BIOPHYSICS

LESSON 37
CHROMATOGRAPHY

What is Chromatography mobile phase is typically a solvent moving through the column
welcome to the wonderful world of chromatography! What is which carries the mixture to be separated. This can either be a
chromatography? Well, quite simply, it is a broad range of liquid or a gas, depending on the type of process. The
physical methods used to separate and or to analyze complex stationary phase is usually a viscous liquid coated on the surface
mixtures. The components to be separated are distributed of solid particles which are packed into the column as discussed
between two phases: a stationary phase bed and a mobile phase above, although the solid particles can also be taken as the
which percolates through the stationary bed. The Russian stationary phase. In any case, the partitioning of solutes
botanist Mikhail Tswett is credited with the original between the stationary and mobile phases lead to the desired
development of a separation technique that we now recognize separations.
as a form of chromatography. In 1903 he first time reported the
1. Feed Injection
separation of a mixture of plant pigment using a column of
The feed is injected into the mobile phase. The mobile phase
calcium carbonate.
flows through the system by the action of a pump (older
How Does It Work analytical chromatorgraphy used capillary action or gravity to
A mixture of various components enters a chromatography move the mobile phase).
process, and the different components run through the system
at different rates. These differential rates of migration as the
2. Separation in the Column
As the sample flows through the column, its different
mixture moves over adsorptive materials provide separation.
components will adsorb to the stationary phase to varying
Repeated sorption/desorption acts that take place during the
degrees. Those with strong attraction to the support move
movement of the sample over the stationary bed determine the
more slowly than those with weak attraction. This is how the
rates. The smaller the affinity a molecule has for the stationary
components are separated.
phase, the shorter the time spent in a column.
The basis of all forms of chromatography is the partition or 3. Elution from the Column
distribution coefficient(Kd), which describes the way in which a After the sample is flushed or displaced from the stationary
compound distribute itself between two immiscible phases. phase, the different components will elute from the column at
For two such phases A and B the value for this coefficient is a different times. The components with the least affinity for the
constant at a given temperature and is given by the expression. stationary phase (the most weakly adsorbed) will elute first,
while those with the greatest affinity for the stationary phase
So, Why Is It So Special?
(the most strongly adsorbed) will elute last.
In any chemical or bioprocessing industry, the need to separate
and purify a product from a complex mixture is a necessary and 4. Detection
important step in the production line. Today, there exists a wide The different components are collected as they emerge from the
market of methods in which industries can accomplish these column. A detector analyzes the emerging stream by measuring
goals. Chromatography is a very special separation process for a a property which is related to concentration and characteristic of
multitude of reasons! First of all, it can separate complex chemical composition. For example, the refractive index or ultra-
mixtures with great precision. Even very similar components, violet absorbence is measured.
such as proteins that may only vary by a single amino acid, can
be separated with chromatography. In fact, chromatography can
purify basically any soluble or volatile substance if the right
adsorbent material, carrier fluid, and operating conditions are
employed. Second, chromatography can be used to separate
delicate products since the conditions under which it is
performed are not typically severe. For these reasons,
chromatography is quite well suited to a variety of uses in the
field of biotechnology, such as separating mixtures of proteins.
Chromatography - Basic Operation
The process of a chromatographic separation takes place within
a chromatography column. This column, made of glass or
metal, is either a packed bed or open tubular column. A packed
bed column contains particles which make up the stationary
phase. Open tubular columns are lined with a thin film
stationary phase. The center of the column is hollow. The

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Figure: explains the very basic operation of column
chromatography.
Types of Chromatography
Thin layer Chromatography (TLC)
Gas Chromatography
Liquid Chromatography
Ion Exchange Chromatography
Affinity Chromatography
• Thin Layer Chromatography (TLC) is an extremely useful
technique for monitoring reactions. It is also used to
determine the proper solvent system for performing
separations using column chromatography. TLC uses a
stationary phase, usually alumina or silica, that is highly polar
(standard) or non-polar (reverse phase). The mobile phase is
a solvent whose polarity you will choose. In most lab
applications, you will use standard phase silica plates. You
will apply your reaction mixture in solution to the plate and
then “run” the plate by allowing a solvent (or combination
of solvents) to move up the plate by capillary action.
Depending on the polarity of the components of the
mixture, different compounds will travel different distances
up the plate. More polar compounds will “stick” to the polar
silica gel and travel short distances on the plate. Non-polar
substances will spend more time in the mobile solvent phase
and travel larger distances on the plate. The measure of the
distance a compound travels is called the Rf value. This
number, between zero and one, is defined as the distance the
compound moved from the baseline (where it was originally
spotted) divided by the distance the solvent front moved
from the baseline. The actual run on the slica plate is given
in the figure below. Here, the details of the TLC running is
described below.
Steps for TLC:
1. Cut TLC plates. Usually silica plates are bought as square
glass pieces that must be cut using a diamond tipped glass
cutter and following a template. Before scoring the glass, use
a ruler and a pencil to lightly mark baselines on the silica side
of the plate (be careful not to remove any silica from the
plate). Using a sharp glass cutter and a ruler as a guide, you
should have no problem scoring the glass. Once the entire
plate is scored, you can thenbreak the glass into individual
pieces. (In the beginning this may be frustrating, but after
some practice, you should become comfortable with this
technique.)
2. Determine an appropriate solvent system. Your
compounds will travel different distances up the plate
depending on the solvent you choose. In non-polar solvents
like pentane and hexane, most polar compounds will not
move, while non-polar compounds will travel some distance
up the plate. In contrast, polar solvents will usually move
non-polar compounds to the solvent front and push the
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polar compounds off of the baseline. A good solvent
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system is one that moves all components of your mixture
off the baseline, but does not put anything on the solvent
front - Rf values between 0.15 and 0.85. This is not always
possible, but should be your goal when running a TLC. (For
column chromatography the correct solvent system should
give an Rf between 0.2 and 0.3.) Now, which solvents to
pick? Here is a list of some standard solvents and their
relative polarity (from LLP):
Very polar additives:
Methanol > Ethanol > Isopropanol
Moderately polar additives:
Acetonitrile > Ethyl Acetate > Chloroform >
Dichloromethane > Diethyl Ether > Toluene
Non-polar additives:
Cyclohexane, Petroleum Ether, Hexane, Pentane
Common solvent combinations:
Ethyl Acetate/Hexane : 0–30% most popular combination,
sometimes tough to remove solvents completely on rotary
evaporator
Ether/Pentane: 0–40% very popular, easy to remove on the
rotary evaporator
Ethanol/Hexane or Pentane: 5–30% useful for very polar
compounds
Dichloromethane/Hexane or Pentane : 5–30% sometimes
useful when other mixtures fail
3. Fill TLC chamber with 1–2 mL of the desired solvent
system. Place a large piece of cut filter paper in the chamber as
well.
4. Spot the compound on the baseline of the TLC plate. We
will use commercial spotters, but spotters can be pulled from
hot Pasteur pipets (you may see this in your UROP). If you
are monitoring a reaction, make sure to spot the starting
material, the reaction mixture, and a co-spot of both.
5. Run the TLC. Let the solvent go about 90% of the way up
the plate.
6. Remove the plate from the chamber and mark the
solvent front immediately with a pencil. You will use this
to calculate the Rf.
7. Let the solvent dry off of the plate.
8. Visualize the TLC using non-destructive technique(s).
The best non-destructive method is the UV lamp. Place your
plate under the UV lamp and circle any UV active spots with
your pencil. Although we won’t do this in 5.301, another
popular non-destructive method is staining with iodine.
(You might see this in your UROP.)
9. Visualize the TLC using a destructive method. This will
be critical for compounds that are not UV-active. There are
several varieties of stains that are very useful and will be
available to you in 5.301. To use the stain, pick up the dried
TLC plate with a pair of tweezers and dip it into the stain,
making sure to cover the area from the baseline to the
solvent front. Completely dry the back of the plate with a
paper towel. Place on a hot plate and watch the development
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 of the spots. Remove the TLC plate from the heat once the
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spots are visible and before the background color obscures
the spots.
10. Revise your choice of solvent system based on the
results of your initial TLC. Make the solvent system more
polar if you want a larger Rf or make it less polar if you
want to decrease the Rf. Also, if there is “streaking” of your
compound on the plate -basically you see large streaks
instead of sharp circles - your sample is probably too
concentrated. Try diluting your sample and running the TLC
again. If this doesn’t work, you will have to move to a
different solvent system.
11. Label your TLC, calculate the Rf for each spot and draw a
picture of it in your notebook. 49
Figure: Left side of the picture shows the actual run of the
samples on the TLC plate. The calculated migration values are
drawn in the graph on the left. The sample is loaded on the
lower line, which migrated according the polarity of the
substance. For the spot ‘A’ the migration value is calculated by
the densitometer trace formula.
• Gas Chromatography makes use of a pressurized gas
cylinder and a carrier gas, such as helium, to carry the solute
through the column. The most common detectors used in
this type of chromatography are thermal conductivity and
flame ionization detectors. There are three types of gas
chromatography that will be discussed here: gas adsorption,
gas-liquid and capillary gas chromatography.
Gas adsorption chromatography involves a packed bed
comprised of an adsorbent used as the stationary phase.
Common adsorbents are zeolite, silica gel and activated
alumina. This method is commonly used to separate
mixtures of gases.
Gas-liquid chromatography is a more common type of
analytical gas chromatography. In this type of column, an
inert porous solid is coated with a viscous liquid which acts
as the stationary phase. Diatomaceous earth is the most
common solid used. Solutes in the feed stream dissolve into
the liquid phase and eventually vaporize. The separation is
thus based on relative volatilities.
Capillary gas chromatography is the most common analytical
method. Glass or fused silica comprise the capillary walls
which are coated with an absorbent or other solvent. Because
of the small amount of stationary phase, the column can
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contain only a limited capacity. However, this method also
yields rapid separation of mixtures.
The mobile phase in gas chromatography is generally an inert
gas. The stationary phase is generally an adsorbent or liquid
distributed over the surface of a porous, inert support.
• Liquid Chromatography:There are a variety of types of liquid
chromatography. There is liquid adsorption chromatography
in which an adsorbent is used. This method is used in large-
scale applications since adsorbents are relatively inexpensive.
There is also liquid- liquid chromatography which is
analogous to gas-liquid chromatography. The three types
that will be considered here fall under the category of
modern liquid chromatography. They are reverse phase, high
performance and size exclusion liquid chromatography, along
with supercritical fluid chromatography.
Reverse phase chromatography is a powerful analytical tool
and involves a hydrophobic, low polarity stationary phase
which is chemically bonded to an inert solid such as silica.
The separation is essentially an extraction operation and is
useful for separating non-volatile components.
High performance liquid chromatography (HPLC) is similar
to reverse phase, only in this method, the process is
conducted at a high velocity and pressure drop. The column
is shorter and has a small diameter, but it is equivalent to
possessing a large number of equilibrium stages.
Size exclusion chromatography, also known as gel
permeation or filtration chromatography does not involve
any adsorption and is extremely fast. The packing is a porous
gel, and is capable of separating large molecules from smaller
ones. The larger molecules elute first since they cannot
penetrate the pores. This method is common in protein
separation and purification.
Supercritical fluid chromatography is a relatively new analytical
tool. In this method, the carrier is a supercritical fluid, such as
carbon dioxide mixed with a modifier. Compared to liquids,
supercritical fluids have solubilities and densities have as
large, and they have diffusivities and viscosities quite a bit
larger. This type of chromatography has not yet been
implemented on a large scale.
• Ion exchange chromatography: is commonly used in the
purification of biological materials. There are two types of
exchange: cation exchange in which the stationary phase
carries a negative charge, and anion exchange in which the
stationary phase carries a positive charge. Charged molecules
in the liquid phase pass through the column until a binding
site in the stationary phase appears. The molecule will not
elute from the column until a solution of varying pH or
ionic strength is passed through it. Separation by this
method is highly selective. Since the resins are fairly
inexpensive and high capacities can be used, this method of
separation is applied early in the overall process.
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Figure: Shows the attraction of mobile anion phase towards the
stationaly cation phase.
Affinity Chromatography: An intriguing chromatographic
technique based on the natural specificity of some biopolymers
is affinity chromatography.
There are a number of proteins and other biological
macromolecules that complex with some other biological entity
with a high degree of specificity. This fact is made use of in
product recovery operations via the use of affinity
chromatography. A very good example of purifying the
enzymes is given in the diagrammatic form in Figure4.
Suppose a certain biomolecule (a) is attached to a solid used to
pack a chromatographic column. Now consider a molecule (b)
in solution, which has a specific affinity for (a). It is but natural
that (b) will want to get out of solution and bind to (a), right?
It’s this attraction of (b) for (a) which is defined as the partition
coefficient ‘K’. Now since ‘K’ for (b) is going to be much higher
than that of any other proteins in solution, it will bind to the
column while the rest of the complex solution will merely pass
through the column with insignificant amounts of non-specific
binding occuring.
Figure 4: Diagram of purification of enzyme of by affinity
column chromatography
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Here are few examples of affinity column chromatography:
BIOPHYSICS
ENZYME + INHIBITOR <=> ENZYME-INHIBITOR
COMPLEX
ANTIBODY + ANTIGEN —> ANTIBODY-ANTIGEN
PRECIPITATE
LECTIN + CELL WALL ——> LECTIN-CELL-WALL
COMPLEX
Fig: Molecular diagram about the functioning of the affinity
chromatography
With the advent of Monoclonal Antibody production, which
allows the synthesis of a single type of antibody with a very
high specific binding constant to its corresponding antigen (a
particular protein or other molecule), the preparation of affinity
columns has become not only routine, but commercial. With
such ‘immunosorbent’ column seperation, some important
practical and theoretical differences arise compared to more
conventional forms of chromatographic resolution. They are as
follows:
1. The dominant cost in the process is the antibody needed to
make the immunosorbent column. Generally speaking, this
is much more costly than the antigen-containing broth itself.
As a result,
2. A small column of repeated, high capacity use is required.
3. Elution of the adsorbed product requires breaking the
antigen-antibody complex. Now this means that denaturing
conditions must be employed. Since the antibodies
themselves are proteins too, loss of some antibody binding
affinity typically occurs, resulting in gradual loss of column
capacity.
4. A first cycle on a new column gives poorer recovery than
successive operations, apparently due to some irreversible
binding.
5. A major economic goal in designing any affinity
chromatography setup is determination of optimal elution
buffer wash volumes and concentrations.
Now let us look at a specific example where affinity
chromatography is used, namely, in the purification of human
leukocyte interferon made in E. coli.
Proteins synthesized in genetically engineered organisms and
intended for injection into animals must be stringently purified.
Pyrogens from E. coli, including the outer envelope
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 lipopolysaccharide (LPS) must be removed or inactivated. Hence
BIOPHYSICS

product recovery operations such as affinity chromatography are

an important step in the manufacturing process.
Given below is a schematic representation of the typical steps
involved in processing human leukocyte interferon produced by
recombinant DNA techniques. This will give you an idea of
where exactly affinity chromatography is usually involved in the
realm of bioprocessing.

HUMAN LEUKOCYTE INTERFERON

|
E. coli EXTRACTION BY MECHANICAL BREAKAGE
|
POLYETHELYNEIMINE PRECIPITATION
|
AMMONIUM SULFATE PRECIPITATION OF
SUPERNATENT
|
DIALYSIS OF PELLET
| Since the sample is separated in the column, different peaks on
*IMMUNOADSORBENTCOLUMN (MONOCLONAL
ANTIBODIES)
| separations of protein mixtures by ion exchange
CATION EXCHANGE CELLULOSE
CHROMATOGRAPHY
Affinity chromatography involves the use of packing which has
been chemically modified by attaching a compound with a
specific affinity for the desired molecules, primarily biological
compounds. The packing material used, called the affinity
matrix, must be inert and easily modified. Agarose is the most
common substance used, in spite of its cost. The ligands, or
“affinity tails”, that are inserted into the matrix can be genetically
engineered to possess a specific affinity. In a process similar to
ion exchange chromatography, the desired molecules adsorb to
the ligands on the matrix until a solution of high salt
concentration is passed through the column. This causes
desorption of the molecules from the ligands, and they elute
from the column. Fouling of the matrix can occur when a large
number of impurities are present, therefore, this type of
chromatography is usually implemented late in the process.
Presentation of Results
Till now you have studied the basics of chromatography its
types and the functioning and now you will be able to see the
results in the form of graphs which is called as the
chromatogram. Here a example of the chromatogram is. The
figure below will help you to follow along in our discussion.
the chromatogram correspond to different components in the
sample mixture. The chromatograms above show the results of
chromatography. The lettered peaks correspond to different
proteins (A = ovalbumin, B = conalbumin, C = cytochrome c,
D = lysozyme). The separation corresponding to the
chromatogram on the left was performed at pH 5.85, while the
one on the right was performed at pH 6.5. It is evident that
operation conditions such as pH and temperature have a
significant effect on the output.
Analysis of the result:
• The level of complexity of the sample is indicated by the
number of peaks which appear.
• Qualitative information about the sample composition is
obtained by comparing peak positions with those of
standards.
• Quantitative assessment of the relative concentrations of
components is obtained from peak area comparisons.
• Column performance is indicated by comparison with
standards.
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:
1. identification of a “target protein” which may have
therapeutic value

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2. identification of the “target gene” — the gene responsible

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for encoding the target protein
3. isolation of the target gene
4. insertion of the target gene into a host cell (such as E. coli)
which will both grow well, and continue to
5. produce the protein product encoded for by the target gene
6. separation of the target protein from the many other host
cell proteins
7. large scale production of the target protein under controlled
manufacturing conditions
8. large scale testing for efficacy as a medicine
9. 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 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).

Notes

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