CHROMATOGRAPHY

Chromatography is the science which is studies the separation of molecules based

on differences in their structure and/or composition. In general, chromatography involves
moving a preparation of the materials to be separated - the "test preparation" - over a
stationary support. The molecules in the test preparation will have different interactions
with the stationary support leading to separation of similar molecules. Test molecules
which display tighter interactions with the support will tend to move more slowly through
the support than those molecules with weaker interactions. In this way, different types of
molecules can be separated from each other as they move over the support material.

Chromatographic separations can be carried out using a variety of supports,

including immobilized silica on glass plates (thin layer chromatography), volatile gases
(gas chromatography), paper (paper chromatography), and liquids which may incorporate
hydrophilic, insoluble molecules (liquid chromatography).

Chromatography theory
Chromatography is a separation method that exploits the differences in partitioning
behavior between a mobile phase and a stationary phase to separate the components in a
mixture. Components of a mixture may be interacting with the stationary phase based on
charge, relative solubility or adsorption. There are two theories of chromatography, the
plate and rate theories.

How it works

In all chromatography there is a mobile phase and a stationary phase. The

stationary phase is the phase that doesn't move and the mobile phase is the phase that
does move. The mobile phase moves through the stationary phase picking up the
compounds to be tested. As the mobile phase continues to travel through the stationary
phase it takes the compounds with it. At different points in the stationary phase the
different components of the compound are going to be absorbed and are going to stop
moving with the mobile phase. This is how the results of any chromatography are gotten,
from the point at which the different components of the compound stop moving and
separate from the other components.

In paper and thin-layer chromatography the mobile phase is the solvent. The
stationary phase in paper chromatography is the strip or piece of paper that is placed in
the solvent. In thin-layer chromatography the stationary phase is the thin-layer cell. Both
these kinds of chromatography use capillary action to move the solvent through the
stationary phase.

1
Retention

The retention is a measure of the speed at which a substance moves in a chromatographic

system. In continuous development systems like HPLC or GC, where the compounds are
eluted with the eluent, the retention is usually measured as the retention time Rt or tR, the
time between injection and detection. In interrupted development systems like TLC the
retention is measured as the retention factor Rf, the run length of the compound divided
by the run length of the eluent front:

The retention of a compound often differs considerably between experiments and

laboratories due to variations of the eluent, the stationary phase, temperature, and the
setup. It is therefore important to compare the retention of the test compound to that of
one or more standard compounds under absolutely identical conditions.

What is the Retention Factor, Rf ?

The retention factor, Rf, is a quantitative indication of how far a particular compound
travels in a particular solvent. The Rf value is a good indicator of whether an unknown
compound and a known compound are similar, if not identical. If the R f value for the
unknown compound is close or the same as the Rf value for the known compound then
the two compounds are most likely similar or identical.

The retention factor, Rf, is defined as:

Rf = distance the solute (D1) moves divided by the distance traveled by the solvent front
(D2)

2
R f = D1 / D 2
where

D1 = distance that color traveled,

measured from center of the band of color to

the point where the food color was applied

D2 = total distance that solvent traveled

PARTITION CHROMATOGRAPHY
Simple Theory of Partition Chromatography
In this chromatography there are two physically distinguishable compounds, a
mobile phase and a stationary phase. Molecular species separate because they differ in
their distribution between these two phases. The relative movement of each molecule is
result of a balance between a driving force (the movement of the mobile phase) and the
retarding forces. The stationary phase is the sorbent. If the sorbent is a liquid held
stationary by a solid, the solid is called the support or matrix. The mobile phases is the
solvent or developer and the components in the mixture to be separated constitute then
solute.

3
 The theory of partition chromatography is that, in general, if two phases are in
contact with one another and if one or both phases contain a solute, the solute will
distribute itself between the two phases. This is called partitioning and is described by
the partition coefficient (K), the ratio of the concentrations of the solute in the two
phases.

Techniques in Partition Chromatography

Partition chromatography will be described in terms of the operation of a column
– that is, a tube filled with a sorbent and a solvent. A solution containing the solute is
layered on the top of the sorbent and allowed to enter the sorbent. The solvent is hen
allowed to pass continually through the column. Although he sorbent and solvent within
the column are certainly continuous from he top of the column to the bottom, the column
can e thought of as consisting of a large number of individual layers (theoretical plates),
each containing he two phases. Consider 256 identical molecules that distribute
themselves equally between the two phases, one stationary and one mobile and a column
with 18 plates. In the uppermost plate (the origin) the 256 molecules are distributed so
that 128 are in each phase. When the mobile 128 molecules from plate 1 enter the second
plate, they redistribute 64 and 64 in that plate, the 128 remaining in plate 1 also
redistribute 64 and 64, as shown in figure.

After 20 successive transfers, the situation shown in the graph is achieved. The
distributions of these two kinds of molecules are different and a substantial fraction of the
molecules have separated. As the number of the theoretical plates increased (i.e. if the
column length is increased), greater separation will result.

4
Materials
Partition chromatography can be carried out in columns by using a matrix that
does not adsorb the solutes. Common supporting materials are diatomaceous earth (e.g.
Celite), silica gel, cellulose powder and certain cross – linked dextrans (e.g. Sephadex
LH20). The stationary phase is created by suspending the support or washing the column
with the appropriate sorbent. Typical stationary-phase materials are hydrophobic solvents
such as benzene for the separation of nonpolar materials or hydrophilic solvents such as
an alcohol, for polar materials. Typical mobile phases are alcohols or amides for ye
nonpolar material or water for polar substances. The stationary phase materials are liquid.

Types of partition chromatography

The most common types of partition chromatography are

1) Paper Chromatography
2) Thin – layer chromatography
3) Gas-liquid chromatography
4) Gel chromatography

Special types of partition chromatography are

5
 1) Liquid chromatography (LC)
2) Hi Performance Liquid chromatography (HPLC)
3) Size exclusion chromatography
4) Column chromatography

PAPER CHROMATOGRAPHY

This is an older technique which involves placing a small spot of sample solution
onto a strip of chromatography paper. The paper is placed into 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 allows the calculation of an Rf value and can be compared to standard
compounds to aid in the identification of an unknown substance.

Techniques in Paper Chromatography

In paper chromatography there is no effluent and substances are distinguished by
their relative positions in the paper after the solvent has moved a given distance.

A tiny volume of a solution of the mixture to be separated is placed at a marked

spot on a strip or sheet of paper (Fig) and allowed to dry. This spot defines the origin. The
paper is then placed in closed chamber and one end is immersed in a suitable solvent (the
mobile phase). Capillarity draws the solvent through the paper, dissolves the sample as it
passes the origin and moves the components in direction of flow. After the solvent front
has reached a point near the other end of the paper, the sheet is removed and dried. The
spots which may or may not be visible are then detected and their positions marked. The

6
relative distance traveled by a spot and by the solvent front is called the Rf. Values of Rf
depend on the substance, he paper and the solvent.

• RETARDATION FACTOR,

RF = sample spot distance

solvent front distance

• VARIATIONS IN COLOUR of spots when sprayed (developed) with reagent (e.g.

ninhydrin for amino acids.)

Paper chromatograms can be developed by either ascending or descending solvent

flow. Descending chromatography has two advantages: (1) it is faster because gravity
aids in the flow and (2) for quantitative separations of materials with very small Rf
values, which therefore require long runs, the solvent can run off the paper. Its only
disadvantages is the care with which the apparatus must be assembled because dirt or
poor contact where the paper passes over the support bar can result in the inhomogeneous
flow and consequent streaking.

7
 A particularly useful variant is two-dimensional paper chromatography. In this
method, after chromatography has carried out in a single direction, the paper is dried and
then rechromatographed at right angles to the original direction of flow, using a different
solvent system. (FIG) In this way substances that fail to separate in he first solvent can
often be separated in the second.

Detection and Identification of spots

Spots in paper chromatograms can be detected by their color, by their
fluorescence, by the chemical reactions that take place after the paper has been sprayed
with various reagents, or by the radioactivity. Identification is usually based on
comparison with standards of known Rf or by elution. Elution is accomplished by cutting
out the spot and soaking the paper in the appropriate solvent.

Fingerprinting: A special application of paper chromatography to

study of Proteins.
An important problem in molecular biology is to identify an isolated protein or to
identify the site of an amino acid change in a mutant protein. The fingerprint technique
developed by Vernon Ingram allows this to be done relatively simply.

8
 The fingerprint of a mutant protein with a single amino acid change differs from
that of a wild type protein. If the amino acid change does not affect the site of cleavage
by the protease, a single spot in the fingerprint will disappear and one new spot will
appear. If it does not affect the site of cleavage, several spots will be altered. By eluting
the original and mutant spots and determining the amino acid composition of each, the
amino acid change can be determined.

The fingerprinting technique has had several important applications in molecular

biology, among which are:

Proof that a Protein (A) is a product of cleavage of a larger Protein (B)

If all spots in a fingerprint of protein A are found in protein B, it is likely that
protein A is a part of the amino acid sequence of protein B. Hence, either protein B is
made from protein A by the addition of amino acids or protein A is derived from protein
B by hydrolysis.

Fig: A paper chromatogram in which four amino acids have been separated.

Identification of the amino acids inserted by various suppressor transfer

RNAs

Certain mutations cause the premature termination of amino acid sequence in

proteins. This termination is reversed by the presence of suppressor tRNA molecules,
which insert an amino acid at the site of premature termination and thereby allow
continued synthesis to the natural terminus. A protein results that has a single amino acid

9
replacement. Fingerprints of such proteins allow the identification of the amino acid
inserted by each of he known suppressor tRNAs.

Identification of base changes produced by particular mutagens

It is possible to identify base changes that produce particular mutations because,

the genetic code is well known. Suppose that, a mutagen is found to produce frequent
changes from phenylalanine to isoleucine. The DNA triplets corresponding to
phenylalanine are AAA and AAG and to isoleucine are TAA, TAG and TAT. Because
most mutations change only a single base, the particular mutagen would have to lead to
frequent replacement of A with T.

THIN –LAYER CHROMATOGRAPHY

Simple theory of TLC:

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.

In thin-layer chromatography, the stationary phase is a layer (0.25-05) of sorbent

spread uniformly over the surface of a glass or plastic plate. The TLC plate is placed in a
chamber containing the solvent and
developed by ascending
chromatography.

10
 After the solvent front has almost reached the top, the plate is removed from the
chamber and dried. Spots are usually located as in paper chromatography by natural
color, fluorescence or by spraying various reagents that react with the substances in the
spot to produce color. Commonly used sprays are ninhydrin for amino acids, rhodamine
B for lipids, antimony chloride for steroids and terpinoids, H2SO4 plus heating for almost
any organic substance; K2MnO4 in H2SO4 for hydrocarbons, anisaldehyde in H2SO4 for
carbohydrates, bromine vapor for olefins and so forth. Materials can be eluted from he
chromatogram by scraping off the sorbent and eluting the powder with suitable solvent. A
typical thin- layer
chromatogram is shown in
figure:

11
 Fig: A typical thin-layer chromatogram

Advantages of TLC:
Thin – layer chromatography is widely used because, compared with paper or column
chromatography, it offers the following advantages:

1) Greater resolving power because spots are smaller

2) Greater speed of separation and has higher resolution.
3) A wider choice of materials as sorbent
4) Easy detection of spots and
5) Easy isolation of substances from chromatogram.

TLC will be used to examine the composition of various common over-the-counter

medications. These medications are classified into one or more basic categories:
analgesics (pain relieving), antipyretic (fever reduction), anti-inflammatory (reduces
swelling), or uricosuric (relieves symptoms of arthritis and gout). The best known of
these is aspirin, but several other chemically similar compounds are (or were) also used.
Among these are phenacetin, salicylamide and acetaminophen.

12
 Caffeine is sometimes added to these formulations to overcome drowsiness. A few
other compounds such as N-cinnamylephedrine (cinnamedrine) and diphenylpyrilene are
included for other therapeutic effects, such as antispasmodic or slight sedative action. In
addition to the active ingredients, the tablets of these drugs contain starch, lactose, and
other substances that act as binders and permit rapid solution, and sometimes also
inorganic bases. The objective of this experiment is to identify an unknown drug tablet by
a TLC comparison with standard compounds.

The main uses of TLC

1. Monitoring reactions. Simplest and quickest way to monitor a reaction
Reaction should be chromatographed against starting-materials (and a co-spot). Allows
you to follow the progress of the reaction, and to assess the best time for work-up.

2. Indicate identity of a compound. The unknown is compared with a known

materials. Each substance is spotted separately and also together (co-spot).

3. Indication of purity. Diasteroisomers can usually be distinguished

(But not always).

4. For flash chromatography.

a. TLC is used to determine the solvent system and quantity of silica required
b. TLC is used to monitor the column fractions

13
 GAS – LIQUED CHROMATOGRAPHY
Gas chromatography (GC) is based on a partition equilibrium of analyses between
a solid stationary phase and a mobile gas. 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,
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.

Simple Theory of Gas Liquid Chromatography

A gas chromatograph is a chemical analysis instrument for separating chemicals
in a complex sample. A gas chromatograph uses a flow-through narrow tube known as
the column, through which different chemical constituents of a sample pass in a gas
stream (carrier gas, mobile phase) at different rates depending on their various chemical
and physical properties and their interaction with a specific column filling, called the
stationary phase. As the chemicals exit the end of the column, they are detected and
identified electronically. The function of the stationary phase in the column is to separate
different components, causing each one to exit the column at a different time (retention
time). Other parameters that can be used to alter the order or time of retention are the
carrier gas flow rate, and the temperature.

In a GC analysis, a known volume of gaseous or liquid analyte is injected into the

"entrance" (head) of the column, usually using a micro syringe (or, solid phase micro
extraction fibers, or a gas source switching system). Although the carrier gas sweeps the
analyte molecules through the column, this motion is inhibited by the adsorption of the
analyte molecules either onto the column walls or onto packing materials in the column.
The rate at which the molecules progress along the column depends on the strength of
adsorption, which in turn depends on the type of molecule and on the stationary phase
materials. Since each type of molecule has a different rate of progression, the various
components of the analyte mixture are separated as they progress along the column and
reach the end of the column at different times (retention time).

14
 A detector is used to monitor the outlet stream from the column; thus, the time at
which each component reaches the outlet and the amount of that component can be
determined. Generally, substances are identified by the order in which they emerge
(elute) from the column and by the retention time of the analyte in the column.

Methods
The method is the collection of conditions in which the GC operates for a given
analysis. Method development is the process of determining what

15
conditions are adequate and/or ideal for the analysis required. Conditions which can be
varied to accommodate a required analysis include inlet temperature, detector
temperature, column temperature and temperature program, carrier gas and carrier gas
flow rates, the column's stationary phase, diameter and length, inlet type and flow rates,
sample size and injection technique. Depending on the detector(s) (see below) installed
on the GC, there may be a number of detector conditions that can also be varied. Some
GCs also include valves which can change the route of sample and carrier flow, and the
timing of the turning of these valves can be important to method development.

Identification of compounds from the Detector output

Each of the detectors indicates the amount of material emerging from the column
as a function of time. However, there is no direct indication of the identify of the material
producing a particular peak nor of he amount in the peak. There are various means of
identifying peaks. For example, if the substances present in a mixture are known in
advance, peaks may be identified by preparing a duplicate sample containing a small
amount of an added known substance to the mixture and rechromatographing the
mixture.

16
Advantages of Gas-liquid chromatography
The separation in gas-liquid chromatography is excellent. Sensitivity and speed
are extraordinary, with 10-12 gram being detectable for many substances. Because the
rapidity of development of chromatograms depends on the rate of diffusion between the
mobile and stationary phases and because the diffusion rate of gases is much greater than
that of liquids, the gas chromatogram can be run approximately one thousand times as
fast as that produce by liquid column chromatography. Hence separation is frequently
achieved in less then a minute. Furthermore, by using a nondestructive detector and
condensing the samples at the collection end, it is possible to use gas-liquid
chromatography preoperatively. Large preparative instruments can purify gram quantities
of material.

Application
In general, substances that vaporize below ca. 300 °C (and therefore are stable up
to that temperature) can be measured quantitatively. The samples are also required to be
salt-free; they should not contain ions. Very minute amounts of a substance can be
measured, but it is often required that the sample must be measured in comparison to a
sample containing the pure, suspected substance.

Various temperature programs can be used to make the readings more meaningful;
for example to differentiate between substances that behave similarly during the GC
process.

Professionals working with GC analyze the content of a chemical product, for

example in assuring the quality of products in the chemical industry; or measuring toxic
substances in soil, air or water. GC is very accurate if used properly and can measure Pico
moles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in
gaseous samples.

17
 In practical courses at colleges, students sometimes get acquainted to the GC by
studying the contents of Lavender oil or measuring the ethylene that is secreted by
Nicotiana benthamiana plants after artificially injuring their leaves. These GC analyses
are done rather quickly (1 to 15 minutes per sample) and therefore suited for such
courses.

One example of the use of gas chromatography is in the study of the selectivity of
Fischer-Tropsch synthesis catalysts. The outlet from this process contains a number of
light gases including H2, CO, CO2 and CH4, as well as heavier parafinic and olefinic
hydrocarbons (C2-C40+). In a typical experiment, a packed column is used to separate
the light gases, which are then detected with a TCD. The hydrocarbons are separated
using a capillary column and detected with an FID.

A complication with light gas analyses that include H2 is that He, which is the
most common and most sensitive inert carrier (sensitivity is proportional to molecular
mass) has an almost identical thermal conductivity to hydrogen (it is the difference in
thermal conductivity between two separate filaments in a Wheatstone

Bridge type arrangement that shows when a component has been eluted). For this
reason, dual TCD instruments are used with a separate channel for hydrogen that uses
nitrogen as a carrier are common.

Argon in often used when analysing gas phase chemistry reactions such as F-T
synthesis so that a single carrier gas can be used rather than 2 separate ones. The
sensitivity is less but this is a tradeoff for simplicity in the gas supply.

18
 GEL CHROMATOGRAPHY
Gel chromatography (or molecular sieve chromatography) is a special type of
partition chromatography in which separation is based on molecular size.

Simple Theory of Gel Chromatography

The basis of gel chromatography is quite simple. A column is prepared of tiny
particles of an inert substance that contains small pores. If a solution containing
molecules of various dimensions is passed through the column, molecules larger than the
pores move only in the space between the particles and hence are not retarded by the
column material. However molecules smaller than the pores diffuse in and out of
particles with a probability that increases with decreasing molecular size; in this way,
they are slowed down in their movement down the column. As long as the material of
which the particles are made (i.e. the gel) does not absorb the molecules, the probability
of penetration is the principal factor determining the rate of movement through the
column. Hence, molecules are eluted from the column in order to decreasing size or, if
the shape is relatively constant (e.g. globular or rod like), decreasing molecular weight.

In detailed analysis of the mechanism of gel chromatography, it is clear that this

steric effect, although the principal factor, does not alone explain the chromatographic
behavior of all molecules. Another important factor is the charge of the molecule,
although this is only manifested at very low ionic strength when highly charged small
molecules seem to be excluded from the pores even though the size is sufficient. This is
probably due to electrostatic repulsion between the molecules, this limiting the number of
molecules in a pore at any given time. At very low ionic strength, there are also
apparently adsorptive effects with some types of gel.

Materials of Gel Chromatography

A gel is a three dimensional network whose structure is usually random. The gels
used as molecular sieves consist of cross – linked polymers that are generally inert, do
not react or bid with the material being analyzed, and are uncharged. The space within the
gel is file with liquid and this liquid occupies most of the gel volume.

The gel currently in use is of three types:

a) Dextran
b) Agarose
c) Polyacrylamide
They are use for aqueous solution.

19
Dextran:
Dextran is a polysaccharide composed of glucose residues and produced by the
fermentation of sucrose by the microorganism Leuconostoc mesenteroides. It is prepared
with various degrees of cross – linking to control pore size and is supplied in the form of
dry beads of various degrees of fineness that swell when water is added. Swelling is the
process by which the pores become filled with the liquid to be used as eluant. It is
commercially available under the trade name Sephadex.

Agarose:
Obtain from certain seaweeds, is a linear polymer of D – galactose and 3, 6 –
anhydro – 1 – galactose and forms a gel that is held together without cross links by
hydrogen bonds. It is dissolved in boiling water and forms a gel when cooled. The
concentration of the material in the gel determines the size of the pores – which are much
larger than those of Sephadex. This makes it useful for the analysis or separation of large
globular proteins or long, linear molecule such as DNA. Agarose is useless as a solid gel

20
because the flow rate is too low; so it is supplied as wet beads called Sepharose and Bio –
gel.

Polyacrylamide:
Polyacrylamide gels are prepared by cross linking acrylamide with N,
N’-methylene-bis-acerylamide. Again, the pore size is determined by the degree of cross
linking. These gels differ from Dextran and Agarose gels in that they contain a polar,
carboxylamide group on alternate carbon atoms, but their separation properties are much
the same as those of the dextrans. Polyacrylamide gels, which are marketed as Bio-gel P,
seem to be as useful as dextrans, although they have used less frequently. They do have
advantages over the dextrans in that they are commercially available in a wider range of
pore sizes.

Fig: Mechanism of gel chromatography

Red particles – sample molecules smaller than the pore size
Light green particles – sample molecules bigger than red particles but equal to the pore size
Green particles – bigger than all particles and also from the pore size.

Advantages of Gel Chromatography

For the separation of molecules whose molecular weights differ, gel chromatography
is unsurpassed for the following reasons:

1. Because the chromatographic behavior of almost all substances in gels is independent

of temperature, pH, ionic strength, and buffer composition, separations can be carried
out under virtually all conditions. For very labile materials (e.g., enzymes), this
means that the conditions for maximum stability can be maintained.
2. Because there is virtually no adsorption, very labile substances are not affected by the
chromatography. For example, some enzymes are inactivated or altered by binding to
adsorbent surfaces or ionic – exchange resins.

21
3. There is less zone spreading than with other chromatographic techniques.
4. The elution volume is related in a simple manner to molecular weight.

Application of Gel Chromatography

1. In the chemical synthesis of various reagents, it is usually necessary to separate the
product from the reactants. For example, in preparing fluorescent antibodies by
reacting antibody with fluorescein isothiocyanate, the conjugated protein must be
separated from unreacted dye. This can be done with Sephadex, using gels that pass
large proteins in the void volume. The unreacted protein is not separated from the
conjugation protein but, for the fluorescent antibody technique, this is usually
unnecessary.

2. In the assay of enzymes or the determination of cofactor requirements, the enzyme

preparation sometimes contains inhibitors of small molecular size or the cofactors
themselves. Also, I physical studies of some molecules (e.g. in fluorescence
spectroscopy), interfering substances may be present. Such small molecules are easily
removed with the Dextran or Polyacrylamide gels.

3. Similarly there are frequently contaminants of large molecular size in mixtures being
assayed for small molecules. He small – pore dextrans are useful in such cases. Also
protein must often be freed to nucleic acids; this can sometimes be done by using an
Agarose gel, which impedes all proteins and passes nucleic acids in the void volume.
4. The most common use of gel chromatography is in the purification of proteins. To
purify a protein from a cell extract, it is usually necessary to use a sequence of
separation procedures based on such parameters as solubility in certain solutions,
chare, molecular weight, and so forth. The step in which size separation takes place
almost invariably uses gel chromatography.

Gel Chromatography is also a valuable analytical tool. The determination of

molecular weight mentioned previously is an important example of this. Other examples
are:

1. In studying RNA metabolism, various fractions of RNA are usually distinguished by

zone centrifugation or even better by Polyacrylamide gel electrophoresis. Gel
chromatography with Agarose is also of great use.

2. Plasma protein fractions must often be determined quantitatively in the diagnosis of

certain human diseases. This can be done directly with Dextran gels and as been
developed as a reliable test for macro and hyperglobulinemia.
3. The tritium exchange method for examining protein or DNA structure requires that
the macromolecule be rapidly separated from 3H2O. This can be done in about ten
seconds using charged gels because the 3H2O is strongly retarded in all gels. IF the

22
 smallest pore size is used, the macromolecule comes through rapidly in the void
volume or shortly thereafter.

4. Gel chromatography can be used to study biding between proteins and small
molecules either by separating the product and he reactants or by passing protein
through a column equilibrated with the small molecule. A simple calculation allows
the determination of binding constants. The great value of gel chromatography in
studies of chemical equilibrium is that a gel column can be operated over a wide
range of concentrations, pH, ionic strength, and temperature e because the pore size
of the el is unaffected by the factors.

LIQUID CHROMATOGRAPHY

23
 Liquid chromatography (LC) is an analytical chromatographic technique that is
useful for separating ions or molecules that are dissolved in a solvent. If the sample
solution is in contact with a second solid or liquid phase, the different solutes will interact
with the other phase to differing degrees due to differences in adsorption, ion-exchange,
partitioning, or size. These differences allow the mixture components to be separated
from each other by using these differences to determine the transit time of the solutes
through a column.

Simple theory of Liquid Chromatography

Simple liquid chromatography consists of a column with a fritted bottom that
holds a stationary phase in equilibrium with a solvent. Typical stationary phases are:
solids (adsorption), ionic groups on a resin (ion-exchange), liquids on an inert solid
support (partitioning), and porous inert particles (size-exclusion). The mixture to be
separated is loaded onto the top of the column followed by more solvent. The different
components in the sample mixture pass through the column at different rates due to
differences in their partioning behavior between the mobile liquid phase and the
stationary phase. The compounds are separated by collecting aliquots of the column
effluent as a function of time.

Application
Conventional LC is most commonly used in preparative scale work to purify and
isolate some components of a mixture. It is also used in ultra trace separations where
small disposable columns are used once and then discarded. Analytical separations of
solutions for detection or quantification typically use more sophisticated high-
performance liquid chromatography instruments.

HI – PERFORMANCE LIQUID CHROMATOGRAPHY

(HPLC)

24
 High performance liquid chromatography (HPLC) is a form of column
chromatography used frequently in biochemistry and analytical chemistry. The analyte is
forced through a column (stationary phase) by a liquid (mobile phase) at high pressure,
which decreases the time the separated components remain on the stationary phase and
thus the time they have to diffuse within the column. 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.

General characteristics of reversed phase chromatography

• Broad scope which allows sample types with a wide range of polarities and
molecular weights to be separated.
• General rapidity of mobile phase column equilibration during methods
development and gradient regeneration.
• General ease of use.
• Applicability to separation of ionic or ionizable compounds by manipulating
secondary chemical equilibrium such as ionization control and ion pairing in the
aqueous mobile phase.
o Buffering the mobile phase in the pH range from 2 to 5 with one of the
common buffers, the ionization of the weak acids can be suppressed or
controlled allowing them to be retained in their neutral form. Similarly
weak bases can be retained in their neutral form at pH 7-7.5.
o For strong acids and bases ionization control cannot be employed because
the stability of alkyl bonded phases is diminished below pH 2 and above
pH 7.5. Highly hydrophilic weak acids and bases often remain difficult to
retain with ionization control. In such cases ion pair reversed phase
chromatography can be used. In this method, counterions (species of
opposite charge to the solutes) thereby regulate the retention. Typically
alkyl amines or tetra alkyl amines are added to ion pair with acids whereas
alkyl sulfates, sulfonates, or phosphates are used to ion pair with bases.
• The possibility of special selectivity such as structural or steric are achievable by
specific mobile phase additives:
o Metal ions are capable of binding to organic compounds in a very
selective method which is used for ligand exchange chromatography. The
selectivity generated in these metal ion phase systems is based in part on
differences of the solute (ligand) binding strength to the metal ion. An
alternate approach is the addition of various chelating agents (4-
dodecyldiethylene-triamine - C12 dien) in combination with a metal ion.
The type and strength of the metal chelate complex-solute binding can be
greatly varied depending upon the chemical environment surrounding the
metal ion as determined by the chelating agent added.

HPLC Instrumentation

25
 Solvents must be degassed to eliminate formation of bubbles. The pumps provide
a steady high pressure with no pulsating, and can be programmed to vary the composition
of the solvent during the course of the separation. The liquid sample is introduced into a
sample loop of an injector with a syringe. When the loop is filled, the injector can be
injecting the sample into the stream by placing the sample loop in line with the mobile
phase tubing. The different types of HPLC columns are described below. The presence of
analytes in the column effluent is recorded by detecting a change in refractive index, UV-
VIS absorption at a set wavelength, fluorescence after excitation with a suitable
wavelength, or electrochemical response.

Columns -- Conventional liquid chromatography uses plastic or glass columns

that can range from a few centimeters to several meters. The most common lengths are
10-100 cm, with the longer columns finding use for preparative-scale separations.

High-performance liquid chromatography (HPLC) columns are stainless steel

tubes, typically of 10-30 cm in length and 3-5 mm inner diameter. Short, fast analytical
columns, and guard columns, which are placed before an analytical column to trap junk
and extend the lifetime of the analytical column, are 3-10 cm long.

26
 Fig: Picture of an HPLC column

The applicability of chemiluminescence reactions as a means of detecting compounds in

liquid chromatography (LC) is based to a large degree on post column reactions. A
primer on liquid chromatography (and high performance LC) can be found here;
however, a brief description follows. This describes, in the main, HPLC chromatographic
systems.

Components of High Performance Liquid Chromatography

Liquid phase samples (mixtures) are injected onto an LC column usually using a
syringe and specially devised injection valve. The sample is swept onto the
chromatographic column by the flowing mobile phase and chromatographic separation
occurs as the mixture travels down the column. Normal HPLC detectors detect the elution
of a compound from the end of the column based on some physical characteristic such as
ultraviolet light absorption, ability to fluoresce, or the difference in index of refraction
between the analyte and the mobile phase itself. The majority of HPLC systems work this
way.

An example schematic of an HPLC system is shown below:

Need for HPLC Chemiluminescence’s Detection

The use of chemiluminescence detection for HPLC comes from the need to detect
compounds either very sensitively (at very low concentrations) or very selectively, that is,
a target compound that must be determined in the presence of co-eluting compounds that
just can not be successfully separated from the analyte.

27
 Since chemiluminescence derives from the generation of light cause by a
chemical reaction, there is no source lamp light that must be filtered out (as in the case of
fluorescence detection) in order to detect the analyte emission. This means that the
photons coming from the de-exciting analyte molecule are detected against a black
background, and this detection can be accomplished by a photomultiplier which can
detect a large percentage of the emitted photons.

28
Methods of HPLC Post Column Chemiluminescence Detection

IF a target analyte can be determined via HPLC chemiluminescence then it

probably has one of three characteristics: 1) it either chemiluminesces when mixed with a
specific reagent; 2) it catalyzes chemiluminescence between other reagents; or 3) is
suppresses chemiluminescence between other reagents. Examples of all three will be
given below using the well explored luminol reaction.

Luminol based chemiluminescence’s detection

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants like

hydrogen peroxide (H2O2) in the presence of a base and a metal catalyst to produce an
excited state product (3-aminophthalate, 3-APA) which gives off light at approximately
425 nm. If luminol is the target analyte (seldom) then a schematic of a post column
detector based on its solution phase reaction would look like this:

In this case one reagent pump would send a solution containing a dissolved metal
ion like copper(II) or iron(III) to the mixer at the end of the LC column, while the other
reagent pump would send a solution containing the oxidant such as H 2O2 or hypochlorite
(another oxidant) and a base.

Depending on the catalyst used (which basically controls the time necessary for
maximum light emission to develop and the decay profile of that emission) the distance
from the mixer to the detection cell is carefully determined to allow for the most sensitive
detection-in this case the detection of luminol arriving from the LC column where it
could have been separated from interfering compounds.

More realistically, some important chemical species can be derivatized using

luminol itself or luminol like reagents that can be detected in the same or similar ways.

29
Detection based on luminol suppression

What follows is a method of chemiluminescence detection in which the

suppression of a background chemiluminescence signal could be used to determine a
compound that elutes from the LC column. For instance, many organic molecules will
complex metal cations and thereby make them less available as catalysts in the luminol
reaction. This is a nifty way to determine the concentration of the organic molecule: Mix
a constant concentration of a metal cation, luminol, base, and an oxidant. This will create
a baseline light signal that is relatively constant.

With the LC column output fed into the mixer, the amount of light detected will
decrease when an organic analyte (which can complex with the metal ion) elutes from the
column.

30
 The amount of light decrease depends directly on the amount of the analyte. This
is true as long as the amount of metal cation is not completely complexed. At this point
the light decrease will no longer be linearly related to the amount of organic analyte.

Basically the same schematic seen above is seen here with the metal catalyst
coming from the first reagent pump and feeding into a second mixer placed upstream of
the first mixer.

This is to allow the eluting organic molecules (e.g., analytes like amino acids) to
have time to tie up the metal catalyst before they are mixed with the other reagents.

The second reagent pump adds luminol, base and oxidant. When that
metal/organic complex gets to the second mixer and ultimately to the detection cell, the
baseline light intensity will drop off. Voila! An "antisignal"-proportional to the amount of
the (analyte) organic molecules eluting from the column.

31
 SIZE – EXCLUTION CHROMATOGRAPHY
Size exclusion chromatography (SEC) is also known as gel permeation
chromatography or gel filtration chromatography and separates particles on the basis
of size.

Smaller molecules enter a porous media and take longer to exit the column,
whereas larger particles leave the column earlier. It is generally a low resolution
chromatography and thus it is often reserved for the final, "polishing" step of 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.

32
Separation principle:

giving chromatogram as below

33
Mild, non-denaturing conditions - very suitable for separating proteins of different
molecular masses.

Also used for:

• De-salting or buffer exchange of, protein solutions

• Determination of Molecular mass of biological macromolecules - calibrate
column with similar molecules of known molecular mass

34
Quaternary structure usually remains intact.

Types of matrix for forming stationary phase:

• Cross-linked Dextran polymer (Sephadex G-10 to G-200)

• Cross-linked Polyacrylamide (Bio-gel P-2 to P-300)
• Agarose - the largest pore size

COLUMN CHROMATOGRAPHY

Column chromatography encompasses a number of techniques based around

utilizing a vertical glass column filled with some form of solid support, with the sample
to be separated placed on top of this support. The rest of the column is filled with a
solvent which, under the influence of gravity, moves the sample through the column.
Similarly to other forms of chromatography, differences in rates of movement through the
solid medium are translated to different exit times from the bottom of the column for the
various elements of the original sample.

35
 In 1978, W. C. Still introduced a modified version of column chromatography
called flash column chromatography (flash). 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

The protein separation actually occurs on a chromatography column that contains

a charged stationary phase. Oppositely charged proteins bind to the stationary phase and
like charged or uncharged proteins wash through the column with the void volume. The
mobile phase travels through the column, carrying unbound proteins with it. The bound
proteins are then eluted from the column by increasing the salt concentration in the
mobile phase.

36
 The chromatography column is the heart of the separation process. For protein
separations the column usually contains porous beads of a hydrophilic polymer, such as
cellulose or some other type of carbohydrate polymer. The surface of the polymer beads
is chemically modified to give it properties that would make it suitable for various types
of chromatography: ion exchange, molecular exclusion, hydrophobic interaction or
affinity. The appropriate stationary phase is suspended in the desired mobile phase and
poured into the chromatography column.

Once the stationary phase has been fully equilibrated with the mobile phase, the
protein sample can be introduced onto the column. The separation then occurs based on
the attraction between the protein, the stationary phase and the mobile phase. For
example, a positively charged protein would bind to a negatively charged stationary
phase when the mobile phase has a low ionic strength (see Salts). An increase in the salt
concentration may displace the protein from the stationary phase when positive ions in
the mobile phase compete with the protein for binding sites on the stationary phase.

In column chromatography, the stationary phase, a solid adsorbent, is placed in a

vertical glass (usually) column and the mobile phase, a liquid, is added to the top and
flows down through the column (by either gravity or external pressure). Column
chromatography is generally used as a purification technique: it isolates desired
compounds from a mixture.

The mixture to be analyzed by column chromatography is applied to the top of the

column. The liquid solvent (the eluant) is passed through the column by gravity or by the

37
application of air pressure. An equilibrium is established between the solute adsorbed on
the adsorbent and the eluting solvent flowing down through the column. Because the
different components in the mixture have different interactions with the stationary and
mobile phases, they will be carried along with the mobile phase to varying degrees and a
separation will be achieved. The individual components, or eluant, are collected as the
solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how the solvent
flows down the column. If the solvent is allowed to flow down the column by gravity, or
percolation, it is called gravity column chromatography. If the solvent is forced down
the column by positive air pressure, it is called flash chromatography, a "state of the
art" method currently used in organic chemistry research laboratories The term "flash
chromatography" was coined by Professor W. Clark Still because it can be done in a
“flash."

The Adsorbent
Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the
organic chemist for column chromatography. These adsorbents are sold in different mesh
sizes, as indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400”
are a couple examples. This number refers to the mesh of the sieve used to size the silica,
specifically, the number of holes in the mesh or sieve through which the crude silica
particle mixture is passed in the manufacturing process. If there are more holes per unit
area, those holes are smaller, thus allowing only smaller silica particles go through the
sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles.

Adsorbent particle size affects how the solvent flows through the column. Smaller
particles (higher mesh values) are used for flash chromatography; larger particles (lower
mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used
for gravity columns and 230–400 mesh for flash columns.

Alumina is used more frequently in column chromatography than it is in TLC.

Alumina is quite sensitive to the amount of water which is bound to it: the higher its
water content, the less polar sites it has to bind organic compounds, and thus the less
“sticky” it is. This stickiness or activity is designated as I, II, or III, with I being the most
active. Alumina is usually purchased as activity I and deactivated with water before use
according to specific procedures. Alumina comes in three forms: acidic, neutral, and
basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.

Silica gel and alumina are the only column chromatography adsorbents used in
the CU organic chemistry teaching labs; please refer to the references for information on
other column chromatography adsorbents.

38
The Solvent
The polarity of the solvent which is passed through the column affects the relative
rates at which compounds move through the column. Polar solvents can more effectively
compete with the polar molecules of a mixture for the polar sites on the adsorbent surface
and will also better solvate the polar constituents. Consequently, a highly polar solvent
will move even highly polar molecules rapidly through the column. If a solvent is too
polar, movement becomes too rapid, and little or no separation of the components of a
mixture will result. If a solvent is not polar enough, no compounds will elute from the
column. Proper choice of an eluting solvent is thus crucial to the successful application of
column chromatography as a separation technique. TLC is generally used to determine
the system for a column chromatography separation. The choice of a solvent for the
elution of compounds by column chromatography is covered in the Chromatography
Overview section.

Often a series of increasingly polar solvent systems are used to elute a column. A
non-polar solvent is first used to elute a less-polar compound. Once the less-polar
compound is off the column, a more-polar solvent is added to the column to elute the
more-polar compound.

Analysis of Column Eluant

If the compounds separated in a column chromatography procedure are colored,
the progress of the separation can simply be monitored visually. More commonly, the
compounds to be isolated from column chromatography are colorless. In this case, small
fractions of the eluant are collected sequentially in labeled tubes and the composition of

39
each fraction is analyzed by thin layer chromatography. (Other methods of analysis are
available; this is the most common method and the one used in the organic chemistry
teaching labs.)

ADSORPTION CHROMATOGRAPHY
Adsorption chromatography is probably one of the oldest types of
chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto
the surface of a stationary solid phase. The equilibration between the mobile and
stationary phase accounts for the separation of different solutes.

Simple theory of Adsorption Chromatography

Consider a solid surface containing a wide varity of binding sites – for example,
regions that are electron – rich (negatively charged), electron – poor (positively charged),
nonpolar and so forth, and a liquid containing solute in contact with the surface. If
binding is reversible, the number of molecules bound to the surface will depend on the
solute concentration. This dependency is shown in figure. Curves of this sort are called
adsorption isotherms. The most common is the convex curve – that is, binding sites with
high affinity are filled first so that additional amounts of solute are bound less tightly. The
binding isotherm is a characteristic of a particular molecule and sorbent.

40
 If a given concentration of a molecule is applied to the surface and solvent is
allowed to flow across the surface, a fixed amount will bind ad the remainder will move
along. The advancing material will bind with two differences: (1) the retarding force is
binding or adsorption and (2) the fraction bound is not a constant fraction but decreases
with decreasing concentration.

The rate at which the substance moves is related to the strength of binding – that
is, the tighter the binding, the slower the movement. Clearly then, molecules can be
separated if they have different adsorption isotherms.

Materials
Solute in liquid (or gas) phase interacts with adsorption sites on solid surface
(finely divided particles for maximum surface area).

Material Substance separated

Alumina Small organic molecules, Proteins

Silica Gel Sterols, amino acids

Activated carbon Peptides, amino acids, carbohydrates

Calcium phosphate gel Proteins, Polynucleotide

Hydroxyapatite Nucleic acids

Polar groups on solid form dipolar interactions (e.g. hydrogen bonds) with sample
dissolved (usually) in organic solvent. Elute by increasing polarity of the solvent (e.g. if
using acetonitrile CH3CN, add methanol (CH3OH)) --> competing bonds with adsorption
sites. Gradient elution useful (also for ion-exchange and partition chromatography).

41
Types of Adsorption
Chromatography
Adsorption chromatography uses a mobile liquid phase and a solid stationary
phase. Separation is either in columns or on thin layers.

An important variation of adsorption chromatography is ion – exchange

chromatography. This differs mainly in that the composition of the mobile phase such
that, as the material is being applied to the adsorbent, the solute becomes immobilized.
Migration does not begin until a new mobile phase is added.

42
 Fig : A simple fraction collector

Operation of columns
The chromatogram is developed by flowing a solvent (the mobile phase) through
the column. The process is called eluting the column. As different substances move
through the column, they separate and appear in the effluent when particulars volume of
material, both solid and liquid in the column is called the bed volume. The volume of the
mobile phase is the void, retention or hold up volume.

Fig: Operation of a column showing the loading of the column and various stages of
elution.

43
 The liquid leaving the column (the eluent) is usually collected as discrete
fractions, using an automatic collector. The separated components are then found and
identified by testing aliquots of each fraction – for example, spectral measurements,
chemical tests, radioactivity and so forth. In cases in which analysis is by the absorption
of light, an automatic, continuously recording spectrophotometer is used.

ION-EXCHANGE CHROMATOGRAPHY
Ion exchange chromatography (IEC) is applicable to the separation of almost any
type of charged molecule, from large proteins to small nucleotides and amino acids. It is
very frequently used for proteins and peptides, under widely varying conditions.
However, for amino acids standardized conditions are used. In protein structural work the
consecutive use of gel permeation chromatography (GPC) and IEC is quite common.

Basic Principles of Ion Exchange Chromatography

In ion exchange chromatography, the stationary phase of the column has a charge
(either + or -). A mixture of proteins is added to the column and everything which has the
same charge passes through the column due to electrostatic repulsion. If the charge of the
matrix is positive, it will bind negatively charged molecules. This technique is called
anion exchange. If the matrix is negatively charged, it will bind positively charged
molecules (cation exchange). Thus, a scientist picks the resin to use based on the
properties of the protein of interest. During the chromatography, the protein binds to the
oppositely charged beads. Contaminating proteins which have the same net charge as the
matrix can be separated from the bound proteins by washing the column with buffer.
Proteins which remain bound to the matrix can be differentially eluted by increasing the
salt concentration or by altering the pH of the mobile phase. Ion exchange containing
diethyl aminoethyl (DEAE) or carboxymethyl (CM) groups is frequently used in
biochemistry.

44
 The
ionic properties of both DEAE and CM are dependent on pH, but both are sufficiently
charged to work well as ion exchangers within the pH range 4 to 8 where most protein
separations take place.

Proteins are made up of twenty common amino acids. Some of these amino acids
possess side groups ("R" groups) which are either positively or negatively charged.

A comparison of the overall number of positive and negative charges will give a
clue as to the nature of the protein. If the protein has more positive charges than negative
charges, it is said to be a basic protein. If the negative charges are greater than the
positive charges, the protein is acidic.

45
 When the protein contains a predominance of ionic charges, it can be bound to a
support that carries the opposite charge. A basic protein, which is positively charged, will
bind to a support which is negatively charged.

An acidic protein, which is negatively charged, will bind to a positive support.

The use of ion-exchange chromatography, then, allows molecules to be separated based
upon their charge.

Families of molecules (acidic, basics and neutrals) can be easily separated by this
technique. This is perhaps the most frequently used chromatographic technique used for
protein purification.

46
Retention by attraction between groups on stationary phase with opposite charge to
sample molecules. Stationary phase = insoluble, but solvent permeable polymer matrix
(e.g. cellulose) chemically modified to introduce ionizable groups (e.g. -COOH).

Elute by

• Change of pH to neutralize charged group on either solute or stationary phase.

• Increase [salts] (especially polyvalent) in eluant buffer --> Displace by competing
ions.
• pH or salt gradient to enhance separation.

Ion-exchange media are classified according to whether the attached ionizable

group is strongly or weakly acidic or basic --> determines the usable pH range

Medium Nature pH Applications

(X = matrix) range
Anion exchangers
X-CH2N+(CH3)3 strong 2 - 11 nucleotides
X-CH2NH+(CH3)2 intermediate 2-7 organic acids
(CH3CH2)2 weak 3-6 proteins
X-CH2CH2NH+
diethylamino-ethyl (DEAE)
Cation exchangers
X-SO3- strong 2 - 11 amino acids
X-COO- intermediate 6 - 10 peptides
X-CH2COO- weak 7 - 10 proteins
carboxymethyl (CM)

47
Note DEAE-cellulose and CM-cellulose popular for chromatographic separation of
proteins - mild, non-denaturing procedure.

pH and Selectivity in Ion Exchange Chromatography

The property of a protein which governs its adsorption to an ion exchanger is the
net surface charge. Since surface charge is the result of weak acidic and basic groups of
protein; separation is highly pH dependent. Going from low to high pH values the surface
charge of proteins shifts from a positive to a negative charge surface charge.

The pH vs. net surface curve is an individual property of a protein, and

constitutes the basis for selectivity in IEC. At a pH value below its isoelectric point a
protein (+ surface charge) will adsorb to a cation exchanger (-) such as one containing
CM-groups. Above the isoelectric point protein (- surface charge) will adsorb to a anion
exchanger (+), e.g. one containing DEAE-groups.

Ionic Strengths and Selectivity in Ion Exchange Chromatography

As in all forms of liquid chromatography, conditions are employed that permit the
sample components to move through the column with different speeds. At low ionic
strengths, all components with affinity for the ion exchanger will be tightly adsorbed at
the top of the ion exchanger and nothing will remain in the mobile phase. When the ionic
strength of the mobile phase is increased by adding a neutral salt (e.g., NaCl), the salt

48
ions will compete with the protein and more of the sample components will be partially
desorbed and start moving down the column. Increasing the ionic strength even more
causes a larger number of the sample components to be desorbed, and the speed of the
movement down the column will increase. The higher the net charge of the protein, the
higher the ionic strength needed to bring about desorption. At a certain high level of ionic
strength, all the sample components are fully desorbed and move down the column with
the same speed as the mobile phase. Somewhere in between total adsorption and total
desorption one will find the optimal selectivity for a given pH value of the mobile phase.
Thus, to optimize selectivity in ion exchange chromatography, a pH value is chosen that
creates sufficiently large net charge differences among the sample components. Then, an
ionic strength is selected that fully utilizes these charge differences by partially desorbing
the components. The respective speed of each component down the column will be
proportional to that fraction of the component which is found in the mobile phase.

Gradient Elution
Very often the sample components vary so much in their adsorption to the ion
exchanger that a single value of the ionic strength cannot make the slow ones pass
through the column in a reasonable time. In such cases, a salt gradient is applied. This
will bring about a continuous increase of ionic strength in the mobile phase. Such a
gradient will gradually desorbs the sample components in the order of increasing net
charge, until all the components are fully desorbed. At this point, though, we have already
separated the components of the sample. Thus a salt gradient compresses a chromatogram
so as to elute components with widely different adsorptive properties within a reasonable
time. In fact, most IEC experiments utilize a salt gradient. If it is necessary to selectively
increase resolution somewhere within the gradient, but still to elute the slow components
within a reasonable time, a section of lower gradient slope is built into the gradient so
that it covers that part of the chromatogram where increased resolution is desired. This is
called the adapted gradient technique and requires an advanced programmable gradient
device.

Application of Ion-exchange Chromatography

In principle, any substance that is charged can be chromatographed on an ion exchanger.
Resin exchangers are most useful for small organic molecules and can even be used to
separate metallic ions (e.g. Ca2+ from Mg2+). Proteins and polysaccharides are best used
with the cellulose, Dextran and Polyacrylamide exchangers. The Dextran and
Polyacrylamide exchangers have also been widely used for the separation of nucleotides,
amino acids and other biologically important small molecules.

49
 AFFINITY CHROMATOGRAPHY

Theory of Affinity Chromatography

Affinity chromatography is a type of chromatography that makes use of a specific
affinity between a substance to be isolated and a molecule that it can specifically bind (a
ligand). The column material is synthesized by covalently coupling a binding molecule
(which may be a macromolecule or a small molecule) to an insoluble matrix. The column
material s the specifically able to absorb from the solution the substance to be isolated.
Elution is accomplished by changing the conditions to those in which binding does not
occur.

This is the most selective type of chromatography employed. It utilizes the

specific interaction between one kind of solute molecule and a second molecule that is
immobilized on a stationary phase. For example, the immobilized molecule may be an
antibody to some specific protein. When solutes containing a mixture of proteins are
passed by this molecule, only the specific protein is reacted to this antibody, binding it to
the stationary phase. This protein is later extracted by changing the ionic strength or pH.

50
Requirements:

Several requirements must be met for success in affinity chromatography

1) The matrix should be a substance that does not itself adsorb molecules to any
significant extent.
2) The ligand must be coupled without altering its binding properties.
3) A ligand should be chosen whose binding is relatively tight because, although
weak binding will enhance retardation, it may not be adequate for separation to
result.
4) It should be possible to elute without destroying the sample.

51
 The most useful matrix material is Agarose because it exhibits minimal
adsorption, maintains good flow properties after coupling, and tolerates the extremes of
pH and ionic strength as well as 7.0 M guanidium chloride and urea, which are often
needed for successful elution. It is possible to purchase Agarose to which are covalently
coupled either reagents for coupling proteins, membranes and steroids or concanavalin A,
a ready-to-use adsorbent for polysaccharides and glycoprotein containing α-D-mannosyl
and α-D-glucosyl residues (e.g. cell membranes and whole cells), is an additional benefit.

Use of Affinity Chromatography

The major use of affinity chromatography to date has been the purification of
proteins, membranes and polysaccharides. Examples of its use are as follows:

Purification of Proteins
This is usually done with Sepharose to which the substance that is being
transported is coupled (e.g. thyroxin binding globulins, estradiol-binding proteins and
hormone and drug receptors).

Affinity Chromatography Method

Affinity chromatography is designed to purify a particular protein from a mixed sample.

Figure 1. Loading affinity column.

52
Figure 2. Proteins sieve through Figure 3. Proteins interact with
affinity ligand with some binding
matrix of affinity beads.
loosely and others tightly.

Figure 4. Wash off proteins that do not bind.

53
 Figure 5. Wash off proteins that bind loosely.

Figure 6. Elute proteins that bind tightly to ligand and collect purified protein
of interest.

Purification of enzymes:
The substrate, a tight binding inhibitor, or a cofactor can be coupled to the matrix.
If a mixture of proteins or even a crude cell extract is passed through the column, only
materials that bid remain on the column. In some cases, enzymes can be substantially
purified directly from very complex mixtures.

Purification of Antibodies

54
 This has been accomplished mainly with cyanogens bromide Sepharose to which
has been coupled various antigens such as proteins, viruses or bovine serum albumin
coupled with haptens; it is the method of choice for antibody purification.

Purification of Membranes and Particles containing known substances

Membranes to which a hormone binds can be purified using Sepharose coupled
with that hormone; influenza virus, which contains neuraminidase on its surface, has also
been purified using Sepharose to which inhibitors of neuraminidase are coupled.

Purification of Glycoprotein
This is efficiently done with concanavalin A-Sepharose.

Separation of Specific Animal Cells

This has been done using coupled agglutinins, such as concanavalin A, wheat
germ agglutinin or phytohemagglutinin. For example, some virus induced tumor cells can
be separated fro normal cells because the tumor cells bind more tightly to concanavalin A
Sepharose.

Chromatography and Biotechnology

This discussion of chromatography will focus on the separation of proteins into relatively
homogeneous groups because proteins are often the target molecules which must be
purified for use as "biopharmaceuticals" or medicines. It is important to remember,
however, that chromatography can also be applied to the separation of other important
molecules including nucleic acids, carbohydrates, fats, vitamins, and more.

One of the important goals of biotechnology is the production of the therapeutic

molecules known as "biopharmaceuticals," or medicines. There are a number of steps that
researchers go through to reach this goal:

• identification of a "target protein" which may have therapeutic value

• identification of the "target gene" -- the gene responsible for encoding the target
protein
• isolation of the target gene
• insertion of the target gene into a host cell (such as E. coli) which will both grow
well, and continue to produce the protein product encoded for by the target gene
• separation of the target protein from the many other host cell proteins
• large scale production of the target protein under controlled manufacturing
conditions
• large scale testing for efficacy as a medicine
• marketing of a new medicine
• Many different disciplines, including microbiology, molecular biology, chemistry,
and others, are required to complete the steps listed above to bring a protein from

55
 the "scientifically interesting" state to that of a full-fledged drug to be used in
treating a specific disease. This discussion will focus on the work and tools of the
chromatographer.

Chromatographers use many different types of chromatographic techniques in

biotechnology as they bring a molecule from the initial identification stage to the stage of
a becoming a marketed product. The most commonly used of these techniques is liquid
chromatography, which is used to separate the target molecule from undesired
contaminants (usually host-related), as well as to analyze the final product for the
requisite purity established with governmental regulatory groups (such as the FDA).

56