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Chromatography – An Overview

Chromatography is the
separation of a mixture of
compounds into its individual

©Mehau Kulky/Science Photo Library/Getty Images

components based on their
relative interactions with an inert
matrix. However,
chromatography is more than a
simple technique, it is an
important part of science
encompassing chemistry,
physical chemistry, chemical

Figure 1 These colored bands represent the separation of different

engineering, biochemistry and
chemicals by the process of chromatography. The process uses a solvent
to move the initial sample across some substrate (such as paper). The
cutting through different fields. It is
different physical properties of different chemicals will cause them to move
at different rates and separate. worth to be mentioned here that
the IUPAC definition of
chromatography is "separation of sample components after their distribution between
two phases". Biomolecules are purified using chromatography techniques that separate
them according to differences in their specific properties.
History and Discovery of Chromatography
Source: http://www.vipfaq.com/Mikhail%20Tsvet.html

Mikhail Tsvet (1872-1919), a Russian botanist,

discovered chromatography in 1901 during his
research on plant pigments. According to M. Tsvet: "An
essential condition for all fruitful research is to have at
one's disposal a satisfactory technique". He
discovered that he could separate colored leaf pigments
by passing a solution through a column packed
with adsorbent particles. Since the pigments
separated into distinctly colored bands as
represented in Figure 2 Mikhail Tsvet (1872-
1919), a Russian botanist,
Figure 3, he named discovered chromatography in
the new method 1901 during his research on
plant pigments.
(chroma – color, graphy –writing). Tswett emphasized later that colorless substances
can also be separated using the same principle. The separation results from the
differential migration of the compounds contained in a mobile phase through a column
uniformly packed with the stationary matrix. A mobile phase, usually a liquid or gas, is
used to transport the analytes through the stationary phase while the matrix, or
stationary phase, is generally an inert solid or gel and may be associated with various
moieties, which interact with the analyte(s) of interest. Interactions between the analytes
and stationary phase are non-covalent and can be either ionic or non-ionic in nature
depending on the type of chromatography being used. Components exhibiting fewer
interactions with the stationary phase pass through the column more quickly than those
that interact to a greater degree.

© 2013 Moustafa and Morsi

Figure 3 Schematic diagram of the principles of chromatography as discovered by Tswett (1901)

Tswett’s initial experiments involved direct visual detection and did not require a means
of quantitation. Nowadays, chromatography is not only a separation technique. In most
versions, it is hyphenated analytical techniques combining the separation with the
identification and quantitative determination of the separated components. In this form,
chromatography has become the most widely used technique in the chemical analysis
of complex mixtures. Many versions of chromatography are used. The various
chromatographic techniques are subdivided according to the physical state of these two
phases, the mobile and the stationary phases. These are: liquid chromatography
including high performance, ion, micellar, electrokinetic, thin-layer, gel-permeation, and
countercurrent versions; gas chromatography and supercritical fluid chromatography.
Various forms of chromatography can be used to separate a wide variety of
compounds, from single elements to large molecular complexes. By altering the
qualities of the stationary phase and/or the mobile phase, it is possible to separate
compounds based on various physiochemical characteristics. Among these
characteristics are size, polarity, ionic strength, and affinity to other compounds.
Chromatography also permits a great flexibility in the technique itself. The flow of the
mobile phase might be controlled by gravity, pressure, capillary action and electro-
osmosis; the separation may be carried out over a wide temperature range and sample
size can vary from a few atoms to many kilograms. Also, the shape of the system in
which the separation takes place can be varied, using columns of various length and
diameter or flat plates. Through all this, evaluation chromatography has been
transformed from an essentially batch technique into an automated instrumental
method. Through its continuous growth, chromatography became the most widely used
analytical separation technique in chemistry and biochemistry. Thus, it is not
exaggeration to call it the technique of the 20th Century.

Column Chromatography

Column chromatography is one of

the most useful techniques for
purifying compounds. This
technique utilizes a stationary

Copyright © 2019 American Chemical Society

phase, which is packed in a
column, and a mobile phase that
passes through the column. This
technique exploits the differences in
polarity between compounds,
allowing the molecules to be facilely
separated. The two most common
stationary phases for column
chromatography are silica gel (SiO2)
and alumina (Al2O3), with the most commonly used mobile phases being organic
solvents. The solvent(s) chosen for the mobile phase are dependent on the polarity of
the molecules being purified. Typically, more polar compounds require more polar
solvents in order to facilitate the passage of the molecules through the stationary phase.
Once the purification process has been completed the solvent can be removed from the
collected fractions using a rotary evaporator to yield the isolated material.

Copyright © 2017 Science Squared - all rights reserved

The sample mixture is placed on the top of the column and absorbed onto the top of the
stationary phase. Subsequently, the mobile phase is applied to the column and used to
elute the mixture through the stationary phase. Column chromatography exploits a
molecule's polarity to separate the compounds. The difference in polarity leads to
variances in the rate at which the molecules travel through the column, which effectively
separates the compounds from one another. The mobile phase is collected in small
fractions in test tubes as it elutes off the column, thus allowing for the isolation and
purification of the compounds. Lastly, the solvent is removed using a rotary evaporator
to yield the isolated compound(s).

Column chromatography's versatility and convenience has made it one of the most
widely used techniques for purifying compounds. Unlike recrystallization (another
commonly used purification technique) compounds purified with column
chromatography do not have to be solid. Column chromatography is also capable of
isolating a number of compounds from a mixture. Another advantage of column
chromatograph is that very little needs to be known about the compound's physical
properties in order to use this purification method, making this technique very valuable
when synthesizing or isolating novel compounds, in which little is known about the
Figure 5 Sample Separation by Column Chromatography
Biomolecules are purified using purification techniques that separate according to
differences in specific properties, as shown in Figure 6.

Property Technique
Biorecognition(ligand specificity) Affinity Chromatography
Charge Ion Exchange Chromatography
Size Gel Filtration Chromatography or (SEC)
Hydrophobicity Hydrophobic Interaction Chromatography
Reversed Phase Chromatography

© 2007, GE Healthcare Handbook

Figure 6 Schematic drawing of separation principles in chromatography purification. From left to right SEC, HIC, IEX, AC, and RPC.
Common Acronyms and Abbreviations

AC - affinity chromatography

AIEX - anion exchange chromatography

ATP - adenosine triphosphate

AU - absorbance units

BEH - bridged ethyl hybrid

BSA - bovine serum albumin

CF - chromatofocusing

CIEX - cation exchange chromatography

DNA - deoxyribonucleic acid

EDTA - Ethylenediaminetetra acetic acid

ELISA - Enzyme-linked immunosorbent assay

GF - Gel Filtration (also referred to as SEC, size exclusion chromatography)

HCl - hydrochloric acid

HIC - hydrophobic interaction chromatography

HIV - human immnunodeficiency virus

HMW - high molecular weight

HPLC - high-performance liquid chromatography

IEF - isoelectric focusing

IEX - ion exchange chromatography

IGF-1 - insulin-like growth factor 1

IgG - immunoglobulin G

IgM - immunoglobulin M
IMAC - immobilized metal affinity chromatography

IU - inhibitor activity units

IUPAC - International Union of Pure and Applied Chemistry

LMW - low molecular weight

NaCl - sodium chloride

PAGE - polyacrylamide gel electrophoresis

PBS - phosphate-buffered saline

pI - isolectric point

psi - pounds per square inch

PVDF - polyvinylidene fluoride

RI - refractive index

RNA - ribonucleic acid

RPC - reversed phase chromatography

SDS - sodium dodecyl sulfate

SEC - size exclusion chromatography (also referred to as GF, gel filtration)

ssDNA- single-stranded DNA

ssRNA- single-stranded RNA

SR - solvent resistant

TLC - Thin Layer Chromatography

Chromatography terminology

Adapter Often used for the movable end pieces of columns; contains filter, flow
distributor, and possibility to connect tubing.

Adsorption The process of interaction between the solute (for example, a protein) and
the stationary phase.

Affinity chromatography A group of methods based on various types of specific

affinities between target molecule(s), for example, a protein and a specific ligand
coupled to a chromatography medium.

Asymmetry (asymmetry factor) Factor describing the shape of a chromatographic


Backpressure The pressure drop across a column and/or a chromatography system.

Band broadening The widening of a zone of solute (for example, a protein) when
passing through a column or a chromatography system. Gives rise to dilution of the
solute and reduces resolution. Also often called peak broadening or zone broadening.

Binding Adsorption. The process of interaction between a solute (for example, a

protein) and the stationary phase.

Binding buffer Buffer/solution/eluent used for equilibration of the column before sample

Binding capacity The maximum amount of material that can be bound per ml of
chromatography medium. See also Dynamic binding capacity.

Capacity factor The degree of retention of a solute (for example, a protein) relative to
an unretained peak.

Chromatofocusing Method that separates proteins on the basis of pI.

Chromatogram A graphical presentation of detector response(s) indicating the

concentration of the solutes coming out of the column during the purification (volume or

Chromatography From Greek chroma, color, and graphein, to write.

Chromatography medium/media The stationary phase, also called resin. The

chromatography medium is composed of a porous matrix that is usually functionalized
by coupling of ligands to it. The matrix is in the form of particles (beads) or, rarely, a
single polymer block (monolith).
CIP (cleaning-in-place) Common term for cleaning chromatography columns and/or
systems with the purpose of removing unwanted/ nonspecifically bound material.

Column Usually column hardware packed with chromatography medium.

Column equilibration Passage of buffer/solution through the chromatography column

to establish conditions suitable for binding of selected sample components. For
example, to establish correct pH and ionic strength, and ensure that proper counter ions
or counter ligands are present.

Column hardware The column tube and adapters. All pieces of the column except the
chromatography medium/the packed bed.

Column hardware pressure The pressure inside the column. Column hardware
pressure that is too high can break the column.

Column packing Controlled filling of the column hardware with chromatography

medium to obtain a packed bed.

Column volume The geometrical volume of the column interior/the chromatography

bed. Counter ion Ion of opposite charge that interacts with an ion exchange
chromatography medium after the column equilibration. The counter ion is displaced by
a protein that binds to the ion exchanger. If a high concentration of the counter ion is
applied, it will compete with the bound protein and elute it from the chromatography

Counter ligand Substances that interact with ligands of a chromatography medium and
can be displaced by a solute (for example, protein) binding to the ligand.

Dead volume The volume outside the packed chromatography bed. Can be column
dead volume or chromatography system dead volume. The dead volume contributes to
band broadening.

Degassing Removal of dissolved air from buffers/solutions.

Desorption Elution Release or removal of bound substances from the chromatography


Efficiency Measured as number of theoretical plates. High efficiency means that sharp
peaks will be obtained.
Effluent The mobile phase leaving the column (= eluate).

Eluate The mobile phase leaving the column (= effluent).

Eluent The buffer/solution used during chromatography (= mobile phase).

Elution buffer Buffer/solution used for elution (desorption) of bound solutes (for
example, proteins) from a column.

Elution volume The volume of buffer/solution (eluent) required to elute the solute for
example, a protein (= retention volume).

Elution time The time required for elution of a solute (protein) (= retention time).

Flow rate Volumetric flow (ml/min) or linear flow rate (cm/h). Measurement of flow
through a column and/or chromatography system.

Flowthrough Material passing the column during sample loading (without being

Frit Type of deep filter often used at top and bottom of columns.

Gel filtration (GF) Size-exclusion chromatography. Separates solutes (for example,

proteins) according to size.

Gradient elution Continuous increased or decreased concentration of a substance (in

the eluent) that causes elution of bound solutes (for example, proteins).

Hydrophobic interaction chromatography (HIC) Method based on the hydrophobic

interaction between solutes (for example, proteins) and the chromatography medium in
the presence of high salt concentration.

Hydroxyapatite chromatography Mixed-mode ion exchange chromatography method.

Immobilized metal ion affinity chromatography (IMAC) Method based on the affinity
of proteins with His, Cys, or Trp amino residues on their surface and metal ions on the
chromatography medium.

Ion exchange chromatography (IEX) Method based on electrostatic interactions

between solutes (for example, proteins) and chromatography medium.

Isocratic elution Elution of the solutes without changing the composition of the
buffer/solution (eluent).

Ligand The specific molecular group that is coupled to the matrix to give some decided
function to the chromatography medium.

Ligand density Related to ligand concentration. The distribution of ligands on the

surfaces (also surfaces inside pores) of the chromatography matrix.

Linear velocity The flow rate normalized by the column cross section (cm/h).
Mass transfer Movement of a solute (for example, a protein) in and out of the
stationary phase. Important factor for column efficiency.

Matrix The matrix is the nonfunctional base for the chromatography medium. The
matrix has a porous structure that provides a large surface that can be modified with
ligands that introduce possibilities for protein binding.

Mobile phase The fluid (buffer/solution) carrying the solutes during chromatography (=

Peak broadening Same as band broadening.

Peak capacity The number of peaks that can be separated using a chromatography

Peak tailing Broadening at the end of a peak due to additional delay of a fraction of the
solute. Results in increased asymmetry factor.

Pore Cavity in a chromatography matrix.

Pore volume The total volume of the pores in a chromatography medium.

Pressure over the packed bed The pressure drop across the packed bed upon
passage of solution through the column. Caused by flow resistance in the packed bed.

Recovery The relative amount of target protein that is retrieved after purification
compared with amount loaded on the column.

Resin The term is sometimes used instead of the more generic term, chromatography

Resolution Measurement of the ability of a packed column to separate two solutes


Retention volume Same as elution volume.

Retention time Same as elution time.

Reversed phase chromatography (RPC) Method based on hydrophobic interactions

between solutes (sample components) and ligands coupled to the chromatography
medium. Organic modifiers (for example, acetonitrile) in the eluent are used for elution.

Sample The material loaded on the chromatography column/medium, or to be

analyzed. Sample application Applying/loading sample on the column.
Sample loading Loading/applying sample on the column.

Sample volume Usually the volume of the sample loaded on the chromatography

Selectivity Measure of the relative retention of two solutes in a column. Related to the
distance between two peaks.

Solute The dissolved substance (for example, a protein) in, for example, the mobile

Stationary phase Often called resin, chromatography beads, chromatography material,

chromatography medium or media.

Step gradient elution Stepwise increase in concentration of the substance that affects
elution of bound solutes.

Void volume The elution volume of solutes that do not enter the pores or interact with
the chromatography medium, thus passing between the beads in the packed bed.

Wash Wash step. Removal of unbound or weakly bound material from a column after
the sample loading.

Wash buffer Buffer/solution used for washing the column after sample loading.

Wash volume Volume of buffer/solution used for the wash step.

Yield Amount of target protein (or other solute) obtained after a purification step, or after
the entire purification (multiple steps).

Zone broadening Same as peak broadening.

Size Exclusion
Mori and Barth (1999)
mentioned in their book that by far
the most popular and convenient
method of determining the average
molecular weight and the MWD of a
polymer is size exclusion
chromatography (SEC). SEC has
essentially supplanted many

© 2007, GE Healthcare Handbook

classical MW techniques for routine
measurements. SEC separates on
the basis of molecular hydrodynamic
volume or size, rather than by
enthalpic interactions with the

Figure 7 Process of SEC. (A) Schematic picture of a bead with an

stationary phase or packing surfaces,
electron microscopic enlargement. (B) Schematic drawing of sample
as is the case with other modes of
molecules diffusing into bead pores. (C) Graphical description of
liquid chromatography, such as
separation: (i) sample is applied on the column; (ii) the smallest
molecule (yellow) is more delayed than the largest molecule (red);
adsorption, partition, or ion exchange.
(iii) the largest molecule is eluted first from the column. Band
broadening causes significant dilution of the protein zones during
In SEC, the polymer is dissolved in an
chromatography. (D) Schematic chromatogram.
appropriate solvent and is injected
into a column packed with porous particles of fairly defined pore size.
Mori and Barth (1999) also stated that the SEC mobile phase is generally the
same solvent used to dissolve the polymer. As the polymer elutes through the column,
molecules that are too large to penetrate the pores of the packing elute in the interstitial
or void volume of the column.
“As the molecular size of the polymer decreases with respect to the average
pore size of the packing, polymer molecules penetrate into the pores and access
greater pore volume and, as a result, elute at a later time. Small molecules, which can
freely diffuse into and out of the pores and can sample the total pore volume, elute at
the total elution volume of the column. High-molecular-weight material elutes first from
an SEC column, followed by low-molecular-weight components,” Barth mentioned.
Since SEC is a relative and not an absolute molecular weight technique,
columns must be calibrated with polymer standards of known molecular weight, or an
online light scattering detector must be used. When compared to other modes of HPLC,
the instrumentation requirements for SEC are somewhat simpler since mobile phase
gradients are not used; however, computer support for data acquisition and processing
is critical. Depending on the application, high-temperature capability, for example,
150°C may be required. Although any concentration-sensitive detector can be used in
SEC, the differential refractometer is the most widely utilized (Cooper, 1989).
Column calibration is of utmost concern in SEC, and a number of different
approaches have been developed, including the use of molecular-weight sensitive
detectors. Column and mobile phase selection are obviously essential to the success of
an SEC separation (Mori & Barth, 1999).

On the other hand, Shandilya in 2013 stated that:

Size-exculsion chromatography (SEC), also called gel filtration or gel-permeation
chromatogaphy (GPC), uses porous particles to separate molecules of different sizes. It is
generally used to separate biological molecules, and to determine molecular weights and
molecular weight distributions of polymers. It is usually applied to large molecules or
macromolecular complexes such as proteins and industrial polymers. When an aqueous
solution is used to transport the sample through the column, the technique is known as Gel-
filtration chromatography organic solvent is used as a mobile phase, the technique is known
as Gel-permeation chromatography. The separation of molecules is called fractionation. Size
of pores in beads determines the exclusion limit (what goes through the beads and what goes
around the beads).

History and Discovery

Source: http://soft-matter.seas.harvard.edu
Source: https://www.nobelprize.org

© Uppsala University

Proponents of SEC. from left to right: Richard L. M. Synge,Arne Tiselius, and J.W. McBain.

The concept of size-based separations by chromatography was first speculated

by Synge and , based on the observation that small molecules could be excluded from
the small pores of zeolites as a function of their molecular size. The term “molecular
sieve,” coined by J. W. McBain to describe this property of zeolites, was subsequently
used to describe the technique commonly known today as size-exclusion
chromatography. Over the years, SEC has been known by a number of other names,
such as exclusion chromatography, steric-exclusion chromatography, restricted-
diffusion chromatography, liquid-exclusion chromatography, gel-filtration
chromatography, and gel-permeation chromatography. The first examples of size-based
separations by liquid chromatography were noted by Wheaton and Bauman in their
work on ion-exclusion chromatography. They observed that various nonionic species
could be separated on ion-exchangers by a size-based mechanism. Similarly, R. T.
Clark demonstrated the separation of sugar alcohols on a strong cation exchange resin.
Lindqvist and Storgårds reported the first separation of biomolecules by a size-
exclusion process, where they separated peptides from amino acids on a column
packed with starch. Subsequently, Lathe and Ruthven performed extensive
characterizations on columns packed with potato or maize starch, which demonstrate
very low adsorption of proteins. Using a column packed with maize starch, they were
able to separate a variety of compounds including proteins and peptides by the
“molecular sieve” effect.
In the 1970s, derivatized porous silica became the predominant chromatographic
stationary phase media due to its superior mechanical strength, non-swelling nature and
inertness over a fairly wide range of conditions. The utility of porous silica for SEC was
explored, as the greater mechanical strength provided a means to further improve
performance by reducing particle size. As a size-exclusion medium for proteins, it
suffered from strong ionic interactions due to the acidic surface silanols. To mitigate
these interactions, both surface modifications and mobile phase additives were
employed. Surface modifiers include glyceropropylsilane and N-
acetylaminopropylsilane. However, these functional groups are non-ideal as they exhibit
significant hydrophobic interactions with proteins. The most commonly used surface
modifier today is a diol functional group, which has minimal hydrophobic interactions.
However, even with high coverage, a significant concentration of surface silanols still
remains. To further diminish interactions with these residual silanols, high ionic strength
mobile phases are typically required.
More recently, porous hybrid organic/inorganic particleshave been employed as
the base particle for size-exclusion chromatography. The bridged ethyl hybrid (BEH)
particles, surface modified with diol groups, provide a significant reduction in silanol
activity, thus requiring lesser amounts of salt additives to minimize the ionic interactions
with proteins. In addition, the high mechanical strength of BEH particles enables a
reduction in particle size to 1.7 μm, providing gains in chromatographic

A mixture of molecules dissolved in liquid (the
mobile phase) is applied to a chromatography
Source: tutkiabiotech.blogspot.com

column which contains a solid support in the form of

microscopic spheres, or "beads" (the stationary
phase). The mass of beads within the column is
often referred to as the column bed. The beads act
as "traps" or "sieves" and function to filter small

Figure 8 Principle of SEC

molecules which become temporarily trapped within the pores. Larger molecules pass
around or are "excluded" from the beads. Large sample molecules cannot or can only
partially penetrate the pores, whereas smaller molecules can access most or all pores.
Thus, large molecules elute first, smaller molecules elute later, while molecules that can
access all the pores elute last from the column. Particles of different sizes will elute
(filter) through a stationary phase at different rates (Shandilya, 2013).

1. Semi-permeable, porous beads with well-defined range of pore sizes.
2. Beads are crosslinked polymers.
3. Degree of crosslinking is controlled carefully to yield different pore sizes.
4. Smaller pore sizes are used for rapid desalting of proteins for protein purification.
5. Intermediate pore sizes are used to separate relatively small proteins.
6. Very large pore sizes are used for purification of biological complexes.
7. Stationary phase used for gel exclusion chromatography include dextran
(SephadexTM), polyacrylamide and dextran- polyacrylamide (SephacryTM).
8. Each is available with a variety of different ranges of pore size in the beads,
permitting separation of macromolecules of different size.

1. The liquid used to dissolve the biomolecules to make the mobile phase is usually
called a buffer.
2. The mixture of biomolecules dissolved in the buffer is called the sample.
3. The choice of mobile phase to be used in any separation will depend on the type of
separation to be achieved and component to be separated.
4. The most common eluents in for polymers that dissolve at room temperature. (e.g.-
Tetrahydrofuran, Chloroform, Dimethyl formamide).

The solvents used for mobile phase of SEC are limited to those follows following
 The solvent must dissolve the sample completely.
 The solvent has different properties with solute in the eluent: typically with solvent
refractive index (RI) solvent must not degrade the sample during the viscosity of
eluent will gradually increase over times.
 The solvent is not corrosive to any components of the equipment.


1. High purity of solvent is recommended.
2. Filter mobile phase solvents using 0.5 micron filter to remove any particular
impurities such as dusts, insoluble salts.
3. Antioxidant is added to trichlorobenzene to keep solvent stable in high temperature.
4. Other additives eliminate adsorption or interaction of solutes with column packing

 The sample solutions should be less than 2 mg/mL.
 Samples with broad molecular weight distribution may require higher
 It is recommended to filter the sample solutions before injecting into columns in
order to get rid of clogging and excessively high pressure problems.
 Agitation and filtration is required to remove insoluble impurities. Do not agitate and
filter samples that contain very high MW (>1 million).

Taken from the books of Switzer & Garrity (1999) and Synder, Kirkland,& Glajch (1997):
1.) You will prepare your column the week before the start of the lab as the gel needs
time to settle.
2.) Obtain the SEC gel (G-100) from your instructor. It will already be soaking in 0.05
M pH 7 TRIS-HCl buffer with 0.1 M NaCl. This allows the beads to swell to their full size
before pouring.
3.) Obtain a column from your instructor and plug the bottom with glass wool.
4.) Close the stop cock. Swirl the gel in the Erlenmeyer flask and pour it down a glass
rod. If you have an air bubble, place parafilm on the top of the column, remove the
column from the stand. Invert the column a number of times to redisperse the gel and
then allow to resettle. If the air bubble is near the top of the column, you may be able to
remove it with your glass rod.
5.) Pour the column at least 30-40 cm high.
6.) Open the stop cock and allow the buffer to pull through the column to just above
the level of the gel.
7.) Close the stop cock. Cover the top with parrafilm and place in the refrigerator until
the following week.


1.) Remove the column from the fridge and clamp it to the stand in a buret clamp.
2.) Obtain 250 mL of each of Blue dextran and DNP-amino acid in phosphate buffer.
3.) Mix the two samples to give a total volume of 0.5 mL.
4.) DO NOT LET THE COLUMN RUN DRY. Make sure that the level of the buffer at
the top of the column is very close to the top level of the gel. If not, drain the buffer until
it reaches the top of the gel.
5.) Using a long Pasteur pipette, add your 0.5 mL sample to the top of the gel with the
stopcock closed. Do not disturb the gel at the top of the column.
6.) Place a graduated cylinder under the stopcock. Open the stopcock and allow the
sample to move onto the column. Close the stop cock.
7.) Apply 1 mL of buffer to the top of the column.
8.) Drain into the graduated cylinder.
9.) Add 5-10 mL of buffer to the top of the column and drain into the graduated
10.) Continue collecting volume into a graduated cylinder until the first appearance of
blue color (from blue dextran) is seen to elute from the column. Record this total
volume as accurately as possible (Vo).
11.) Collect all the blue dextran into the graduated cylinder. Remember to keep filling
the buffer at the top of the column gently—do not disturb the level of the column)
12.) After all the blue dextran is past, record the total volume at this point.
13.) In a new graduated cylinder, collect volume from the time the blue dextran stops
until the yellow DNP-amino acid elutes. Record this volume. Drain the column until all
yellow dextran is removed. Vt is the total volume collected (blue dextran plus DNP-
amino acid).
14.) Close the stopcock.


1.) Obtain a mixture of two protein standards. Record the name of the standards and
their molecular weight. (Myoglobin at 16,500 and bovine serum albumin at 67,000)
2.) Your elution order will be from largest to smallest. The proteins were chosen to try
to cover the size exclusion range of the gel we are using.
3.) Apply 1 mL of the standard to the top of the column, again, taking care not to
overly disturb the top of the column.
4.) Allow the sample to migrate onto the column by opening the stopcock. The eluting
volume can be collected into a graduate cylinder up until 1 mL before the Vo determined
in Part 1.
5.) Add 1 mL of buffer and collect the eluate.
6.) Add 5-10 mL at a time to the top of the column, taking care that it does not dry out.
7.) Collect your eluate in 1 mL marked test tubes beginning 1 mL before the Vo.
8.) Continue collecting fractions until you react Vt.
9.) Analyze your fractions on the UV-VIS. Be sure to run a blank of your buffer at 280


1.) Absorbance vs. Volume and determine the volume where each of the three
standard proteins eluted. Plot absorbance vs. volume for the unknown protein to
determine where the unknown elutes.
2.) Plot MW vs. Vr/Vo for each protein (if you want you can also graph blue dextran
and DNP amino acid.) Fit to a linear line in Excel and obtain the equation of the line.
3.) Calculate Kavg and plot log MW vs. Kavg for the protein samples. Fit to a linear
4.) Use the equations from each plot to determine the molecular weight of the

In a journal, Werner et al., (1994) emphasized that:
SEC is not a high resolution technique; generally the molecules to be separated must differ
by at least two-fold in molecular weight. Therefore, SEC is useful for desalting or removal of
small molecule contaminants from protein samples, determination of the solution subunit
composition of a multimeric protein, and to isolate different multimers from each other. Size
exclusion chromatography is generally used near the end of the purification process for a
protein of interest, for example to desalt the protein or separate the correctly folded native
protein from the denatured protein.

Key Points and Insights

As described in the beginning, size exclusion chromatography is a technique
used to sort molecules by size. However, even for molecules with the same size, some
can have a low molecular weight with a loosely packed structure, while others can have
a high molecular weight with a high-densely packed structure. Depending on the type of
solvent the sample is dissolved in, some structures can spread out, while others can
move closer together. In other words, the molecular weight cannot be determined by
elution position alone.
Therefore, if the substance in the molecular weight marker (a standard sample
with molecular weights known in advance) differs from the substance in the
measurement sample, molecular weights and molecular weight distributions cannot be
calculated accurately. The ideal situation is a molecular weight marker of the same
material as the measurement sample, if available. However, if not, use a molecular
marker that is structurally as similar to the sample as possible or use correction factors,
such as Q-factors (molecular weight per unit chain length), or other techniques to get as
close to appropriate values as possible.
In addition, accurate molecular weight distributions cannot be determined if
there is interaction between the sample components and the packing material (such as
adsorption or ion exchange). Such interaction occurs frequently when using aqueous
gel filtration chromatography. In such cases, a salt is added to the eluent to reduce the
Size exclusion chromatography is based on a relatively simple principle, but can
involve complicated considerations when actually performing analyses or analyzing
separates proteins on the
basis of a reversible
interaction between a

© 2007, GE Healthcare Handbook

protein (or group of
proteins) and a specific
ligand coupled to a
chromatography matrix.
Figure 9 Common terms in affinity chromatography
The technique is ideal for a capture or
intermediate step in a purification protocol and can be used whenever a suitable ligand
is available for the protein(s) of interest. With high selectivity, hence high resolution, and
high capacity for the protein(s) of interest, purification levels in the order of several
thousand-fold with high recovery of active material are achievable. Target protein(s) is
collected in a purified, concentrated form. Biological interactions between ligand and
target molecule can be a result of electrostatic or hydrophobic interactions, van der
Waals’ forces and/or hydrogen bonding. To elute the target molecule from the affinity
medium the interaction can be reversed, either specifically using a competitive ligand, or
non-specifically, by changing the pH, ionic strength or polarity.
History and Discovery
According to the research of Acikara et. Al
(2013), affinity chromatography is one of the oldest
forms of liquid chromatography method. The first
use of the idea of affinity chromatography may be
considered as the isolation of α-amylase by using an
insoluble substrate, starch, in 1910 by Starkenstein.
Similar studies with starch and amylase were carried
out in the 1920s through 1940s by other
Source: http://abicko.avcr.cz

investigators. In another study polygalacturonase

was used as a support and ligand for the adsorption
of alginic acid, the purification of pepsin through the
Figure 10 Emil Starkenstein (1884-1942)
use of edestin, a crystalline protein and the isolation
of porcine elastase with powdered elastin were also performed. Afterwards Willstatter et
al. enriched lipase by selective adsorption onto powdered stearic acid (Roque and
Lowe, 2010). The majority of the previous studies related purification of the enzymes.
However the selective purification of antibodies with biological ligands was
also being conducted. In 1920, it was reported that antibodies can recognize
and bind substances with a specific structure, "antigens" (Hage, D.S., 2006).
This principle is firstly used in order to isolate rabbit
anti-bovine serum albumin antibodies on a specific
immunoadsorbent column consisting bovine serum
albumin coupled to diazotized paminobenzyl-
©2004, I P Hurley Journal of dairy science 87

cellulose. According to this approach, antibodies

were isolated using urease and exhibited that these
antibodies were proteins. Biomolecules purified by
affinity chromatography Antibodies were first purified
using affinity chromatography in 1951 when

Figure 11 Anti-Bovine IgG (whole

molecule)−Peroxidase antibody produced in Campbell et al. used affinity chromatography
to isolate rabbit anti-bovine serum albumin antibodies
(Campbell et al, 1951). For their purification, bovine serum albumin was used as the
affinity ligand on a cellulose support. Two years later, this technique was expanded to
purify mushroom tyrosinase using an immobilized inhibitor of the enzyme (azophenol)
(Lerman, 1953). Since then, affinity chromatography is commonly used to purify
biomolecules such as enzymes, recombinant proteins, antibodies, and other
biomolecules. Affinity chromatography is often chosen to purify biomolecules due to its
excellent specificity, ease of operation, yield and throughput. In addition, affinity
chromatography has the ability to remove pathogens, which is necessary if the purified
biomolecules are to be used in clinical applications. The purity and recovery of target
biomolecules is controlled by the specificity and binding constant of the affinity ligand. In
general, the association constants of affinity ligands used for biomolecule purification
range from 103 – 108 M-1 (Janson, 1984). A common affinity ligand used in these
purifications is an antibody, but other affinity ligands such as biomimetic dye-ligands,
DNA, proteins and small peptides have been used as well.


Affinity chromatography
which is known as a liquid
chromatographic technique for
separation and specific analysis of
biomolecules based on their
© 2016 Caframo lab solutions

biological functions or individual

structures has become increasingly
important and useful separation

Figure 12 Principal steps of affinity chromatography. method in pharmaceutical science,

biochemistry, biotechnology and
environmental science in recent years (Hage, 1999).
This technique is especially known as the most specific and effective technique
for protein purification. (Lowe CR, 2001). Separation of the biomolecules is based on
highly specific biological interactions (molecular recognition) between two molecules,
such as enzyme and substrate. These interactions, which are typically reversible, are
used for purification by placing one of the interacting molecules, referred to as affinity
ligand, onto a solid matrix to create a stationary phase while the target molecule is in
the mobile phase (Zhao et. Al, 2009)

Figure 13.a One way to elute the bound protein is to introduce free ligand that will bind to the
target molecule


Figure 13.b The bound protein can be eluted by introducing another protein that will outcompete
the target protein and bind to the ligand

Affinity chromatography separates proteins on the basis of a reversible

interaction between a protein or group of proteins and a specific ligand coupled to a
chromatography matrix The technique offers high selectivity, hence high resolution, and
usually high capacity for the protein interest. Purification can be in the order of several
thousand-fold and recoveries of active material are generally very high. Affinity
chromatography is unique in purification technology since it is the only technique that
enables the purification of a biomolecule on the basis of its biological function or
individual chemical structure. Purification that would otherwise be time-consuming,
difficult or even impossible using other techniques can often be easily achieved with
affinity chromatography. The technique can be used to separate active biomolecules
from denatured or functionally different forms, to isolate pure substances present at low
concentration in large volumes of crude sample and also to remove specific
contaminants. (GE Healthcare Bio-Sciences, 2007).

© 2011 Elsevier Inc.

Figure 14 Purification Process

Affinity chromatography utilizes specific and irreversible biological interactions

between a ligand covalently coupled to a support material and its complementary target.
The solid support and ligand covalently attached on it, selectively adsorbs the
complementary substance from the sample. The unbound part of the sample is
removed and the purified substance can easily be recovered. Selectivity of the ligand,
recovery process, throughput, reproducibility, stability and economic criteria are some of
the factors that influence the success of affinity chromatography process. Successful
affinity purification requires a certain degree of knowledge and understanding of the
nature of interactions between the target molecule and the ligand to help determine the
selection of an appropriate affinity ligand and purification procedure. Ligands are often
based on biological functional pairs, such as enzymes and substrate, antigens and
antibodies, receptor and hormone, or lectins to sugars chains on proteins. The specific
ligand binds the protein of interest and all non-specific molecules are washed away.
Recovery of the bound protein can be achieved by changing experimental conditions to
favor desorption. The protein is eluted in a specific buffer, either by pH and/or ionic
strength shift or by competitive displacement; a solution is passed through the column
that has a high concentration of free ligand. This is a very efficient purification method
since it relies on the biological specificity of your target protein, such as the affinity of an
enzyme for a substrate.

Traditionally, affinity
chromatography support materials have
consisted of porous support materials
such as agarose, polymethacrylate,
polyacrylamide, cellulose, and silica. All
of these support materials are
commercially available and come in a
range of particle and pore sizes. Some
Copyright © 2019 Elsevier B.V.

supports may be available with common

affinity ligands already immobilized (e.g.
protein A, Cibacron Blue, heparin). Other
types of support materials are being

Figure 15 the principal of affinity chromatography in a developed including nonporous supports,

column format is depicted in this illustration. An
immobilized ligand is attached to an insoluble support membranes, flow-through beads (perfusion
material which is typically beaded in nature and the affinity
resin is packed into a column. The column is equilibrated media), monolithic supports, and
with binding buffer and a sample containing a target
molecule which has affinity for the ligand is passed through expanded-bed adsorbents.
the column. The target molecule binds to the immobilized
ligands and is retained in the column while any not-bound As it is defined above; this technique
material is washed off. An elution buffer is then added to
the column, which disrupts the interaction between the is based on the interactions between
ligand and the target molecule and allows the purified
target to be isolated. specific bioactive substances, so the
ligands are supposed to be originally
biological substances, nevertheless columns with non-biological ligands are also
available and the same term “affinity chroma‐ tography” is used for the techniques
performed by using these ligands. In order to distinguish the techniques according to
the origin of the ligand, affinity chromatography with biological ligands may be termed
as “bio-affinity chromatography” or “bispecific adsorption”.
According to Dubey, theoretically affinity chromatography is capable of giving
absolute purification in a single step. The technique was developed for purification of
enzymes but now affinity chromatography is used for various other purposes like
purification of nucleotides, nucleic acid, immunoglobulin, membrane receptors etc.
(Dubey, V.K.)

1. Centrifuge the Affinity Column briefly (~500xg) for 5 seconds to bring the resin to
the bottom of the column.

2. Open the cap and break off the bottom plug of the column. Place the column in a
2ml tube and centrifuge for 5 seconds to let the buffer drain out.

3. Equilibrate the column: Place the column in a 2ml collection centrifuge tube. Add
0.2ml Affinity Binding Buffer to the column and centrifuge for 5 seconds to let the
buffer drain into the 2ml tube, discard the buffer collected in the collection tube
(the flow through). Repeat this step twice to thoroughly equilibrate the column.

OPTIONAL: These chromatography steps (equilibration, binding, and elution) may also
be performed by simply allowing the buffers to slowly drip into the collection tubes.
However, the drip method will be considerably slower.

4. Place the column in a clean 2ml tube. Carefully load 100µl ASA (Animal Serum
Albumin) to the column. Incubate for 5minutes and then centrifuge for 5 seconds
and collect the flow-through in the collection tube, label this tube Fraction 1.

5. Wash the column 3 times with Affinity Binding Buffer: Place the column in a new
tube. Apply 0.2ml Affinity Binding Buffer to the column. Centrifuge for 5 seconds
and label the flow through Fraction 2. Repeat this step twice in two separate
tubes and label the flowthroughs Fractions 3 and Fraction 4, respectively.

6. Elute the sample using a salt gradient: A salt gradient elution buffer is prepared by
mixing the Affinity Elution Buffer and the Elution Buffer, which has a high
concentration of salt, as in the table below:

7. For gradient elution, place the column into a clean tube. Apply 0.2ml of the lowest
salt concentration buffer (Fraction 5). Centrifuge for 5 seconds and collect the
fraction in a 2ml collection tube.

8. Place the column in a fresh, clean tube and apply the next elution buffer starting
with fraction 6 through to 10 and repeat step 7, until all the elution buffers have
been added. Collect all 6 fractions in 6 separate 2ml tubes.

RED660 Protein Assay

1. A protein assay can be undertaken to determine the distribution of the proteins.

2. Label 10 tubes and transfer 20μl sample from each fraction to the labeled tubes.

3. Mix the RED660 Reagent gently by inverting the bottle several times.

To avoid foaming, DO NOT SHAKE THE BOTTLE

4. Add 1ml RED660 Reagent to each tube and vortex briefly to mix the content.
Incubate the tubes at room temperature for 5 minutes.

If using a spectrophotometer, proceed to step 5. If not, examine the tubes closely, the
stronger the color the higher the concentration of protein.
5. In the meantime, turn on the spectrophotometer to allow it to warm up. Adjust the
wavelength to 660nm.

6. Add 1ml distilled water to a cuvette to zero the absorbance of the

spectrophotometer. Measure the absorbance of each tube and record the values in
the results section.

7. The absorbance can be measured with a microplate reader instead of using a

spectrophotometer. Transfer 250μl from each assay tube to a microtiter plate well. Add
250μl distilled water to a well as reference blank. Read the absorbance at 660nm.

OPTIONAL: The purified protein can also be identified by polyacrylamide gel

electrophoresis. Perform SDS-electrophoresis with crude protein extract and the
purified fractions and examine the distribution of the protein bands.

Affinity chromatography is currently being used for a wide variety of applications
ranging from the study of drug-protein binding interactions to the depletion of high
abundance proteins to enhance the detection/quantification of dilute proteins. Affinity
chromatography can be used to study drug-protein binding interactions. Frontal
analysis, zonal elution, and the Hummel-Dreyer method can be used to measure drug
protein binding constants, to quantify kinetic properties of the various interactions, to
quantify allosteric interactions, and to identify drug binding sites. More information about
the measurement of drug-protein binding constants can be found in two review articles
(Hage, 2002; Hage et al., 2011). Information on quantifying kinetic properties of drug-
protein interactions can be found in a review by (Schiel & Hage, 2009). A discussion on
the quantification of allosteric interactions by affinity chromatography can be seen in an
article by (Chen & Hage, 2004). Additional information on the identification of drug-
binding sites can be found in a review article (Hage & Austin, 2000). When trying to
analyze low abundance proteins, it is often necessary to remove high abundance
proteins prior to analysis. This removal effectively enriches low abundance proteins and
allows more of them to be identified and quantified. Removal of the top 7 or top 14 high-
abundance proteins has been shown to result in a 25% increase in identified proteins
(Tu et al, 2010). Moreover, affinity chromatography is widely used in many ‘omics’
studies (e.g. proteomics, metabolomics and genomics) and is currently used in tandem
with other methods to develop high-throughput screening methods for potential drugs.

Key Points and Insights

Ion Exchange
Classical liquid

Lehninger Principles of Biochemistry, Fifth Edition,© 2008 W.H. Freeman and Company
chromatography based on
adsorption- desorption was
essentially a non-linear
process where the time of
retardation (retention time)
and the quantitative
response depend on the
position on the adsorption
isotherm. Essentially, it was
a preparative technique: the
aim was to obtain the
components present in the
sample in pure form which
could then be submitted to
further chemical or physical
Ion exchange
chromatography (or ion
Figure 16 Ion exchange chromatography technique chromatography, IC) is a
subset of liquid
chromatography which is a process that allows the separation of ions and polar
molecules based on their charge. Similar to liquid chromatography, ion chromatography
utilizes a liquid mobile phase, a separation column and a detector to measure the
species eluted from the column. Ion exchange chromatography can be applied to the
determination of ionic solutes, such as inorganic anions, cations, transition metals, and
low molecular weight organic acids and bases. It can also be used for almost all kinds of
charged molecule including large proteins, small nucleotides and amino acids. The IC
technique is frequently used for the identification and quantification of ions in various
History and Discovery
Beginning about 1947, Spedding
and Powell at Iowa State published a

Source: Hamish Small: Experimenter Extraordinaire LCGC North America

series of papers describing practical
methods for preparative separation of the
rare earths by displacement ion-
exchange chromatography. The same
group then demonstrated the ion-
exchange separation of 14N and 15N
isotopes in ammonia. Beginning in the
1950s. Kraus and Nelson at Oak Ridge
published numerous analytical methods
for metal ions based on separation of

Volume 33, Issue 10

their chloride, fluoride, nitrate or sulfate
complexes by anion chromatography. In
the period from about 1960 to 1980 many
Figure 16 Left to right: W.C Bauman, H. Small, and T. clever chromatographic methods for
Stevens during 1979 Pittsburgh Applied Analytical Chemistry metal ion separations were reported by
Awards. researchers throughout the world and
automatic in-line detection was gradually introduced. A truly innovative method by
Small, Stevens and Bauman at Dow Chemical Co. marked the birth of modern ion
chromatography. Anions, as well as cations, could now be separated quickly and
conveniently by a system of suppressed conductivity detection. A method for anion
chromatography with non-suppressed conductivity detection was published by Gjerde et
al. in 1979. This was followed by a similar method for cation chromatography in 1980.
Ion chromatography as we know it today did not just happen. It was built on a solid
foundation of knowledge that has accumulated over a period of many years. Revisiting
the older ion-exchange chromatography serves not only to pay tribute to some
remarkable accomplishments, but it can also be a learning experience. Trends and
ideas in science tend to run in repeating cycles. Thus, an awareness of older work may
provide inspiration for new research using improved contemporary technology.
Selection of milestones is a rather personal matter. I chose to write about subjects of
which I came to have a firsthand knowledge during my career. The topics selected are
in roughly chronological order and cover the period from about 1945 to 1980. An effort
has been made to explain the chemical principles as well as to recount the major
accomplishments of the various research projects.

Ion exchange chromatography relies on the attraction between oppositely
charged stationary phase, known as an ion exchanger, and analyte. The ion exchanger
consists of an inert support medium coupled covalently to either positive (anion
exchanger) or negative (cation exchanger) functional groups. To these covalently bound
functional groups the oppositely charged ions are bounded (mobile counter ion), which
will be exchanged with like charge ions in the sample having charge magnitude more
than the ions bounded to the matrix. Thus, if anion exchange chromatography is
performed, negatively charged sample components will interact more with the stationary
phase and will be exchanged for like charged ions already bounded to the matrix.
Consider a column having E- Y+ cation exchanger in which E- is negative charged
exchanger and Y+ is the mobile counter ion.
Let X+ be the cation in the sample having charge greater than Y+. The X+ ion can
exchange sites with the counter ion Y+ with satisfying the following relationship:

© 2015, A Journal on Chromatographic Principle

Figure 17 Exchange between Y+ and X+ occurs. Bounded interest of ion (X+) can now be eluted by either
of the two ways: a) by adding component M+ having magnitude of charge more than that of X+ so that M+
will replace X+ and X+ will be eluting out. b) by changing the pH of the solvent (mobile phase so that X+
have no charge and is then unbounded from the matrix and can be eluted out.

The basic process of chromatography using ion exchange can be represented in 5
steps (assuming a sample contains two analytes A & B): eluent loading, sample
injection, separation of sample, elution of analyte A, and elution of analyte B, shown and
explained below. Elution is the process where the compound of interest is moved
through the column. This happens because the eluent, the solution used as the solvent
in chromatography, is constantly pumped through the column. The representative
schemes below are for an anion exchange process. (Eluent ion = , Ion A= , Ion B =
1. The eluent loaded onto the column displaces any anions bonded to the resin and
saturates the resin surface with the eluent anion.
This process of the eluent ion (E-) displacing an anion (X-) bonded to the
resin can be expressed by the following chemical interaction: Resin+-X-+E-
2. A sample containing anion A and anion B are injected onto the column. This
sample could contain many different ions, but for simplicity this example uses just
two different ions as analytes in the sample.
3. After the sample has been injected, the continued addition of eluent causes a
flow through the column. As the sample elutes (or moves through the column),
anion A and anion B adhere to the column surface differently. The sample zones
move through the column as eluent gradually displaces the analytes.
4. As the eluent continues to be added, the anion A moves through the column in a
band and ultimately is eluted first.
This process can be represented by the chemical interaction showing the
displacement of the bound anion (A¯) by the eluent anion (E¯). Resin+-A¯ + E¯
<=> Resin+- E¯ + A¯
5. The eluent displaces anion B, and anion B is eluted off the column.
Resin+-B- + E- <=> Resin+-E- + B

© 2013 Moustafa and Morsi

Figure 18 A schematic diagram of Ion exchange chromatography.

Ion exchange is the most widely used chromatographic method for the
separation and purification of charged biomolecules such as polypeptides, proteins,
polynucleotides, and nucleic acids. Its widespread applicability, high capacity and
simplicity, and its high resolution are the key reasons for its success as a separation
method. It also has several industrial uses such as separation and purification of blood
components such as albumin, recombinant growth factors and enzymes and also,
analytical applications such as quality control and process monitoring.
Ion chromatography is basically a chromatographic method that has become a
routine analytical method. It is regarded as a versatile analytical technique for
separating and quantifying ions. The concept of IC was successively widened with
advancements of the rapid development in separation, column stationary phase, great
variety of detectors, data analysis and hyphenated techniques. Moreover, it could
include other separation methods (e.g., ion interaction and ion exclusion) for
simultaneous separation of analyte components. IC analysis has matured to a well-
established rugged, sensitive and reliable analysis technique for a wide variety of
chemical compounds present in various matrices. On this manner, many papers have
been published during the last few years dealing with new modalities in sample
pretreatment, separation, detection, etc., for improving samples analysis.

Key Points and Insights