Chromatography

The Russian Botanist Mikhail Tswett developed the separation

technique that we call chromatography

In 1903, he successfully separated a mixture of plant pigments using a

column of calcium carbonate

In the process, he became the first scientist to recognize that

chlorophyll was not a single compound.
 Distribution coefficients

The basis for all forms of chromatography is the distribution

or partition coefficients (Kd) which describes the way in
which a compound ( the analyte) is distributed between two
immiscible phases

For two phases A and B, the value for this coefficient is a

constant at a given temperature and is given by

Concentration in phase A/concentration in phase B= Kd

 Principle

Chromatography is a separation method where the analyte is contained within a liquid

or gaseous mobile phase which is pumped through a stationary phase

Usually one phase is hydrophilic and the other lipophilic

The components of the analyte interact differently with these two phases

Depending on their polarity, they spend more or less time interacting with the
stationary phase and are thus retarded to a greater or lesser extent

This leads to the separation of the different components present in the sample.

Each sample component elutes from the stationary phase at a specific time, its
retention time tR.
As the components pass through the detector, their signal is recorded
and plotted in the form of a chromatogram

All chromatographic systems consist of the stationary phase, which may be a solid,
gel, liquid or a solid/liquid mixture that is immobilized and the mobile phase which
may be liquid or gaseous, and which is passed over or through the stationary phase
after the mixture of analytes to be separated is applied to the stationary phase

During the chromatographic separation, the analytes continuously pass back and
forth between the two phases so that differences in their distribution coefficients
result in their separation
 Modes of chromatography
Chromatographic separations may be carried out in one of two modes – in a
column(e.g., ion-exchange chromatography) or on a planar surface (i.e., flat sheet e.g.,
paper or thin-layer chromatography). These can be further sub-divided into liquid
chromatography (LC) and gas chromatography

Column chromatography:

Stationary phase is packed in a glass or metal column

The mixture of analytes is then applied and the mobile phase (called the eluent) is
passed through the column either by gravity feed or by the use of pumping system or
applied gas pressure

This is the most common mode of chromatography

The stationary phase is either coated onto discrete small particles (the matrix) and
packed into the column or applied as a thin film to the inside wall of the column.

As the eluent flows through the column the analytes separate on the basis of their
distribution coefficients and emerge individually in the eluate as it leaves the column
The principle of chromatographic separatio
The sample components interact
differently with the stationary and mobile
and elute at their specific retention
time, tR.
 Thin-layer or planar chromatography

The stationary phase is coated thinly onto a glass, plastic or metal foil plate.

The mixture of analytes is applied as a spot or band near the edge of the coated plate

The mobile liquid phase is passed across the plate (held either horizontally or
vertically) by capillary action, causing the analytes to migrate at characteristic rates
to the opposite end.

It is simple to carry out and has the advantage that multiple samples can be studied
simultaneously
Chromatographic methods can be further classified into gas chromatography
(GC) and liquid chromatography (LC) depending on the nature of the mobile
phase involved.
Gas chromatography can be applied only to gaseous or volatile substances that
are heat-stable.
The mobile phase is usually an inert carrier gas such as nitrogen, hydrogen or
helium and it is pumped through a heated column.
The column can be packed with a silicon oxide based material or is coated with a
polymeric wax.
The sample is vaporised, pumped through the column and the analytes are
detected in the gas stream as they exit the column
Analyte detection is achieved by flame ionization or thermal conductivity.

GC is not used for analyzing biological macromolecules since large molecular

weight compounds such as peptides and proteins are thermally destroyed before
evaporation.

Smaller molecules such as amino acids, fatty acids,peptides and carbohydrates

can be analysed if they are modified chemically to increase their volatility.

Some cell cultures produce volatile metabolites such as aldehydes, alcohols or

ketones.

These can be analysed readily via GC

In liquid chromatography (LC), the sample is dissolved and pumped through a
column containing a stationary phase

LC has wider applicability than GC as it is not restricted to volatile and heat stable
samples

The sample only has to dissolve completely in the mobile phase.

Common detection methods are UV spectroscopy, measurement of refractive index,

fluorescence, electrical conductivity and mass spectrometry.

Modes of operation can be classified as normal and reversed phase chromatography

In normal phase chromatography, the stationary phase consists of a hydrophilic
material such as silica particles and the mobile phase is a hydrophobic organic solvent
such as hexane

In reversed phase chromatography, the stationary phase is hydrophobic and the

mobile phase is a mixture of polar solvents such as water and acetonitrile.

Biomolecules are generally soluble in polar solvents, hence, reversed phase

chromatography is the method of choice for amino acids, peptides, proteins, nucleic
acids and carbohydrates.
 Basic Chromatographic Theory

A typical chromatogram is depicted in the figure below

The peak width, w, is

defined as the
intersection of the
tangents on each
side of the peak
with the baseline.

t0 = zero retention time

tR(A) = retention time of
compound A
tR(B) = retention time of
compound B
 Some important parameters of chromatographic theory

The capacity factor k’ describes the velocity of the analyte relative to the velocity of
the mobile phase

Each compound spends a different amount of time interacting with the mobile and
stationary phase
The average velocity of a sample compound is dependent on how much time it
spends in the mobile phase.
If k’ is much smaller than 1, then the analyte moves too quickly and the elution
time is so short that an exact determination of tR is difficult
If the sample moves too slowly, the separation time is very high
The selectivity factor alpha decribes the relative velocities of the analytes with
respect to each other.
The selectivity describes how well a chromatographic method can distinguish between
two analytes

′
𝑘 𝐵 𝑡 𝑅 ( 𝐵) −𝑡 0
𝛼= ′
=
𝑘 𝐴
𝑡 𝑅 ( 𝐴)−𝑡 0
 The efficiency of a chromatographic separation is crucially dependent on band
broadening. If band broadening is large, peaks can overlap and resolution is lost.

Band broadening for a column of length L is quantitatively expressed in the

concept of height equivalent of a theoretical plate, H or plate numbers, N, the
larger the number of plates N and the smaller H is the better the
chromatographic efficiency

( )
2
𝑡𝑅
𝑙𝑎𝑡𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑁 =16
𝑤
𝐿
𝑎𝑡𝑒 h𝑒𝑖𝑔h𝑡 𝐻 =
𝑁
The plate model supposes that the chromatographic column contains a large number
of separate layers called theoretical plates.

Separate equilibrations of the sample between the stationary and mobile phase occur
in these plates.

The analyte moves down he column by transfer of equilibrated mobile phase from
one plate to the next.
 The parameters that influence band broadening can be approximated by the van
Deemter equation which is valid for gas and liquid chromatography
Van Deemter equation H = A + B/u + C.u

The height H of a theoretical plate is a sum of three terms

A = influence of the column packing on band broadening. It is called Eddy diffusion and
is constant for a given column and independent of the flow rate

B/u describes longitudinal diffusion in or opposed to the direction of flow- it is

inversely related to the flow rate

C.u describes resistance to mass transfer between stationary and mobile phase
and is directly proportional to rate of flow

By plotting H as a function of u, the optimum flow rate for a chromatographic

separation can be determined
A van Deemter plot for the determination of the optimal flow rate
 The ultimate goal of a separation is to achieve a high resolution Rs

Peak resolution can be optimized by increasing the selectivity and minimizing band
broadening
2. [ 𝑡 𝑅 ( 𝐴 ) − 𝑡 𝑅 ( 𝐵 ) ]
𝑅 𝑆=
𝑤 𝐴 +𝑤 𝐵
Baseline resolution is achieved when R=1.5

To obtain high resolution the three terms must be maximized.

The capacity factor k’ influences the resolution.
 Application of liquid chromatography for bioanalysis
In bioanalytical chemistry, chromatography is mainly used for separation, isolation and
purification of proteins from complex sample matrices

In cells proteins occur alongside other compounds such as lipidsand nucleic acids.

In order to be analysed, these proteins must be separated from all the other cell
components.

Then the protein of interest mighthave to be isolated from other proteins and purified
further

Chromatography is an essential part of almost any protein purification strategy

A number of different chromatographic techniques are used for the purification and
analysis of proteins
They can be classified as follows: Reversed phase chromatography, Ion exchange
chromatography, Affinity chromatography, Size exclusion chromatography
 Reversed phase liquid chromatography

In normal phase chromatography the stationary phase is made of polar

materials such as paper, cellulose or silica gel whereas the mobile phase
consisted of non-polar solvents such as hexane or chloroform

In reversed phase chromatography, polar solvents such as water and

acetonitrile are used as the mobile phase whereas the stationary phases are
non- polar. These are prepared by etherification of the polar hydroxyl groups
of the silica gel with long alkyl chains

It is the method of choice for the separation ofsmaller biomolecules such as

peptides, amino acids, carbohydrates and steroids which are solublein
water/acetonitrile mixtures
The separation of proteins cam be problematic as organic solvents such as
acetonitrile can decrease the protein’s solubility and cause denaturation

The stationary phase consists of porous silica particles with non-polar surface
groups obtained from etherification of the initial hydroxyl groups of the silica
particle with silanes containing non-polar hydrocarbon chains
Any chain length from ethy silane (C2) to n-octadecyl silane (ODS)(C18) is used
although octyl silane and ODS are the most commonly used
The mobile phase is based on a polar solvent system consisting of an aqueous
buffer and acetonitrile or methanol.

Gradient elution is employed to increase resolution and shorten separation times

This is achieved by increasing the organic solvent and thus decreasing the mobile
phase polarity and the retention of less polar electrolytes during the separation
process
Solvents can be classified according to their elution strength and polarity
Buffer systems based on ammonium acetate, phosphate or hydrogen carbonate are usually added
at concentrations of about 20mM to adjust the pH of the mobile phase to values between 2 and 8.

Ion pairing reagents can be used at low concentrations, typically 0.1% to increase the
hydrophobicity of the charged analytes

They form ion-pair complexes with the analyte

e.g., anionic ion pairing reagents such as trifluoroacetic acid (TFA) can bind to positively charged
analytes
Cationic ion pairing agents such as tetraalkyl ammonium salts can be used to bind to negatively
charged analytes

These complexes are more retarded by the stationary phase and are thus easier to separate than
the largely unretained charged analytes alonee
•Dextrans are polysaccharides with molecular weights ≥1000 Dalton, which have a linear backbone of α-linked D-
glucopyranosyl repeating units. Three classes of dextrans can be differentiated by their structural features:

Class 1 dextrans contain the α(1→6)-linked D-glucopyranosyl backbone modified with small side chains of D-
glucose branches with α(1→2), α(1→3), and α(1→4)-linkage. The class 1 dextrans vary in their molecular weight,
spatial arrangement, type and degree of branching, and length of branch chains, depending on the microbial
producing strains and cultivation conditions. Isomaltose and isomaltotriose are oligosaccharides with the class 1
dextran backbone structure.
•Class 2 dextrans (alternans) contain a backbone structure of alternating α(1→3) and α(1→6)-linked D-
glucopyranosyl units with α(1→3)-linked branches.
•Class 3 dextrans (mutans) have a backbone structure of consecutive α(1→3)-linked D-glucopyranosyl units with
α(1→6)-linked branches. One and two-dimensional NMR spectroscopy techniques have been utilized for the
structural analysis of dextrans.
Sepharose is a tradename for a crosslinked, beaded-form of agarose, a polysaccharide polymer material
extracted from seaweed. Its brand name is derived from Separation-Pharmacia-Agarose. A common application
for the material is in chromatographic separations of biomolecules

Sephadex: Separation Pharmacia dextran

Ion exchange chromatography
 Affinity Chromatography

Makes use of the higly specific molecular recognition of certain biomolecules

By attaching a specific ligand such as an antigen to the stationary phase material, the
matching antibody can be specifically and reversibly adsorbed
Interactions such as those existing between enzyme and co-enzyme, receptor
protein and hormone, or single strands of oligonucleotides and their matching
counterparts

Affinity chromatography has the highest specificity and selectivity of all

chromatographic methods and is a powerful method for the purification and
isolation of biomolecules even at low concentrations

The target molecule can be picked selectively from complex mixtures such as
blood or serum
The process can be divided into the following steps: (1) sample introduction, (2)
adsorption, (3) washing and (4) desorption

The chromatographic column contains agarose or cellulose beads as a stationary

phase on which ligand molecules have been covalently attached

After addition of the crude sample, those molecules that have an affinity for the
ligand on the beads are adsorbed and retained by the stationary phase

Other substances of the sample with no affinity for the ligand are eluted from the
column

Further washing ensures removal of non-specifically bound components

Next, the adsorbed species have to be eluted from the column.

To achieve desorption, the non-covalent interaction between the

biomolecules must be disrupted

Methods include a pH decrease, an increase in ionic strength, addition of

a denaturing agent such as urea or the addition of an organic solvent –
these sedorption methods are non-specific as they can elute any bound
molecule alike
Specific desorption can be achieved by introducing a species that binds to
the analyte more strongly than the ligand in the stationary phase.

The free ligand competes with the bound ligand on the solid phase for
binding sites on the protein

Once bound to the free ligand, the protein is eluted from the column

The separation mix can then be regenerated for further use

Steps of affinity chromatography
including sample addition, adsorption,
washing
and elution.
Adsorption and specific desorption for affinity
chromatography.
 Size exclusion chromatography

In size exclusion chromatography (SEC), dissolved molecules are separated according to their
size, which is closely related to their molecular weight

This method can be applied for the separation of polymers in non-aqueous solutions and is
also called gel-permeation chromatography (GPC)

It can be used for the separation of biomoleculesin aqueous solution. It is then called gel
filatration chromatography
The chromatographic column is filled with a porous material such as a polymeric gel
or agarose beads with diameters of typically 10-40um.

Separation occurs if the pore size is comparable to the size of the molecules passing
through them

Large molecules cannot enter the pores

They pass the matrix unretained and elute together with the solvent front

Smaller molecules enter the pores and have an average residence time which
depends on the size and shape of the molecule

The smaller the molecule the longer its residence time in the pores and the greater
its retention
Molecules that aremuch smaller than the pore size canenter the pores and have
long residence times

However, there is no differentiation between molecular sizes any more. Therefore,

all these molecules are eluted together after a long retention time
The principle of size exclusion chromatography: Large molecules do
not enter the pores and pass the matrix unretained, very small
molecules spend a long time in the pores, but there is no
differentiation between molecular sizes. Only within a critical size
range is there a relation between residence time and molecular size,
which can be used for separation.
Large molecules are un-retained and eluted first, smaller
molecules are retarded by the pores of the stationary
phase.
Differentiation and separation only occurs over a certain range of molecularsizes, typically
between molecular weights of 2 kDa and 200 kDa, although this can be increased up to
1000 kDa by the use of more specialized gels

This size range is dependent on the sizes of the pores and pore size distribution in the gel
matrix
Molecules are selectively retarded if their molecular weight is
between the exclusion limit and the limit of total permeation.
Immobilized nucleotide and nucleic acid affinity

Poly(U)-sepharose 4B is a chromatography resin for specific and reversible binding of messenger RNAs, reverse
transcriptases, interferons and nucleic acids from plants

Poly(U)-sepharose 4B interacts with mRNA by biospecific hybridization between the synthetic immobilized chain of
polyuridylic acid (poly(U)) and the complementary poly(A) sequence that is a feature of almost all RNA molecules

Some other resins are Oligo(dT) cellulose –for purification of polyadenylated mRNA
A column containing the loosely packed resin particles has a total volume (VT)
made up of three components: the volume of exterior solvent (the void
volume), V0; the solid volume of gel particles, Vg and the internal volume of the
pores of the particles that are accessibke to solvent, Vi

VT = V0 + Vg + Vi

If a solute is added to such a gel column, it will partition between the external
and internal solvent regions.