BCH 204: BIOCHEMICAL METHODS

PRINCIPLES, INSTRUMENTATION, METHODOLOGIES AND APPLICATIONS OF

CHROMATOGRAPHY

The word ‘chromatography’ has its origin in the Greek word ‘Chromo’ meaning colour and
‘graphy’ meaning— to measure; since initially the technique was used to separate coloured
compounds from mixture. The first record of chromatography dates back to 1903 by Russian
Botanist Tswest who used it for separation of plant pigments. The early methods of isolation
and purification of compounds of mixtures were empirical, slow and labourious. With the
advancement in separation procedures over the years, the term Chromatography has come to be
known as the technique used to separate various classes of compounds from mixtures and their
identification/characterization. The technology encompasses a wide range of variants depending
upon the specific requirements of the experiment. There is no single procedure or set of
procedures by which any and every molecule may be isolated but it is easily possible to choose a
sequence of separation steps that will result in a high degree of purification and a high yield. The
general objective is to increase the purity of biological activity of the desired substance per unit
weight, by ridding it of inactive or unwanted materials while at the same time maximizing the
yield. The technique has developed tremendously since its inception and now covers a number of
highly efficient laboratory procedures.

Chromatography is the process for separation of components in a solution by differences in

migration rate as the solution mixture (mobile phase) is passed over or through a stationary
phase. It is a separation process that is achieved by distributing the components of a mixture
between two phases, a stationary phase and a mobile phase. Those components held
preferentially in the stationary phase are retained longer in the system than those that are
distributed selectively in the mobile phase. The separation may make use of one or more of the
following physicochemical principles, depending upon the particular chromatographic system.

NOTE:
 Mobile Phase: is the phase that moves in a specific direction. It may be liquid (as in
Liquid Chromatography, LC and Capillary Electrochromatography, CEC), a gas (as in
Gas Chromatography, GC) or a mixture of fluid called Supercritical fluid (as in
Supercritical-fluid Chromatography, SFC). It consists of the sample being
separated/analyzed and the solvent that moves the sample through the column.
 Stationary Phase: is the substance fixed in place for the chromatography procedure. It is
usually coated on an inert solid support. Examples include the silica layer in Thin Layer
Chromatography.

Principle:
Chromatography is based on the principle of partition of the solute between two
phases/solvents. Since the solutes in the mixture will have different solubility or partition
coefficient between the two solvents/phases, multiple partitioning processes will result in their
separation from each other. A number of chromatographic procedures these days rely on
adsorption of mixture on a solid support/phase followed by differential elution.

1
Physicochemical principle used for chromatographic separations

Type of chromatography Physicochemical principle

Gel filtration Size and shape

Adsorption chromatography Adsorption
Partition chromatography Solubility
Ion exchange chromatography Ionization

Classification of Chromatography
• Chromatographic methods are generally classified according to the physical state of the mobile
phase. This is further sub classified according to how the stationary phase is contained for a
particular chromatographic method.

Classification of chromatography according to the mobile phase

Classification of Chromatography
Chromatography can be classified by various ways:
1. On the basis of interaction of solute to the stationary phase: These are:
i. Adsorption Chromatography: It is probably one of the oldest types of chromatography
around. It utilizes a mobile liquid or gaseous that is adsorbed onto the surface of stationary solid
phase. The equilibration between the mobile and stationary phase accounts for the separation of
different solutes.

2
 ii. Partition Chromatography: This form of Chromatography is based on a thin film formed on
the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile
phase and the stationary phase.

iii. Ion-exchange chromatography: In this type of chromatography, the use of resin (the
stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the
opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Net
charge on the surface of any molecule depends upon the pH. A range of negatively charged
(Cation exchange) as well as positively charged (Anion exchange) resins are available. Most of
these resins are derivatives of polymeric cellulose. When a mixture of charged molecules, e.g.
proteins, is passed through the column carrying the ion-exchange resin (e.g. Cation exchange),
the molecules with greater positive charges interact with the resin and bind tightly compared to
the molecules carrying lesser number of positive charges. The negatively charged molecules may
just be washed out without any binding. The bound molecules may then be eluted out one by one
with an eluting buffer of a suitable pH. A better resolution can be achieved by using a pH
gradient buffer.

iv. Molecular Exclusion Chromatography/ Gel Permeation/ Gel filtration: This type of
chromatography lacks an attractive interaction between the stationary phase and solute. The
liquid or gaseous phase passes through a porous gel which separates the molecules according to
the size. The pores are normally small and exclude the larger solute molecules, but allow smaller
molecules to enter the gel, causing them to flow through a larger volume. This causes the larger
molecules to pass through the column at a faster rate than the smaller ones. In summary, a
number of gels, e.g. sephadex when hydrated, act as molecular sieves. The pores on the surface
of these gels allow smaller molecules to penetrate deeper, whereas larger molecules are left
outside (excluded), hence the name Molecular Exclusion Chromatography. Thus a mixture of
molecules may be separated into its components due to differential retention in the gel-packed
column. The gels are available in different grades (e.g. Sephadex G-25, G-50, G-250, etc.)
depending upon the pore size and hence, suitable for different set of molecules depending on
their molecular weight. The technique is most popular in research laboratories because a large
volume of the sample can be applied and fractions of relatively pure molecules may be easily
collected.

2. On the basis of Chromatographic Bed Shape: The types are:

(i) Paper chromatography: The mixture of amino acids, sugars or some drugs, pigment and
chemicals may be separated by this type of chromatography. Stationary phase in paper
chromatography is the aqueous phase adsorbed on the surface of Whatman No. 1 or Whatman
No. 3 filter paper. This technique involves placing a small dot or line of sample solution onto a
strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent
and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to
travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the
compounds within the mixture travel farther if they are non-polar. More polar substances bond
with the cellulose paper more quickly and therefore do not travel as far.

In paper chromatography, the stationary cellulose phase is more polar than the mobile organic
phase. Amino acids with large non-polar side chains (leucine, phenylalanine, tryptophan, valine,

3
methionine, tyrosine) migrate farther in n-butanol: acetic acid: water (4:1:5) than those with
shorter nonpolar side chains (proline, alanine, glycine) or with polar side chains (threonine,
glutamic acid, serine, arginine, aspartic acid, histidine, lysine, cysteine). This reflect the greater
relative solubility of polar molecules in the hydrophilic stationary phase and on nonpolar
molecules in organic solvents. In summary, Solvent system provides both stationary phase and
mobile phase. For amino acid chromatography, butanol: acetic acid: water in the proportion of
4:1:5 v/v is used as solvent system. Butanol with a little acetic acid acts as the mobile phase and
water and acetic acid forms the stationary phase, which will be held on the paper In clinical
laboratories, paper chromatography is employed for diagnosis of aminoacidurias (e.g.
phenylketonuria, cystinuria, alkaptonuria, etc.).

Ascending paper chromatography

(ii) Thin layer chromatography (TLC): This Chromatography technique is an analytical

chromatography to separate and analyze complex biological or non-biological samples into their
constituents, it is one of the popular techniques for testing the purity of a sample. In this method,
the silica or alumina as a stationary phase is coated onto a glass or aluminium foil as thin layer
and then a sample is allowed to run in the presence of a mobile phase (solvent). In summary,
different grades of silica gel are uniformly layered on glass plates, which are then activated at
high temperature. The sample of lipids, amino acids, drugs, dyes or chemicals may be resolved
by partition between water bound on the silica gel and the organic solvents in the solvent system.
Visualisation of the spots would require specific stains/reagents, e.g. ninhydrin reagent for amino
acids and sulphuric acid for lipids.

(iii). Column chromatography: It is a separation technique in which the stationary bed is within
a tube. The particles of the solid stationary phase or the support coated with a liquid stationary
phase may fill the whole inside volume of the tube (packed column) or be concentrated on or
along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle
part of the tube (open tubular column). Differences in rates of movement through the medium are
calculated to different retention times of the sample.

4
3. Techniques by Physical State of Mobile Phase:

(i) Affinity Chromatography: This is based on selective non-covalent interaction between an

analyte (substance to be separated during chromatography). It works on the principle of mutual
recognition forces between a ligand and receptor. A column of specific affinity may be prepared
by attaching the substance (moiety) that has an inherent ability to bind with the target molecule
to be purified, to a solid support or matrix. A column prepared by coupling an antibody to the
matrix (CM-cellulose) would bind the specific antigen only and the rest of the substances in the
mixture (plasma) will be washed out. The antigen may then be eluted by neutralising the
interaction in the Ag-Ab complex by varying the pH or by using chemicals. A number of
commercially available biological substances and drugs are prepared by using affinity columns.

(ii) Gas Chromatography (GC), also known as Gas-Liquid Chromatography (GLC): It is a

separation technique in which the mobile phase is a gas. It is always carried out in a column,
which is typically ‘packed’ or ‘capillary’, GC is based on a partition equilibrium of analyte
between a solid stationary phase (often a liquid silicon-based material) and a mobile gas (most
often Helium). Gas Chromatography is used to separate a mixture of compounds that are volatile
or can be made volatile. The components in the sample are separated on the basis of partition
between a gaseous mobile phase and a liquid stationary phase. Since the gas used is inert
(nitrogen, helium or argon) and simply carries the molecules through the column, it is called a
carrier gas. The column consists of a non-volatile liquid coated on an inert solid support
(Diatomaceous earth or porous polymer or glass beads). The stationary liquid phase is called
non-selective when the separation is primarily based on the volatility of the components. The
selective liquid phases may be used to separate polar compounds based on their polarity. The
sample is injected through a septum in the form of a gas or the injection port is maintained at
temperature higher than the boiling point of the components so that they may vaporise upon
injection. Sample vapor is swept through the column partially as a gas and partially dissolved in
the liquid phase. Components with higher boiling point will be retained in the liquid phase longer
than the ones with lower boiling points causing their separation. Because elution rate is
dependent upon the temperature, the column is enclosed in controlled temperature oven. The
components of the mixture eluted at different time are measured with the help of (Thermal
conductivity or Flame ionisation) detectors. Flame ionisation detectors are widely used in the
clinical laboratories. They use hydrogen flame to ionise the column effluents, the ions are then
collected by the electrodes and a proportional current is generated making different peaks in the
GLC pattern. Elution time of each component is characteristic and is used for their identification.

(iii) Liquid Chromatography (LC): It is a separation technique in which the mobile phase is a
liquid. It can be carried out either in a column or a plane. The modern state of the art instruments
use high pressure to pump the mobile phase through a tightly packed column and are known as
LHigh Pressure/ Performance Liquid Chromatography (HPLC). Since the detectors in these
instruments are ultraviolet absrptiometers, the purified fractions of the sample mixture can be
recovered. These instruments enjoy the advantages of high speed, high resolution and versatility.
HPLC is being commonly employed for the detection and estimation of hormones (e.g.
epinephrine, norepinephrine, ACTH, etc.), vitamins (e.g. vitamin A, calcitriol, etc.), drugs (e.g.
phenytoin, phenobarbitones, LSD, AZT, etc.) and metabolites (e.g. metanephrines) in clinical
laboratories.

5
Chromatography Techniques and Instrumentation

6
Comparison of different types of Chromatography Technique

7
Applications of Chromatography Technique
• The chromatographic technique is used for the separation of amino acids, proteins and
carbohydrates.
• Purification of proteins, enzymes, nucleic acids, immunoglobulins and membrane receptors.
• Determination of molecular weight of proteins.
• Analysis of drugs, hormones vitamins and brain amines.
• Qualitative and quantitative analysis of complex mixtures.
• In the clinical laboratory for the identification of sugars, amino acids and drugs in serum or
urine.