pass
through the system (from the column inlet to the detector) under set conditions. See also:
Kovat's retention index
 The sample is the matter analysed in chromatography. It may consist of a single
component or it may be a mixture of components. When the sample is treated in the
course of an analysis, the phase or the phases containing the analytes of interest is/are
referred to as the sample whereas everything out of interest separated from the sample
before or in the course of the analysis is referred to as waste.
 The solute refers to the sample components in partition chromatography.
 The solvent refers to any substance capable of solubilizing other substance, and
especially the liquid mobile phase in LC.
 The stationary phase is the substance which is fixed in place for the chromatography
procedure. Examples include the silica layer in thin layer chromatography.

Techniques by chromatographic bed shape

Column chromatography

For more details on this topic, see Column chromatography.

http://en.wikipedia.org/wiki/Image:Columnchromatography.gif
A diagram of a standard column chromatography and a flash column chromatography setup

Column chromatography is a separation technique in which the stationary bed is within a tube.
The particles of the solid stationary phase or the support coated with a liquid stationary phase
may fill the whole inside volume of the tube (packed column) or be concentrated on or along the
inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the
tube (open tubular column). Differences in rates of movement through the medium are calculated
to different retention times of the sample.[2]

In 1978, W. C. Still introduced a modified version of column chromatography called flash

column chromatography (flash).[3] The technique is very similar to the traditional column
chromatography, except for that the solvent is driven through the column by applying positive
pressure. This allowed most separations to be performed in less than 20 minutes, with improved
separations compared to the old method. Modern flash chromatography systems are sold as pre-
packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be
linked with detectors and fraction collectors providing automation. The introduction of gradient
pumps resulted in quicker separations and less solvent usage.

In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed
bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture
broths or slurries of broken cells.

Planar Chromatography

http://en.wikipedia.org/wiki/Image:Chromatography_of_chlorophyll_-_Step_7.jpg
Thin layer chromatography is used to separate components of chlorophyll

Planar chromatography is a separation technique in which the stationary phase is present as or

on a plane. The plane can be a paper, serving as such or impregnated by a substance as the
stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a
glass plate (thin layer chromatography).

Paper Chromatography

Paper chromatography is a technique that involves placing a small dot of sample solution onto a
strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent
and sealed. As the solvent rises through the paper it meets the sample mixture which starts to
travel up the paper with the solvent. Different compounds in the sample mixture travel different
distances according to how strongly they interact with the paper. This paper is made of cellulose,
a polar molecule, and the compounds within the mixture travel farther if they are non-polar.
More polar substances bond with the cellulose paper more quickly, and therefore do not travel as
far. This process allows the calculation of an Rf value and can be compared to standard
compounds to aid in the identification of an unknown substance.
Thin layer chromatography.

Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to

paper chromatography. However, instead of using a stationary phase of paper, it involves a
stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert
substrate. Compared to paper, it has the advantage of faster runs, better separations, and the
choice between different adsorbents. Different compounds in the sample mixture travel different
distances according to how strongly they interact with the adsorbent. This allows the calculation
of an Rf value and can be compared to standard compounds to aid in the identification of an
unknown substance.

Techniques by physical state of mobile phase

Gas chromatography

Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a

separation technique in which the mobile phase is a gas. Gas chromatography is always carried
out in a column, which is typically "packed" or "capillary" (see below) .

Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid

stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium).
The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column)
or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical
chemistry; though the high temperatures used in GC make it unsuitable for high molecular
weight biopolymers or proteins (heat will denature them), frequently encountered in
biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and
industrial chemical fields. It is also used extensively in chemistry research.

Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid.
Liquid chromatography can be carried out either in a column or a plane. Present day liquid
chromatography that generally utilizes very small packing particles and a relatively high pressure
is referred to as high performance liquid chromatography (HPLC).

In the HPLC technique, the sample is forced through a column that is packed with irregularly or
spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile
phase) at high pressure. HPLC is historically divided into two different sub-classes based on the
polarity of the mobile and stationary phases. Technique in which the stationary phase is more
polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is
called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol
mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed
phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and
RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be
noted that the following techniques can also be considered fast protein liquid chromatography if
no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous
Normal Phase Chromatography.

Affinity chromatography.
Affinity chromatography[4] is based on selective non-covalent interaction between an analyte and
specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the
purification of proteins bound to tags. These fusion proteins are labelled with compounds such as
His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification,
some of these tags are usually removed and the pure protein is obtained.

Supercritical fluid chromatography.

Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid
above and relatively close to its critical temperature and pressure.

Techniques by separation mechanism

Ion exchange chromatography

Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is usually
performed in columns but the mechanism can be benefited also in planar mode. Ion exchange
chromatography uses a charged stationary phase to separate charged compounds including amino
acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange
resin that carries charged functional groups which interact with oppositely charged groups of the
compound to be retained. Ion exchange chromatography is commonly used to purify proteins
using FPLC.

Size exclusion chromatography

Size exclusion chromatography (SEC) is also known as gel permeation chromatography

(GPC) or gel filtration chromatography and separates molecules according to their size (or
more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller
molecules are able to enter the pores of the media and, therefore, take longer to elute, whereas
larger molecules are excluded from the pores and elute faster. It is generally a low resolution
chromatography technique and thus it is often reserved for the final, "polishing" step of a
purification. It is also useful for determining the tertiary structure and quaternary structure of
purified proteins, especially since it can be carried out under native solution conditions.

Special techniques
Reversed-phase chromatography
Reversed-phase chromatography is an elution procedure used in liquid chromatography in which
the mobile phase is significantly more polar than the stationary phase.

Two-dimensional chromatography

In some cases, the chemistry within a given column can be insufficient to separate some analytes.
It is possible to direct a series of unresolved peaks onto a second column with different physico-
chemical (Chemical classification) properties. Since the mechanism of retention on this new
solid support is different from the first dimensional separation, it can be possible to separate
compounds that are indistinguishable by one-dimensional chromatography.

Pyrolysis gas chromatography

Fast protein liquid chromatography

For more details on this topic, see Fast protein liquid chromatography.

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography

techniques which are used to purify proteins. Many of these techniques are identical to those
carried out under high performance liquid chromatography.

Countercurrent chromatography

For more details on this topic, see Countercurrent chromatography.

Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the

stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them
to settle into layers and then separating the layers.

Chiral chromatography

Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers,

these have no chemical or physical differences apart from being three dimensional mirror
images. Conventional chromatography or other separation processes are incapable of separating
them. To enable chiral separations to take place, either the mobile phase or the stationary phase
must themselves be made chiral, giving differing affinities between the analytes. Chiral
chromatography HPLC columns (with a chiral stationary phase) in both normal and reversed
phase are commercially available.

UNIT –V FINAL PRODUCT FORMULATION AND FINISHING OPERATIONS

Crystallization is the (natural or artificial) process of formation of solid crystals from a uniform
solution or melt, or more rarely directly from a gas. Crystallization is also a chemical solid-liquid
separation technique, in which mass transfer of a solute from the liquid solution to a pure solid
crystalline phase occurs.

Contents

 1 Process
 2 Crystallization in nature
 3 Artificial methods
o 3.1 Crystal production
o 3.2 Purification
 4 Thermodynamic view
 5 Equipment for Crystallization
 6 References
 7 External links
 8 See also

 9 Gallery

Process
The crystallization process consists of two major events, nucleation and crystal growth.
Nucleation is the step where the solute molecules dispersed in the solvent start to gather into
clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes
stable under the current operating conditions. These stable clusters constitute the nuclei.
However when the clusters are not stable, they redissolve. Therefore, the clusters need to reach a
critical size in order to become stable nuclei. Such critical size is dictated by the operating
conditions (temperature, supersaturation, etc.). It is at the stage of nucleation that the atoms
arrange in a defined and periodic manner that defines the crystal structure — note that "crystal
structure" is a special term that refers to the relative arrangement of the atoms, not the
macroscopic properties of the crystal (size and shape), although those are a result of the internal
crystal structure.

The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical
cluster size. Nucleation and growth continue to occur simultaneously while the supersaturation
exists. Supersaturation is the driving force of the crystallization, hence the rate of nucleation and
growth is driven by the existing supersaturation in the solution. Depending upon the conditions,
either nucleation or growth may be predominant over the other, and as a result, crystals with
different sizes and shapes are obtained (control of crystal size and shape constitutes one of the
main challenges in industrial manufacturing, such as for pharmaceuticals). Once the
supersaturation is exhausted, the solid-liquid system reaches equilibrium and the crystallization
is complete, unless the operating conditions are modified from equilibrium so as to supersaturate
the solution again.
Many compounds have the ability to crystallize with different crystal structures, a phenomenon
called polymorphism. Each polymorph is in fact a different thermodynamic solid state and
crystal polymorphs of the same compound exhibit different physical properties, such as
dissolution rate, shape (angles between facets and facet growth rates), melting point, etc. For this
reason, polymorphism is of major importance in industrial manufacture of crystalline products.

Crystallization in nature

http://en.wikipedia.org/wiki/Image:SnowflakesWilsonBentley.jpg
Snow flakes are a very well known example, where subtle differences in crystal growth
conditions result in different geometries.

There are many examples of natural process that involve crystallization.

Geological time scale process examples include:

 Natural (mineral) crystal formation (see also gemstone);

 Stalactite/stalagmite, rings formation.

Usual time scale process examples include:

 Snow flakes formation (see also Koch snowflake);

 Honey crystallization (nearly all types of honey crystallize).

Artificial methods
For crystallization (see also recrystallization) to occur from a solution it must be supersaturated.
This means that the solution has to contain more solute entities (molecules or ions) dissolved
than it would contain under the equilibrium (saturated solution). This can be achieved by various
methods, with 1) solution cooling, 2) addition of a second solvent to reduce the solubility of the
solute (technique known as anti-solvent or drown-out), 3) chemical reaction and 4) change in pH
being the most common methods used in industrial practice. Other methods, such as solvent
evaporation, can also be used.
Applications:

There are two major groups of applications for the artificial crystallization process: crystal
production and purification.

Crystal production

From a material industry perspective:

 Macroscopic crystal production, for supply the demand of natural-like crystals with
methods that "accelerate time-scale" for massive production and/or perfection:
o ionic crystal production;
o covalent crystal production.
 Tiny size crystals:
o Powder, sand and smaller sizes: using methods for powder and controlled
(nanotechnology fruits) forms.
 Mass-production: on chemical industry, like salt-powder production.
 Sample production: small production of tiny crystals for material
characterization. Controlled recrystallization is an important method to
supply unusual crystals, that are needed to reveal the molecular structure
and nuclear forces inside a typical molecule of a crystal. Many techniques,
like X-ray crystallography and NMR spectroscopy, are widely used in
chemistry and biochemistry to determine the structures of an immense
variety of molecules, including inorganic compounds and bio-
macromolecules.
o Thin film production.

Massive production examples:

 "Powder salt for food" industry;

 Silicon crystal wafer production.
 Production of sucrose from sugar beet, where the sucrose is crystallized out from an
aqueous solution.

Purification

Well formed crystals are expected to be pure because each molecule or ion must fit perfectly into
the lattice as it leaves the solution. Impurities would normally not fit as well in the lattice, and
thus remain in solution preferentially. Hence, molecular recognition is the principle of
purification in crystallization. However, there are instances when impurities incorporate into the
lattice, hence, decreasing the level of purity of the final crystal product. Also, in some cases, the
solvent may incorporate into the lattice forming a solvate. In addition, the solvent may be
'trapped' (in liquid state) within the crystal formed, and this phenomenon is known as inclusion.

Thermodynamic view
The nature of a crystallization process is governed by both thermodynamic and kinetic factors,
which can make it highly variable and difficult to control. Factors such as impurity level, mixing
regime, vessel design, and cooling profile can have a major impact on the size, number, and
shape of crystals produced.

Now put yourself in the place of a molecule within a pure and perfect crystal, being heated by an
external source. At some sharply defined temperature, a bell rings, you must leave your
neighbours, and the complicated architecture of the crystal collapses to that of a liquid. Textbook
thermodynamics says that melting occurs because the entropy, S, gain in your system by spatial
randomization of the molecules has overcome the enthalpy, H, loss due to breaking the crystal
packing forces:

T(Sliquid − Ssolid) > Hliquid − Hsolid

Gliquid < Gsolid

This rule suffers no exceptions when the temperature is rising. By the same token, on cooling the
melt, at the very same temperature the bell should ring again, and molecules should click back
into the very same crystalline form. The entropy decrease due to the ordering of molecules
within the system is overcompensated by the thermal randomization of the surroundings, due to
the release of the heat of fusion; the entropy of the universe increases.

But liquids that behave in this way on cooling are the exception rather than the rule; in spite of
the second principle of thermodynamics, crystallization usually occurs at lower temperatures
(supercooling). This can only mean that a crystal is more easily destroyed than it is formed.
Similarly, it is usually much easier to dissolve a perfect crystal in a solvent than to grow again a
good crystal from the resulting solution. The nucleation and growth of a crystal are under kinetic,
rather than thermodynamic, control.

Equipment for Crystallization

1. Tank crystallizers. Tank crystallization is an old method still used in some specialized cases.
Saturated solutions, in tank crystallization, are allowed to cool in open tanks. After a period of
time the mother liquid is drained and the crystals removed. Nucleation and size of crystals are
difficult to control. Typically, labor costs are very high.

2. Scraped surface crystallizers. One type of scraped surface crystallizer is the Swenson-Walker
crystallizer, which consists of an open trough 0.6m wide with a semicircular bottom having a
cooling jacket outside. A slow-speed spiral agitator rotates and suspends the growing crystals on
turning. The blades pass close to the wall and break off any deposits of crystals on the cooled
wall. The product generally has a somewhat wide crystal-size distribution.

3. Double-pipe scraped surface crystallizer. Also called a votator, this type of crystallizer is used
in crystallizing ice cream and plasticizing margarine. Cooling water passes in the annular space.
An internal agitator is fitted with spring-loaded scrapers that wipe the wall and provide good
heat-transfer coefficients.
4. Circulating-liquid evaporator-crystallizer. Also called Oslo crystallizer. Here supersaturation
is reached by evaporation. The circulating liquid is drawn by the screw pump down inside the
tube side of the condensing stream heater. The heated liquid then flows into the vapor space,
where flash evaporation occurs, giving some supersaturation.The vapor leaving is condensed.
The supersaturated liquid flows down the downflow tube and then up through the bed of
fluidized and agitated crystals, which are growing in size. The leaving saturated liquid then goes
back as a recycle stream to the heater, where it is joined by the entering fluid. The larger crystals
settle out and slurry of crystals and mother liquid is withdrawn as a product.

5. Circulating-magma vacuum crystallizer. The magma or suspension of crystals is circulated out

of the main body through a circulating pipe by a screw pump. The magma flows though a heater,
where its temperature is raised 2-6 K. The heated liquor then mixes with body slurry and boiling
occurs at the liquid surface. This causes supersaturation in the swirling liquid near the surface,
which deposits in the swirling suspended crystals until they leave again via the circulating pipe.
The vapors leave through the top. A steam-jet ejector provides vacuum.

Freeze drying

Freeze drying (also known as lyophilization or cryodessication) is a dehydration process

typically used to preserve a perishable material or make the material more convenient for
transport. Freeze drying works by freezing the material and then reducing the surrounding
pressure and adding enough heat to allow the frozen water in the material to sublime directly
from the solid phase to gas.

Contents

 1 Freeze drying protectants

 2 The freeze-drying process
o 2.1 Freezing
o 2.2 Primary drying
o 2.3 Secondary drying
 3 Properties of freeze-dried products
 4 Applications of freeze-drying
o 4.1 Pharmaceutical and biotechnology
o 4.2 Food industry
o 4.3 Technological industry
o 4.4 Other uses
 5 Freeze-drying equipment
 6 See also
 7 References

 8 External links

Freeze drying protectants

Similar to cryoprotectants, some molecules protect freeze dried material. Known as
lyoprotectants, these molecules are typically polyhydroxy compounds such as sugars (mono-, di-,
and polysaccharides), polyalcohols, and their derivatives. Trehalose and sucrose are natural
lyoprotectants. Trehalose is produced by a variety of plant, fungi, and invertebrate animals that
remain in a state of suspended animation during periods of drought (also known as
anhydrobiosis).

The freeze-drying process

There are three stages in the complete freeze-drying process: freezing, primary drying, and
secondary drying.

Freezing

The freezing process consists of freezing the material. In a lab, this is often done by placing the
material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is
cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger-scale,
freezing is usually done using a freeze-drying machine. In this step, it is important to cool the
material below its eutectic point, the lowest temperature at which the solid and liquid phase of
the material can coexist. This ensures that sublimation rather than melting will occur in the
following steps. Larger crystals are easier to freeze dry. To produce larger crystals the product
should be frozen slowly or can be cycled up and down in temperature. This cycling process is
called annealing. However, in the case of food, or objects with formerly living cells, large ice
crystals will break the cell walls. As discovered by Clarence Birdseye, when food is frozen at
−40 °C to −45 °C or below, then it tastes better. Usually, the freezing temperatures are between
−50 °C and −80 °C. The freezing phase is the most critical in the whole freeze drying process,
because the product can be spoiled if badly done.

Amorphous (glassy) materials do not have a eutectic point, but do have a critical point, below
which the product must be maintained to prevent melt-back or collapse during primary and
secondary drying.

Large objects take a few months to freeze dry. Primary drying

During the primary drying phase the pressure is lowered (to the range of a few millibars) and
enough heat is supplied to the material for the water to sublimate. The amount of heat necessary
can be calculated using the sublimating molecules’ latent heat of sublimation. In this initial
drying phase about 95% of the water in the material is sublimated. This phase may be slow (can
be several days in the industry), because if too much heat is added the material’s structure could
be altered.

In this phase, pressure is controlled through the application of partial vacuum. The vacuum
speeds sublimation making it useful as a deliberate drying process. Furthermore, a cold
condenser chamber and/or condenser plates provide a surface(s) for the water vapour to re-
solidify on. This condenser plays no role in keeping the material frozen; rather, it prevents water
vapor from reaching the vacuum pump, which could degrade the pump's performance.
Condenser temperatures are typically below −50 °C (−60 °F).

It's important to note that in this range of pressure, the heat is mainly brought by conduction or
radiation, the convection effect can be considered as insignificant.

Secondary drying

The secondary drying phase aims to sublimate the water molecules that are adsorbed during the
freezing process, since the mobile water molecules were sublimated in the primary drying phase.
This part of the freeze-drying process is governed by the material’s adsorption isotherms. In this
phase, the temperature is raised higher than in the primary drying phase, and can even be above 0
°C, to break any physico-chemical interactions that have formed between the water molecules
and the frozen material. Usually the pressure is also lowered in this stage to encourage
sublimation (typically in the range of microbars, or fractions of a pascal). However, there are
products that benefit from increased pressure as well.

After the freeze drying process is complete, the vacuum is usually broken with an inert gas, such
as nitrogen, before the material is sealed.

At the end of the operation, the final residual humidity in the product is around 1% to 4%, which
is extremely low.

Properties of freeze-dried products

If a freeze-dried substance is sealed to prevent the reabsorption of moisture, the substance may
be stored at room temperature without refrigeration, and be protected against spoilage for many
years. Preservation is possible because the greatly reduced water content inhibits the action of
microorganisms and enzymes that would normally spoil or degrade the substance.

Freeze drying also causes less damage to the substance than other dehydration methods using
higher temperatures. Freeze drying does not usually cause shrinkage or toughening of the
material being dried. In addition, flavours and smells generally remain unchanged, making the
process popular for preserving food. Unfortunately, water is not the only chemical capable of
sublimation and the loss of other volatile compounds such as acetic acid (responsible for the
smell of vinegar) and alcohols can yield undesirable results.

Freeze-dried products can be rehydrated (reconstituted) much more quickly and easily because
the process leaves microscopic pores. The pores are created by the ice crystals that sublimate,
leaving gaps or pores in its place. This is especially important when it comes to pharmaceutical
uses. Lyophilization can also be used to increase the shelf life of some pharmaceuticals for many
years.

Applications of freeze-drying
Pharmaceutical and biotechnology

Pharmaceutical companies often use freeze drying to increase the shelf life of products, such as
vaccines and other injectables. By removing the water from the material and sealing the material
in a vial, the material can be easily stored, shipped and later reconstituted to its original form for
injection.

Food industry

http://en.wikipedia.org/wiki/Image:SpaceIceCream.jpg
A package of Freeze-dried ice cream, sold as a novelty item.

Freeze drying is used to preserve food and make it very lightweight. The process has been
popularized in the forms of freeze-dried ice cream, an example of astronaut food. It is also
popular and convenient for hikers because the reduced weight allows them to carry more food
and reconstitute it with available water. Instant coffee is sometimes freeze dried, despite high
costs of freeze dryers. The coffee is often dried by vaporization in a hot air flow, or by projection
on hot metallic plates. Freeze dried fruit is used in some breakfast cereal. However, the freeze-
drying process is used more commonly in the pharmaceutical industry.

Technological industry

In chemical synthesis, products are often lyophilized to make them more stable, or easier to
dissolve in water for subsequent use.

In bioseparations, freeze drying can also be used as a late-stage purification procedure, because it
can effectively remove solvents. Furthermore, it is capable of concentrating substances with low
molecular weights that are too small to be removed by a filtration membrane.

Freeze-drying is a relatively expensive process. The equipment is about three times as expensive
as the equipment used for other separation processes, and the high energy demands lead to high
energy costs. Furthermore, freeze drying also has a long process time, because the addition of too
much heat to the material can cause melting or structural deformations. Therefore, freeze drying
is often reserved for materials that are heat-sensitive, such as proteins, enzymes, microorganisms,
and blood plasma. The low operating temperature of the process leads to minimal damage of
these heat-sensitive products.

Other uses

Recently, some taxidermists have begun using freeze drying to preserve animals, such as pets.

Organizations such as the Document Conservation Laboratory at the United States National
Archives and Records Administration (NARA) have done studies on freeze drying as a recovery
method of water-damaged books and documents. While recovery is possible, restoration quality
depends on the material of the documents. If a document is made of a variety of materials, which
have different absorption properties, expansion will occur at a non-uniform rate which could lead
to deformations. Water can also cause mold to grow or make inks bleed. In these cases, freeze
drying may not be an effective restoration method.

Advanced ceramics processes sometimes use freeze drying to create a formable powder from a
sprayed slurry mist. Freeze drying creates softer particles with a more homogenous chemical
composition than traditional hot spray drying, but it is also more expensive.

In high altitude environments, the low temperatures and pressures can sometimes produce
natural mummies by a process of freeze-drying.

Freeze-drying equipment

http://en.wikipedia.org/wiki/Image:Benchtop_freeze_dryer.JPG
Benchtop manifold freeze dryer.

There are essentially three categories of freeze dryers: rotary evaporators, manifold freeze dryers,
and tray freeze dryers.

Rotary freeze dryers are usually used with liquid products, such as pharmaceutical solutions and
tissue extracts.
http://en.wikipedia.org/wiki/Image:Lyophilizer_tray_type.jpg
Unloading trays of freeze-dried material from a small cabinet-type freeze dryer

Manifold freeze dryers are usually used when drying a large amount of small containers and the
product will be used in a short period of time. A manifold dryer will dry the product to less than
5% moisture content. Without heat only primary drying (removal of the unbound water) can be
achieved. A heater needs to be added for secondary drying, which will remove the bound water
and will produce a lower moisture content.

http://en.wikipedia.org/wiki/Image:Production_freeze_dryer.JPG
Production freeze dryer

Tray freeze dryers are more sophisticated and are used to dry a variety of materials. A tray freeze
dryer is used to produce the driest product for long-term storage. A tray freeze dryer allows the
product to be frozen in place and performs both primary (unbound water removal) and secondary
(bound water removal) freeze drying, thus producing the driest possible end-product. Tray freeze
dryers can dry product in bulk or in vials. When drying in vials, the freeze dryer is supplied with
a stoppering mechanism that allows a stopper to be pressed into place sealing the vial before it is
exposed to the atmosphere. This is used for long term storage, such as vaccines.