Downstream Process Technology – BT 402

Lecturer: Dr. Maulishree Agrahari

SAP: Thirtha Poovaiah

Lecture Number 21-22: Liquid liquid extraction and Distillation

Lecture Number 36-37: Precipitaion methods with salts: Salting in and salting
out, ammonium sulphate and PEG
Lecture Number 42-43: Adsorptive chromatography: TLC, PC, Normal Phase
and GC
Lecture Number 44: HPLC
Lecture Number 45: Gel permeation chromatography
Lecture Number 46: Ion exchange chromatography
Lecture Number 47: Membrane Chromatography
Lecture Number 48: Electro- chromatography
Lecture Number 49: Electrophoretic Separation: Native and SDS PAGE
Lecture Number 50: Capillary Electrophoresis
Lecture Number 51: Dialysis
Lecture Number 52: Crystallization

Liquid Liquid Extraction (LLE)

Principle

Liquid liquid extraction is a separating process that is based on the distribution of

one or more components between two immiscible liquids. Generally, one of the
liquid phases is water and the other an organic solvent. Liquid liquid extraction is
also known as solvent extraction, is especially suited for the processing of large
capacities of feed. For this reason, LLE is frequently used in oil refining industry.

The key principle underlying in LLE is often that the solubility differs significantly
between the liquid, producing thermodynamic driving force for transfer from one
phase to another.

Feed phase contains a component, i, which is to be removed. Addition of a

second phase (solvent phase) which is immiscible with feed phase but
component i is soluble in both phases. Some of component i (solute) is
transferred from the feed phase to the solvent phase. After extraction the feed
and solvent phases are called the raffinate (R) and extract (E) phases
respectively.
Normally one of the two phases is an organic phase while the other is an
aqueous phase. Under equilibrium conditions the distribution of solute i over the
two phases is determined by the distribution law. After the extraction the two
phases can be separated because of their immiscibility. Component i is then
separated from the extract phase by a technique such as distillation and the
solvent is regenerated. Further extractions may be carried out to remove more
component i. Liquid liquid extraction can also be used to remove a component
from an organic phase by adding an aqueous phase.

The success of LLE depends upon the difference in solubility of a compound in

various solvents. For a given compound, solubility differences between solvents
is quantified as the "distribution coefficient"

When a compound is shaken in a separatory funnel with two immiscible solvents,

the compound will distribute itself between the two solvents. Normally one
solvent is water and the other solvent is a water-immiscible organic solvent.

At a certain temperature, the ratio of concentrations of a solute in each

solvent is always constant. And this ratio is called the distribution
coefficient, K.

when solvent1 and solvent2 are immiscible liquids (By convention the organic
solvent is (1) and water is (2) )

Extraction Procedure
 Shake the separatory funnel vigorously.

Now, shake the funnel vigorously for a few seconds. Release the pressure, then
again shake vigorously. About 30 sec total vigorous shaking is usually sufficient
to allow solutes to come to equilibrium between the two solvents.
Solvent Selection
For the selection of a suitable solvent, one has to consider not only the
extraction selectivity, but also the ease of handling and regeneration and
production cost. Some of the factors are:

Factors to be considered:

• Selectivity
• Distribution coefficient
• Insolubility of solvent
• Recoverability of solute from solvent
• Density difference between liquid phases
• Interfacial tension
• Chemical reactivity
• Cost
• Viscosity, vapour pressure
• Flammability, toxicity
Distillation

Distillation is a method of separating chemical substances based on differences

in their volatilities in a boiling liquid mixture. Distillation usually forms part of a
larger chemical process, and is thus referred to as a unit operation. Distillation is
defined as the process of heating a liquid until it boils. Capturing and cooling the
resultant hot vapours and collecting the condensed vapours

Types of distillation include simple distillation, fractional distillation and

destructive distillation (usually, a material is heated so that it decomposes into
compounds for collection).

Simple distillation involves the evaporation of a mixture of liquids followed by

the condensation of the resulting vapour. The purpose of distillation is to
separate the different types of liquids, by using the differences in their boiling
points. That is, low boiling point liquids become more concentrated in
the condensate, while high boiling point liquids get left behind in the original
vessel.

Fractional Distillation is a method of obtaining the performance of a multiple

simple distillations within a single pass process. This method is adopted to
separate two or more miscible liquids whose boiling points are quite different.
The liquid with the lower boiling point is heated first so that it evaporates and
after the vapours pass from through the condenser, they fall into a container as
liquid. After this the liquid with the next higher boiling point evaporates and
again is collected in a separate container. Gradually, all the solutions are
separately collected in the same manner. That is different volatile 'fractions' are
collected as they are produced

This method is adopted to obtain both the solute and the solvent from a solution.
Here the solute is a soluble solid. Distillation actually involves both evaporation
as well as condensation. Let us take the example of a salt solution. The solution
is taken in a distillation flask and heated so that the solvent slowly starts
evaporating. The distillation flask is attached to a Leibig’s condenser which has a
lower tube for the inlet of cool water and an upper tube for the outlet of water.
The circulation of cold water in the condenser helps to cool the vapours from the
solution and they form the distillate which is collected in a separate container
attached to the condenser. The complete solution is not allowed to evaporate in
this case but when a little is left, it is heated separately in an open container
leaving it to evaporate completely. The remaining residue is the solute i.e. salt
and the distillate collected is the solvent i.e. water.

Dalton's law states that the total vapour pressure is the sum of the vapour
pressures of each individual component in the mixture. When a multi-component
system is heated, the vapour pressure of each component will rise, thus causing
the total vapour pressure to rise. When the total vapour pressure reaches the
ambient pressure, boiling occurs and liquid turns to gas throughout the bulk of
the solution. Note that a given mixture has one boiling point, when the
components are mutually soluble.
Repeating the distillation process on the collected liquid to improve the purity of
the product is called double distillation. Although the term is most commonly
applied to liquids, the reverse process can be used to separate gases by
liquefying components using changes in temperature and/or pressure.

Applications: The application of distillation can roughly be divided in four

groups: laboratory scale, industrial distillation, distillation of herbs for perfumery
and medicinal (herbal distillate) and food processing. The latter two are distinct
from the former two, in that in the distillation is not used as a true purification
method, but more to transfer all volatiles from the source materials to the
distillate.

• Distillation is used for many commercial processes, such as production of

gasoline, distilled water, xylene, alcohol, paraffin, kerosene, and many
other liquids.
• It is used to separate crude oil into more fractions for specific uses such as
transport, power generation and heating.
• Water is distilled to remove impurities, such as salt from sea water.
• Air is distilled to separate its components - notably oxygen, nitrogen and
argon - for industrial use.
• Distillation of fermented solutions has been used since ancient times to
produce distilled beverages with a higher alcohol content.
Precipitation Methods with Salts: Salting In and Salting Out

The solubility of protein depends on, among other things, the salt concentration
in the solution. At low concentrations, the presence of salt stabilizes the various
charged groups on a protein molecule, thus attracting protein into the solution
and enhancing the solubility of protein. This is commonly known as salting-in.

However, as the salt concentration is increased, a point of maximum protein

solubility is usually reached. Further increase in the salt concentration implies
that there is less and less water available to solubilize protein. Finally, protein
starts to precipitate when there are not sufficient water molecules to interact
with protein molecules. This phenomenon of protein precipitation in the presence
of excess salt is known as salting-out.

Many types of salts have been employed to effect protein separation and
purification through salting-out. Of these salts, ammonium sulphate has been the
most widely used chemical because it has high solubility and is relatively
inexpensive. Because enzymes are proteins, enzyme purification can be carried
out by following the same set of procedures as those for protein, except that
some attention must be paid to the consideration of permanent loss of activity
due to denaturation under adverse conditions.

There are two major salting-out procedures. In the first procedure, either a
saturated salt solution or powdered salt crystals are slowly added to the protein
mixture to bring up the salt concentration of the mixture. For example, the salt
concentration reaches 25% saturation when 1 ml of the saturated salt solution is
added to 3 ml of the salt-free protein solution; 50% for 3 ml added; 75% for 9 ml
added; and so on. The precipitated protein is collected and categorized
according to the concentration of the salt solution at which it is formed. This
partial collection of the separated product is called fractionation. For example,
the fraction of the precipitated protein collected between 20 and 21% of salt
saturation is commonly referred to as the 20-21% fraction. The protein fractions
collected during the earlier stages of salt addition are less soluble in the salt
solution than the fractions collected later.

Whereas the first method just described uses increasing salt concentrations, the
following alternative method uses decreasing salt concentrations. In this
alternative method, as much protein as possible is first precipitated with a
concentrated salt solution. Then a series of cold (near 0ºC) ammonium sulphate
solutions of decreasing concentrations are employed to extract selectively the
protein components that are the most soluble at higher ammonium sulphate
concentrations. The extracted protein is recrystallized and thus recovered by
gradually warming the cold solution to room temperature. This method has the
added advantages that the extraction media may be buffered or stabilizing
agents be added to retain the maximum enzyme activity. The efficiency of
recovery typically ranges from 30 to 90%, depending on the protein. The
recrystallization of protein upon transferring the extract to room temperature
may occur immediately or may sometimes take many hours. Nevertheless, very
rarely does recrystallization fail to occur. The presence of fine crystals in a
solution can be visually detected from the turbidity.
Chromatography
Chromatography (from Greek "color" and graphein "to write") is the collective
term for a set 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
based on differential partitioning between the mobile and stationary phases.
Subtle differences in a compound's partition coefficient result in differential
retention on the stationary phase and thus changing the separation.

Chromatography separates the components of a mixture by their distinctive

attraction to the mobile phase and the stationary phase.
• Compound is placed on stationary phase
• Mobile phase passes through the stationary phase
• Mobile phase solubilizes the components
• Mobile phase carries the individual components a certain distance through
the stationary phase, depending on their attraction to both of the phases

Chromatography may be preparative or analytical. The purpose of preparative

chromatography is to separate the components of a mixture for further use (and
is thus a form of purification). Analytical chromatography is done normally with
smaller amounts of material and is for measuring the relative proportions of
analytes in a mixture.

Chromatography is used to:

• Analyze – examine a mixture, its components, and their relations to one
another
• Identify – determine the identity of a mixture or components based on
known components
• Purify – separate components in order to isolate one of interest for
further study
• Quantify – determine the amount of the a mixture and/or the
components present in the sample

Types of Chromatography
• Liquid Chromatography – separates liquid samples with a
liquid solvent (mobile phase) and a column composed of solid
beads (stationary phase)
 • Gas Chromatography – separates vaporized samples with a carrier gas
(mobile phase) and a column composed of a liquid or of solid beads
(stationary phase)

• Paper Chromatography – separates dried liquid samples with a liquid

solvent (mobile phase) and a paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid samples with a liquid

solvent (mobile phase) and a glass plate covered with a thin layer of
alumina or silica gel (stationary phase)

Applications
• Pharmaceutical Company – determine amount of each chemical found in
new product
• Hospital – detect blood or alcohol levels in a patient’s blood stream
• Law Enforcement – to compare a sample found at a crime scene to
samples from suspects
• Environmental Agency – determine the level of pollutants in the water
supply
Manufacturing Plant – to purify a chemical needed to make a
product

Paper Chromatography
Paper chromatography is one of the earliest known molecular separation
techniques. Paper chromatography is an analytical chemistry technique for
separating and identifying mixtures that are or can be coloured,
especially pigments. This can also be used in secondary or primary colours in ink
experiments. This method has been largely replaced by thin layer
chromatography, however it is still a powerful teaching tool

Principle
• Capillary Action – the movement of liquid within the spaces of a porous
material due to the forces of adhesion, cohesion, and surface tension. The
liquid is able to move up the filter paper because its attraction to itself is
stronger than the force of gravity.

• Solubility – the degree to which a material (solute) dissolves into a solvent.

Solutes dissolve into solvents that have similar properties. (Like dissolves
like) This allows different solutes to be separated by different
combinations of solvents.

Separation of components depends on both their solubility in the mobile phase

and their differential affinity to the mobile phase and the stationary phase.

Thin Layer Chromatography

Chromatography is a sophisticated method of separating mixtures of two or

more compounds. The separation is accomplished by the distribution of the
mixture between two phases: one that is stationary and one that is moving.
Chromatography works on the principle that different compounds will have
different solubilities and adsorption to the two phases between which they are to
be partitioned.

Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two

phases are a solid (stationary phase) and a liquid (moving phase). Thin layer
chromatography is done exactly as it says - using a thin, uniform layer of silica
gel or alumina coated onto a piece of glass, metal or rigid plastic. Solids most
commonly used in chromatography are silica gel (SiO 2 x H2O) and alumina
(Al2O3 x H2O). Both of these adsorbents are polar, but alumina is more so. Silica is
also acidic. Alumina is available in neutral, basic, or acidic forms.

Thin Layer Chromatography (TLC) is a sensitive, fast, simple and inexpensive

analytical technique. It is a micro technique; as little as 10 -9g of material can be
detected, although the sample size is from 1 to 100x10-6 g.

TLC involves spotting the sample to be analyzed near one end of a sheet of glass
or plastic that is coated with a thin layer of an adsorbent. The sheet, which can
be the size of a microscope slide, is placed on end in a covered jar containing a
shallow layer of solvent.

When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in
a covered beaker. It is important that the solvent level is below the line with the
spot on it.

As the solvent slowly travels up the plate, the different components of the dye
mixture travel at different rates and the mixture is separated into different
coloured spots.
As the solvent rises by capillary action up through the adsorbent, differential
partitioning occurs between the components of the mixture dissolved in the
solvent the stationary adsorbent phase. The more strongly a given component of
a mixture is adsorbed onto the stationary phase, the less time it will spend in the
mobile phase and the more slowly it will migrate up the plate

When the solvent front gets close to the top of the plate, the plate is removed
from the beaker and the position of the solvent is marked with another line
before it has a chance to evaporate.These measurements are then taken is order
to calculate the Rf value. The Rf value, or retention factor, of each spot can be
determined by dividing the distance traveled by the product by the total distance
travelled by the solvent (the solvent front). These values depend on the solvent
used, and the type of TLC plate, and are not physical constants

The Rf value for each dye is then worked out using the formula:

For example, if the red component travelled 1.7 cm from the base line while the
solvent had travelled 5.0 cm, then the Rf value for the red dye is:

Visualization
The spots can be visualized using a non destructive method such as exposing
the plate to UV light. The previously invisible spots are visible under UV light.

In some cases, it may be possible to make the spots visible by reacting them
with something which produces a coloured product. A good example of this is in
chromatograms produced from amino acid mixtures.

The chromatogram is allowed to dry and is then sprayed with a solution

of ninhydrin. Ninhydrin reacts with amino acids to give coloured compounds,
mainly brown or purple.

In another method, the chromatogram is again allowed to dry and then placed in
an enclosed container (such as another beaker covered with a watch glass) along
with a few iodine crystals.
The iodine vapour in the container may either react with the spots on the
chromatogram, or simply stick more to the spots than to the rest of the plate.
Either way, the substances you are interested in may show up as brownish spots.

The following are some common uses of Thin-Layer Chromatography:

1. To determine the number of components in a mixture.

2. To determine the identity of two substances.
3. To monitor the progress of a reaction.
4. To determine the effectiveness of a purification.
5. Used in ink identification
6. Determination of organophosphorus compounds in pesticide

Gas Chromatography
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 based on differential partitioning between the mobile and stationary
phases.

Gas chromatography (GC) is a powerful and widely used tool for the separation,
and identification of components in a mixture. GC also known as Vapor-Phase
Chromatography (VPC) and Gas-Liquid Partition Chromatography (GLPC). By
separating the sample into individual components, it is easier to identify
(qualitate) and measure the amount (quantitate) of the various sample
components

Principle of GC
Gas chromatography (GC) is based on partition equilibrium of analyte between a
solid stationary phase (often a liquid silicone-based material) and a mobile gas
(most often Helium). Partitioning process carried out between Moving Gas Phase
and Stationary Liquid Phase

To be suitable for GC analysis, a compound must have sufficient volatility and

thermal stability. If all or some of a compound or molecules are in the gas or
vapor phase at 400-450°C or below, and they do not decompose at these
temperatures, the compound can probably be analyzed by GC.
Working
One of the gases (called the carrier gas) flows into the injector, through the
column and then into the detector. A sample is introduced into the injector
usually with a syringe or an exterior sampling device. The injector is usually
heated to 150-250°C which causes the volatile sample solutes to vaporize. The
vaporized solutes are transported into the column by the carrier gas. The column
is maintained in a temperature controlled oven. The solutes travel through the
column at a rate primarily determined by their physical properties, and the
temperature and composition of the column. The various solutes travel through
the column at different rates. The fastest moving solute exits (elutes) the column
first then is followed by the remaining solutes in corresponding order. As each
solute elutes from the column, it enters the heated detector. An electronic signal
is generated upon interaction of the solute with the detector. The size of the
signal is recorded by a data system and is plotted against elapsed time to
produce a chromatogram.

The ideal chromatogram has closely spaced peaks with no overlap of the peaks.
Any peaks that overlap are called coeluting. The time and size of a peak are
important in that they are used to identify and measure the amount of the
compound in the sample. The size of the resulting peak corresponds to the
amount of the compound in the sample. A larger peak is obtained as the
concentration of the corresponding compound increases. If the column and all of
operating conditions are kept the same, a given compound always travels
through the column at the same rate.

Gas Chromatogram for methyl esters

Retention time
Thus, a compound can be identified by the time required for it to travel through
the column (called the retention time). Retention time (tR) is the time it takes a
solute to travel through the column. The retention time is assigned to the
corresponding solute peak. The retention time is a measure of the amount of
time a solute spends in a column. It is the sum of the time spent in the stationary
phase and the mobile phase.

For a given set of constant conditions (carrier gas, flow rate of carrier gas,
column temperature, column length, liquid phase, injection port temperature),
the retention time of any compound is always constant. Retention Time is similar
to the “Retardation Factor, Rf” in Thin Layer Chromatography.

The identity of a compound cannot be determined solely by its retention time. A

known amount of an authentic, pure sample of the compound has to be analyzed
and its retention time and peak size determined. This value can be compared to
the results from an unknown sample to determine whether the target compound
is present (by comparing retention times) and its amount (by comparing peak
sizes). If any of the peaks overlap, accurate measurement of these peaks is not
possible. If two peaks have the same retention time, accurate identification is not
possible. Thus, it is desirable to have no peak overlap or co-elution

Response

Mixtureof knowncompounds

Octane
Decane
1.6min=RT

Hexane

GCRetentionTimeonCarbowax-20(min)

Response

UnknowncompoundmaybeHexane

1.6min=RT

RetentionTimeonCarbowax-20(min)

Applications
 • Separation and analysis of organic compounds
• Testing purity of compounds
• Determine relative amounts of components in mixture
• Compound identification
• Isolation of pure compounds

Advantages
1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - µl

Disadvantage
1. Material has to be volatilized at 250 C without decomposition

High Performance Liquid Chromatography

High-performance liquid chromatography (or high-pressure liquid
chromatography, HPLC) is a chromatographic technique that can separate a
mixture of compounds and is used in biochemistry and analytical chemistry to
identify, quantify and purify the individual components of the mixture.
HPLC typically utilizes different types of stationary phases, a pump that moves
the mobile phase(s) and analyte through the column, and a detector that
provides a characteristic retention time for the analyte. The detector may also
provide other characteristic information (i.e. UV/Vis spectroscopic data for
analyte if so equipped).

Analyte retention time varies depending on:

• the strength of its interactions with the stationary phase,
• the ratio/composition of solvent(s) used, and
• the flow rate of the mobile phase.

High performance liquid chromatography is basically a highly improved form of

column chromatography. Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high pressures of up to 400
atmospheres. That makes it much faster.

It also allows the use of a very small sized particle for the column packing
material which gives a much greater surface area for interactions between the
stationary phase and the molecules flowing past it. This allows a much better
separation of the components of the mixture.

There are two variants in use in HPLC depending on the relative polarity of the
solvent and the stationary phase.

Normal phase HPLC

The column is filled with tiny silica particles, and the solvent is non-polar -
hexane, for example. A typical column has an internal diameter of 4.6 mm (and
may be less than that), and a length of 150 to 250 mm.

Polar compounds in the mixture being passed through the column will stick
longer to the polar silica than non-polar compounds will. The non-polar ones will
therefore pass more quickly through the column.
Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to make it
non-polar by attaching long hydrocarbon chains to its surface - typically with
either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a
mixture of water and an alcohol such as methanol.

In this case, there will be a strong attraction between the polar solvent and polar
molecules in the mixture being passed through the column. There won't be as
much attraction between the hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the solution. Polar molecules in the
mixture will therefore spend most of their time moving with the solvent.

Non-polar compounds in the mixture will tend to form attractions with the
hydrocarbon groups because of van der Waals dispersion forces. They will also
be less soluble in the solvent because of the need to break hydrogen bonds as
they squeeze in between the water or methanol molecules, for example. They
therefore spend less time in solution in the solvent and this will slow them down
on their way through the column.That means that now it is the polar molecules
that will travel through the column more quickly.

Reversed phase HPLC is the most commonly used form of HPLC.

HPLC Sample Injectors

The function of the injector is to place the sample into the high-pressure flow in
as narrow volume as possible so that the sample enters the column as a
homogeneous, low-volume plug. To minimize spreading of the injected volume
during transport to the column, the shortest possible length of tubing should be
used from the injector to the column.
When an injection is started, an air actuator rotates the valve: solvent goes
directly to the column; and the injector needle is connected to the syringe. The
air pressure lifts the needle and the vial is moved into position beneath the
needle. Then, the needle is lowered to the vial.

HPLC Column
The column is one of the most important components of the HPLC
chromatograph because the separation of the sample components is achieved
when those components pass through the column. The High performance liquid
chromatography apparatus is made out of stainless steel tubes with a diameter
of 3 to 5mm and a length ranging from 10 to 30cm.

Normally, columns are filled with silica gel because its particle shape, surface
properties, and pore structure help to get a good separation. Silica is wetted by
nearly every potential mobile phase, is inert to most compounds and has a high
surface activity which can be modified easily with water and other agents. Silica
can be used to separate a wide variety of chemical compounds, and its
chromatographic behavior is generally predictable and reproducible.

Type of Detectors
• Absorbance (UV with Filters, UV with Monochromators)
• IR Absorbance

Retention time

The time taken for a particular compound to travel through the column to the
detector is known as its retention time. This time is measured from the time at
which the sample is injected to the point at which the display shows a maximum
peak height for that compound.

Different compounds have different retention times. For a particular compound,

the retention time will vary depending on:

• the pressure used (because that affects the flow rate of the solvent)
• the nature of the stationary phase (not only what material it is made of,
but also particle size)
• the exact composition of the solvent
• the temperature of the column

Applications
• HPLC techniques are also used extensively in the isolation and
characterization of novel proteins that will become increasingly important
in the post-genomic age
• This technique is used for chemistry and biochemistry research analyzing
complex mixtures, purifying chemical compounds, developing processes
for synthesizing chemical compounds, isolating natural products, or
predicting physical properties. It is also used in quality control to ensure
the purity of raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product stability and
monitor degradation.
• In addition, it is used for analyzing air and water pollutants, for monitoring
materials that may jeopardize occupational safety or health, and for
monitoring pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug products, for
identifying confiscated narcotics or to check for adherence to label claims.
Gel Permeation Chromatography

Gel permeation chromatography (GPC) is a term used for when the

separation technique size exclusion chromatography (SEC),that
separates analytes on the basis of size.

Gel permeation chromatography (GPC) is a technique used to separate

molecules based on size differences. It is also referred to as gel filtration
chromatography, or size exclusion chromatography. Molecular separation occurs
in the GPC column. Inside the column there are two phases:

a) The stationary phase consists of an inert gel of porous beads-- so-called

because it does not move in the column

b) A mobile phase, which is the eluent or liquid which is run through the column.

Gel Permeation Chromatography (GPC) is the method most widely used today for
determining the molecular size and molecular weight distribution of high
polymers. Its speed and relative ease of operation have made the determination
of molecular weight distribution a routine analysis in most cases.

Principle
GPC provides a physical means for separating molecules by their size in solution.
There is a distribution of pore sizes within the packing such that small molecules
can enter most of the pores and are therefore retained the longest, while larger
molecules enter fewer pores and are retained a shorter length of time. By proper
calibration of the columns, size (and in most cases molecular weight) can be
deduced from elution volume.

Sample Requirements
The sample may be either a soluble solid or liquid. At least 0.04 g of sample is
required; however, it is preferred that at least 0.2 g be supplied.

Applications
• GPC can be used to analyze soluble compounds and mixtures that fall in
the size range of about 10 to 5,000 Angstroms. This range covers high
 polymers of molecular weight over a million to low molecular weight
polymers additives, such as stabilizers and oils.
• High resolution, low porosity columns are available that can separate
oligomers and other organic compounds, which differ by as little as one
methyl group.
• By combining GPC and intrinsic viscosity data (taken in the same solvent
at the same temperature), a relative measure of long chain branching may
be obtained. Semi-preparative separations of mg (1-10) fractions can be
carried out.

Limitations
• The sample must be soluble in a solvent with which the columns are
compatible. Commonly used organic solvents are toluene,
tetrahydrofuran, trichlorobenzene, and chloroform.

Ion exchange chromatography

Adsorption chromatography depends upon interactions of different types

between solute molecules and ligands immobilized on a chromatography matrix.
The first type of interaction to be successfully employed for the separation of
macromolecules was that between charged solute molecules and oppositely
charged moieties covalently linked to a chromatography matrix. The technique
of ion exchange chromatography is based on this interaction.
.
Ion-exchange chromatography also called as ion chromatography is a process
that allows the separation of ions and polar molecules based on their charge. It
can be used for almost any kind of charged molecule including large proteins,
small nucleotides and amino acids. The solution to be injected is usually called
as ample, and the individually separated components are called analytes. It is
often used in protein purification, water analysis, and quality control. The
reasons for the success of ion exchange are its widespread applicability, its high
resolving power, its high capacity, and the simplicity and controllability of the
method

Separation is obtained since different substances have different degrees of

interaction with the ion exchanger due to differences in their charges, charge
densities and distribution of charge on their surfaces. These interactions can be
controlled by varying conditions such as ionic strength and pH. The differences in
charge properties of biological compounds are often considerable, and since ion
exchange chromatography is capable of separating species with very minor
differences in properties, e.g. two proteins differing by only one charged amino
acid, it is a very powerful separation technique.

In ion exchange chromatography one can choose whether to bind the substances
of interest and allow the contaminants to pass through the column, or to bind the
contaminants and allow the substance of interest to pass through. Generally, the
first method is more useful since it allows a greater degree of fractionation and
concentrates the substances of interest.

Principle
Ion exchange chromatography involves two primary steps, first the binding of a
protein to a charged resin and second the elution or displacement of the protein
from the charges of the resin. Critical to the former are the pH of the buffer used
to equilibrate and load the proteins onto the column. Factors that control the
elution are pH or ionic strength

An ion exchanger consists of an insoluble matrix to which charged groups have

been covalently bound. The charged groups are associated with mobile
counterions. These counter-ions can be reversibly exchanged with other ions of
the same charge without altering the matrix. The stationary phase surface
displays ionic functional groups (R-X) that interact with analyte ions of opposite
charge. It is possible to have both positively and negatively charged exchangers.
This type of chromatography is further subdivided into:

• Cation exchange chromatography

• Anion exchange chromatography

The ionic compound consisting of the cationic species M+ and the anionic
species B- can be retained by the stationary phase.

Cation exchange chromatography

Cation exchange chromatography retains positively charged cations because the
stationary phase displays a negatively charged functional group:

Anion exchange chromatography

Anion exchange chromatography retains anions using positively charged
functional group:

Functional groups used on ion exchangers.

Anion exchangers Functional group

1. Diethylaminoethyl (DEAE) -O-CH2-CH2-N+H(CH2CH3)2

2. Quaternary aminoethyl (QAE) -O-CH2-CH2-N+(C2H5)2-CH2-CHOH-CH3

3. Quaternary ammonium (Q) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+

(CH3)3
Cation exchangers Functional group

1. Carboxymethyl (CM) -O-CH2-COO

2. Sulphopropyl(SP) -O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3

3. Methyl sulphonate (S) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3

-

The matrix usually used is Sephadex, Sepharose, or Cellulose

Applications
• Ion exchange is widely used in the food & beverage, hydrometallurgical,
metals finishing, chemical & petrochemical, pharmaceutical, sugar &
sweeteners, ground & potable water, nuclear, softening & industrial water,
semiconductor, power, and a host of other industries.

• Most typical example of application is preparation of high purity water

for power engineering, electronic and nuclear industries;
i.e. polymeric ormineralic insoluble ion exchangers are widely used
for water softening, water purification, water decontamination etc.

• Ion exchange is a method widely used in household (laundry

detergents and water filters) to produce soft water. This is accomplished
by exchanging calcium Ca2+ and magnesium Mg2+ cations against Na+ or
H+ cations

• In biochemistry it is widely used to separate charged molecules such

as proteins. An important area of the application is extraction and
purification of biologically produced substances such as proteins (amino
acids) and DNA/RNA.

• Ion-exchange processes are used to separate and purify metals, including

separating uranium from plutonium and other actinides, including thorium,
and lanthanum, from each other and the other lanthanides.

• Ion exchangers are used in nuclear reprocessing and the treatment

of radioactive waste

Electrochromatography
Electrochromatography is a chemical separation technique in analytical
chemistry, biochemistry and molecular biology used to resolve and separate
mostly large biomolecules such as proteins. It is a combination of size exclusion
chromatography (gel filtration chromatography) and gel electrophoresis. These
separation mechanisms operate essentially in superposition along the length of a
gel filtration column to which an axial electric field gradient has been added. The
molecules are separated by size due to the gel filtration mechanism and
by electrophoretic mobility due to the gel electrophoresis mechanism.
Additionally there are secondary chromatographic solute retention mechanisms.
Capillary Electrophoresis
Capillary electrophoresis (CE), also known as capillary zone electrophoresis (CZE)
 used to separate ionic species by their charge and frictional forces.
 traditional electrophoresis, electrically charged analytes move in a
conductive liquid medium under the influence of an electric field
Introduced in the 1960s, the technique of capillary electrophoresis (CE) was
designed to separate species based on their size to charge ratio in the interior of
a small capillary filled with an electrolyte

Definition: The differential movement for migration of ions by attraction or

repulsion in an electric field.
Separation of components of a mixture using an electric field
• v=Eq/f
– v = velocity of molecule
– E = electric field
– q = net charge of molecule
– f = friction coefficient
Electrophoresis is used to determine the size, shape, and charge of a molecule.
Different forms of electrophoresis are used for each of these factors
independently or in combination.

Types of Electrophoresis
• Capillary
• Native Polyacrylimide Gel Electrophoresis (PAGE)
• SDS-PAGE
• Slab
• Paper

Principle
• Electrophoresis in a buffer filled, narrow-bore capillaries
• Each capillary is about 25-100 μm in internal diameter
• When a voltage is applied to the solution, the molecules move through the
solution towards the electrode of opposite charge
• Depending on the charge, the molecules move through at different speeds
– Separation is achieved
• A photocathode is then used to measure the absorbencies of the
molecules as they pass through the solution
• The absorbencies are analyzed by a computer and they are represented
graphically
Capillary electrophoresis Apparatus
Equipment
• Capillary tube
 Varied length but normally 25-50 cm
 Small bore and thickness of the silica play a role
 Using a smaller internal diameter and thicker walls help
prevent Joule Heating, heating due to voltage

Electrophoretic Mobility
– The movement of ions solely due to the electric field, potential
difference
– Cations migrate toward cathode
– Anions migrate toward anode
– Neutral molecules do not favor either

Application
• Analysis of carbohydrates
• Analysis of inorganic anions/metal ions
• DNA profiling
• Protein identification

Advantages
• Fast
• Small Sample
• Relatively inexpensive
• Automated
Disadvantages
• Cannot identify neutral species
• Cannot discern shape
Electrophoretic Separation

Electrophoresis
Electrophoresis is the motion of dispersed particles relative to a fluid under the
influence of a spatially uniform electric field.
Principle: These forces (electric and viscous drag) act in opposite directions and
due to the difference in magnitude of the forces, the particle moves. When the
forces are balanced the particle is stationary. This is the principle underlying the
technique of electrophoresis. Consider a particle which is being pulled forward by
the electric force (Eq, where E is the electric field and q is the charge), the other
force called the viscous drag (fv, f is frictional coefficient and v the velocity of the
molecule)

←fv Eq→

(1) Ftot = Fe + Ff = 0, where

(2) Fe = QE (the electric force) and

(3) Ff = -fv (viscous drag)

Native Gel Electrophoresis is a technique used mainly in protein

electrophoresis where the proteins are not denatured and therefore separated
based on their charge-to-mass ratio.

Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS.

While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on
their molecular mass, in native PAGE the mobility depends on both the protein's
charge and its hydrodynamic size.

The electric charge driving the electrophoresis is governed by the intrinsic

charge on the protein at the pH of the running buffer. This charge will, of course,
depend on the amino acid composition of the protein as well as post-translational
modifications such as addition of sialic acids.

Since the protein retains its folded conformation, its hydrodynamic size and
mobility on the gel will also vary with the nature of this conformation (higher
mobility for more compact conformations, lower for larger structures like
oligomers). If native PAGE is carried out near neutral pH to avoid acid or alkaline
denaturation, then it can be used to study conformation, self-association or
aggregation, and the binding of other proteins or compounds.

Thus native gels can be sensitive to any process that alters either the charge or
the conformation of a protein. This makes them excellent tools for detecting
things such as:
 changes in charge due to chemical degradation (e.g. deamidation)
unfolded, "molten globule", or other modified conformations
oligomers and aggregates (both covalent and non-covalent)
binding events (protein-protein or protein-ligand)

These properties, and their relatively high throughput, make native gels
excellent tools for analyzing accelerated stability samples, demonstrating
comparability of different lots or processes, or examining the effects of
excipients.

Another advantage of native gels is that it is possible to recover proteins in their

native state after the separation. Recovery of active biological materials may,
however, need to be done prior to any fixing or staining.

The two main types of native gels used in protein electrophoresis

are polyacrylamide gels and agarose gels.

Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins

ranging in size from 5 to 2,000 kiloDalton due to the uniform pore size provided
by the polyacrylamide gel. Pore size is controlled by controlling the
concentrations of acrylamide and bis-acrylamide powder used in creating a gel.
Care must be used when creating this type of gel, as acrylamide is a potent
neurotoxin in its liquid and powdered form.

Polyacrylamide gels are formed from the polymerization of two compounds,

acrylamide and N,N -methylene- bis-acrylamide (Bis, for short). Bis is a cross-
linking agent for the gels. The polymerization is initiated by the addition of
ammonium persulfate along with either -dimethyl amino-propionitrile (DMAP) or
N,N,N ,N ,- tetramethylethylenediamine (TEMED). The gels are neutral,
hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by
methylene groups.

The other type of gel used is agarose gel. Agarose gels can also be used to
separate native protein. They do not have a uniform pore size, but are optimal
for electrophoresis of proteins that are larger than 200 kDalton.

The molecules being separated (usually proteins) therefore differ in molecular

mass and intrinsic charge and experience different electrophoretic forces
dependent on the ratio of the two. Since the proteins remain in the native state
they may be visualised not only by general protein staining reagents but also by
specific enzyme-linked staining.

SDS PAGE
SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve
hydrophobic molecules but also has a negative charge (sulfATE) attached to it.
Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all
the proteins will be soluablized by the detergent, plus all the proteins will be
covered with many negative charges. So a protein that started out like the one
shown in the top part of figure 1 will be converted into the one shown in the
bottom part of figure 1. The end result has two important features: 1) all proteins
retain only their primary structure and 2) all proteins have a large negative
charge which means they will all migrate towards the positve pole when placed
in an electric field

If the proteins are denatured and put into an electric field, they will all move
towards the positive pole at the same rate, with no separation by size. So the
proteins are placed in an environment that will allow different sized proteins to
move at different rates. The environment provided is polyacrylamide, which is
a polymer of acrylamide monomers. When this polymer is formed, it turns into a
gel and we will use electricity to pull the proteins through the gel so the entire
process is called polyacrylamide gel electrophoresis (PAGE). A
polyacrylamide gel is not solid but is made of a laberynth of tunnels through a
meshwork of fibers (figure 2). Also small molecules can manuver through the
polyacrylamide forest faster than big molecules.

Regardless of the system, preparation requires casting two different layers of

acrylamide between glass plates. The lower layer (separating, or resolving, gel)
is responsible for actually separating polypeptides by size. The upper layer
(stacking gel) includes the sample wells. It is designed to sweep up proteins in a
sample between two moving boundaries so that they are compressed (stacked)
into micrometer thin layers when they reach the separating gel.
Set up

Gel analysis

Figure 5. Top view of an SDS PAGE

after the current has been on for a while
(positive pole at the bottom) and then
turned off. The gel (gray box) has five
numbered lanes where five different
samples of proteins (many copies of each
kind of protein) were applied to the
gel. (Lane 1, molecular weight standards
of known sizes; Lane 2, a mixture of
three proteins of different sizes
with a being the largest and c being the smallest protein; Lane 3, protein a by
itself; Lane 4, protein b by itself; Lane 5 protein c by itself.) Notice that each
group of the three proteins migrated the same distance in the gel whether they
were with other proteins (lane 2) or not (lanes 3-5). The molecular weight
standards are used to measure the relative sizes of the unknow proteins (a, b,
and c)

Dialysis

The concept of dialysis, is partition (separation) of substance between two

solutions by use of semipermeable membrane. Extracorporeal dialysis employs
the artificial kidney (dialyzer) as semipermeable membrane, while
Intracorporeal dialysis employs the peritoneal membrane.

Principle of Dialysis
Diffusion, Ultrafiltration and Osmosis are the basic physical principles of dialysis.
  Diffusion is the net directional movement of molecules occurring from a
solution of higher concentration to a solution of low concentration.
 Ultrafiltration is the movement of solvent across a semipermeable
membrane in response to a pressure difference applied across the
membrane.
 Osmosis the movement of the solvent (e.g. water) from the side of low
concentration to the side of higher concentration.

Working

 Blood flows by one side of a semi-permeable membrane, and a dialysate,

or special dialysis fluid, flows by the opposite side.
 A semipermeable membrane is a thin layer of material that contains
various sized holes, or pores.
 Smaller solutes and fluid pass through the membrane, but the membrane
blocks the passage of larger substances (for example, red blood cells,
large proteins)

Types of Dialysis
There are two primary types of dialysis:
 Hemodialysis
 Peritoneal dialysis
Hemodialysis
 During hemodialysis, blood flows out of the body and by one side of a
semi-permeable membrane.
 Dialysate, the fluid in a dialysis machine, flows by the opposite side of the
membrane.
 Undesired waste in the blood flows into the dialysate, while bicarbonate
(a needed solute that helps in pH balance) flows from the dialysate into
the blood.
 The clean blood is then returned to the body. Removing the harmful waste
and extra salt and fluids helps control blood pressure, pH balance, and
plasma volume, similar to the results of a functioning kidney

Schematic of the principles involved in hemodialysis. Blood flows from the body
into the hemodialysis machine through a filter called a dialyzer. The dialyzer
removes waste from the blood. This blood then re-enters the body.

Peritonial Dialysis
 Peritoneal Dialysis (PD) uses a filter to clean the blood and remove excess
fluids
 With PD, the blood is cleansed inside the body using one of the body’s own
membranes, the peritoneum, as the filter
  In peritoneal dialysis, a sterile solution containing glucose is run through a
tube into the peritoneal cavity, the abdominal body cavity around
the intestine, where the peritoneal membrane acts as a semipermeable
membrane.
 The peritoneal membrane or peritoneum is a layer of tissue containing
blood vessels that lines and surrounds the peritoneal, or abdominal, cavity
and the internal abdominal organs (stomach, spleen, liver, and
intestines).
 In PD, a soft tube called a catheter is used to fill the abdomen with a
cleansing liquid called dialysis solution.
 The peritoneum allows waste products and extra fluid to pass from the
blood into the dialysis solution. The solution contains a sugar called
dextrose that will pull wastes and extra fluid into the abdominal cavity.
These wastes and fluid then leave your body when the dialysis solution is
drained. The used solution, containing wastes and extra fluid, is then
thrown away.
 The process of draining and filling is called an exchange and takes about
30 to 40 minutes. The period the dialysis solution is in the abdomen is
called the dwell time. A typical schedule calls for four exchanges a day,
each with a dwell time of 4 to 6 hours. Different types of PD have different
schedules of daily exchanges.

 Peritoneal dialysis is less efficient than hemodialysis, but because it is

carried out for a longer period of time the net effect in terms of removal of
waste products and of salt and water are similar to hemodialysis.
 Peritoneal dialysis is carried out at home by the patient. It does free
patients from the routine of having to go to a dialysis clinic on a fixed
schedule multiple times per week, and it can be done while travelling with
a minimum of specialized equipment.

Types of PD
 One form of PD, continuous ambulatory peritoneal dialysis (CAPD), doesn’t
require a machine. As the word ambulatory suggests, the patient can walk
around with the dialysis solution in his abdomen.
 Another form of PD, continuous cycler-assisted peritoneal dialysis (CCPD),
requires a machine called a cycler to fill and drain your abdomen, usually
when the patient is asleep. CCPD is also sometimes called automated
peritoneal dialysis (APD).
Crystallization
A crystal is a three-dimensional, periodic assembly of individual molecules that
align themselves in a repeating series of "unit cells". .This is true for small
molecule (e.g. salt) crystals and large molecule (protein) crystals.

.Crystallography (from the Greek words crystallon = cold drop / frozen drop, and
graphein = write) is the experimental science of determining the arrangement of
atoms in solids.

Why do we need to crystallize proteins?

• To determine a protein structure
• To understand the protein’s binding to a ligand / substrate / drug
• To understand the protein’s enzymatic mechanism

Method: X-ray crystallography

• No limits in size, whole viruses and ribosomes have been analyzed crystals
serve to amplify the signal (one crystal contains 109 to 1013 molecules)
• Used to determine the exact position of atoms of a protein
• Used for co-crystallization with ligands
• 80% of all protein structures are determined with X-ray (rest using NMR)

.In order to form a highly ordered, homogeneous crystal lattice, proteins have to
be

• Pure = not containing any contaminations

• Correctly folded
• Homogeneous = not containing any truncated proteins, isomorphs,
incorrectly or differently folded molecules
• Proteins are flexible, have multiple conformations, but in the crystal, you
have typically only one conformation -> so everything that is flexible will
make crystallization difficult
• Not aggregated
• When a ligand is bound, it should be added in excess so that all protein
molecules have a ligand bound to them
• If small tags are used for purification, they need to stay attached or
removed completely but the protein solution should not contain a mixture

Methods to evaluate protein before crystallization

SDS-PAGE: overall estimation of protein size and purity

Can show artifacts for very basic or acidic, glycosylated and membrane proteins
Resolution to several kDaltons, e.g. difference of 6 histidines (removal of tag)
can be detected, not precise but cheap

Size-exclusion chromatography: Size, Purity, Homogeneity

Precision depending on resolution of column (e.g. 50 or 150 kDa), can at the
same time be used as final purifcation step

Mass Spectrometry (MS): Size, purity, modification

Very precise measurement of molecular weight in daltons, also shows
glycosylation,
Phosphorylation

The Phase Diagram

Undersaturation Zone (blue)

A protein and crystallizing agent concentration too low; no crystallization

Supersaturation Zone (brown)

D Precipitation: concentrations too high, precipitates appear
C Nucleation: critical nuclei formation
B Metastable: crystal growth, if nucleation has happened before

First goal is to find the nucleation zone.

Strategies used to right crystallization conditions

Finding the right crystallization conditions is a tedious try-and-error process
Determine the optimal protein concentration for each class of precipitant (salts,
organics, PEGs) using the pre-screening assay

2. Perform an initial screening with broad-range screens. Combine a sparse-

matrix and grid screen approach (e.g. JCSG+ / PACT screen)

3. When promising precipitants have been identified, perform a more

detailed screening using this precipitant (e.g. AmSO4 or PEG)

4. When you obtain a hit, optimize it with a grid around the hit conditions
(vary salt, pH)
Formation of Crystals

• Nucleation (initial start of crystallization) happens at a higher saturation

than crystal growth!

• As protein is used up to form the crystal, the concentration drops, and you
get to clear/metastable zone where crystal growth continues to form nice
large crystals

• If the experiment happens too deep in nucleation zone, many nuclei will
form, leading to a „shower“ of crystals

• If the experiments happens too close to the border of nucleation /

metastable phase, crystal growth stops due to depletion of protein, and
you only get small crystals.

Techniques to reach supersaturation

• _ By concentrating the protein (y-axis of the phase diagram)
• _ By adding different concentrations of a precipitant (x-axis)

• .Precipitants are defined as chemicals that lower the solubility of a protein

in solution. These can be:
• _ Salts (e.g. AmSO4, NaCl) „salting out“ process sometimes salts also help
crystallization by binding specifically to the protein
• _ Organic solvents (e.g. ethanol, MPD…)
• _ PEGs (of different molecular weights)

.Commonly used techniques use concentration and / or precipitation:

• _ Vapor diffusion
• _ Microdialysis / microbatch
• _ Counterdiffusion

Vapour Diffusion

Setup
Mix e.g. 2 μl of protein solution with 2 μl of precipitant solution Over time, water
will evaporate from the drop, increasing the concentration of both protein and
precipitant
When you have the right concentration and combination of chemicals, pH and
temperature, crystals will appear in the drop!

The crystals are visualized using X ray diffraction techniques

• When a crystal diffracts, the proteins electrons absorb the energy of the X-
ray radiation.
• When the excited electrons fall back to the ground state, they emit x-ray
radiation which is now scattered
• When a large number of electrons in a crystal scatters the X-rays,
interference occurs that can be additive or subtractive. Results of additive
interference give rise to detectable signals
• The overall emission pattern of a crystal is a function of its 3-D structure,
or, to be more precise, its electron densities (comparable to the ringing of
a bell, the emitted sound waves are analyzed by the ear to recognize a
sound)
• Crystal is turned in the beam 1-2°per image to get a full dataset