CHROMATOGRAPHY

• Chromatographic methods are used for the separation of components of a

mixture present in a flowing mobile phase, based on their different strengths of
interaction with a stationary phase.

• Stronger interaction with the stationary phase yields a longer retention time,
which are the times between sample introduction and component elution.

UNITS AND DEFINITIONS

• Flow rates may be expressed in linear or volume units. Linear flow rates
refer to the column length traveled by the mobile phase per unit time, and are
usually expressed in centimeter per minute (cm/min). Volume flow rates are
commonly used, and are expressed in milliliters per minute (mL/min). The
time between sample introduction and component elution is the retention time
of the component and is symbolized tr.
The relative retention of two components, 1 and 2, is called α and is defined as
α (alpha)= tr2/tr1

where tr2 > tr1, so that a values are always greater than unity. Under a given set
of experimental conditions, an individual component has a characteristic
capacity factor, k:

k = (tr-tm)/ tm

A large value of k for a given component usually means that a good separation
will be achieved; however, large k values imply long elution times. Values of k
between 2 and 20 are generally considered useful. The capacity factor is
related to the partition coefficient, K, of the solute (S) between the mobile and
stationary phases, where K= [S]stationary/[S]mobile, and to the relative volumes of
stationary and mobile phases at equilibrium, Vs and Vm

k = K (Vs/ Vm)

The relative retention of two components, a, may also be expressed in terms

of K:
PLATE THEORY OF CHROMATOGRAPHY
• Consider a column that is divided into N
segments of equal length, and that each
segment is just long enough to allow
complete equilibration of solute
partitioning between the stationary phase
and the mobile phase, according to its
partition coefficient.

• Each of these segments is called a

theoretical plate, and a column of length
L will have a height equivalent to a
theoretical plate (HETP) given by L/N.

• A ‘‘good’’ column will have large N

values, and a small HETP.

• Columns packed with a small-particle

stationary phase have been shown to
yield higher N values than those packed
with larger particles.
It is important to remember that the plates do not really exist; they are a
figment of the imagination that helps us understand the processes at work in
the column.They also serve as a way of measuring column efficiency, either
by stating the number of theoretical plates in a column, N (the more plates the
better), or by stating the plate height; the Height Equivalent to a Theoretical
Plate (the smaller the better).

If the length of the column is L, then the HETP is

HETP = L / N

The number of theoretical plates that a real column possesses can be found
by examining a chromatographic peak after elution;
                                     

where w1/2 is the peak width at half-height.

The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes

account of the time taken for the solute to equilibrate between the stationary and
mobile phase (unlike the plate model, which assumes that equilibration is infinitely
fast). The resulting band shape of a chromatographic peak is therefore affected
by the rate of elution. It is also affected by the different paths available to solute
molecules as they travel between particles of stationary phase. If we consider the
various mechanisms which contribute to band broadening, we arrive at the Van
Deemter equation for plate height;

HETP = A + B / u + C u

where u is the average velocity of the mobile phase. A, B, and C are factors which
contribute to band broadening.

A - Eddy diffusion

The mobile phase moves through the column which is packed with stationary
phase. Solute molecules will take different paths through the stationary phase at
random. This will cause broadening of the solute band, because different paths
are of different lengths.
B - Longitudinal diffusion

The concentration of analyte is less at the edges of the band than at the center.
Analyte diffuses out from the center to the edges. This causes band broadening. If
the velocity of the mobile phase is high then the analyte spends less time on the
column, which decreases the effects of longitudinal diffusion.

C - Resistance to mass transfer

The analyte takes a certain amount of time to equilibrate between the stationary
and mobile phase. If the velocity of the mobile phase is high, and the analyte has a
strong affinity for the stationary phase, then the analyte in the mobile phase will
move ahead of the analyte in the stationary phase. The band of analyte is
broadened. The higher the velocity of mobile phase, the worse the broadening
becomes.
The resolution of two components may be quantitated using Eq.

Resolution values have been shown to increase with the square root
of column length. Thus, doubling the length of a column will increase
resolution by a factor of (2)1/2.
Figure 14.2 shows the five major classes of chromatography in common use.

Each is based on a unique retention mechanism, and three of these mechanisms

are of particular interest for the separation and quantitation of biological
macromolecules.

Size-exclusion chromatography (SEC), also called gel filtration, separates species

on the basis of molecular size, with small molecules being retained and large
species eluting first. Affinity chromatography involves selective binding
interactions such as antibody–antigen and enzyme–substrate interactions. Ion-
exchange chromatography is used to separate species on the basis of molecular
charge and the distribution of molecular charge; the stationary phase possesses
either positive or negative charge, and interacts only with oppositely charged
solutes. All three of these retention mechanisms may be applied to
macromolecular solutes that are present in aqueous solutions, and function well
under mild conditions of temperature and pH.
HPTLC- High Performance Thin Layer Chromatography
HPTLC- High Performance Thin Layer Chromatography is a sophisticated and
automated form of TLC.
Main Difference of HPTLC and TLC - Particle and Pore size of Sorbents.
TLC
HPTLC

Layer of Sorbent
•100µm •250µm

Efficiency
•High due to smaller particle size generated •Less

Separations
•3 - 5 cm •10 - 15 cm

Analysis Time •Shorter migration distance and the analysis time is

•Slower
greatly reduced

•Wide choice of stationary phases like silica gel for

Solid support •Silica gel , Alumina
normal phase and C8 , C18 for reversed phase
& Kiesulguhr
modes

Development chamber
•New type that require less amount of mobile phase •More amount

Sample spotting
•Auto sampler •Manual spotting

•Use of UV/ Visible/ Fluorescence scanner scans the

Scanning
entire chromatogram qualitatively and quantitatively •Not possible
and the scanner is an advanced type of densitometer
attracting biomolecules with the opposite charge.
Molecule Ligand
Antigen Antibody
Enzyme Substrate
Receptor Ligand
Nucleic Acid Binding Protein Nucleic Acid
Polysaccharide, glycoprotein Lectin
Developing an effective affinity chromatography method involves:

1.Finding a ligand that is specific enough

2.Finding suitable conditions for binding between the target protein and the ligand as
well as release the protein.

Stages in affinity chromatography

The affinity chromatography process can be separated into 3 main stages:

1.Equilibration

2.Application of Sample

3.Elution