II- Chromatographic Methods

• Credit is usually given to Michael Tswett for scientifically establishing the

basic principles of chromatographic separations. When, in 1903, he separate
plant pigments into colored bands on calcium carbonate column (as stationary
phase) with petroleum ether as mobile phase.

Chromatography is combined Greek words

Chroma "color" and graphein "to write"
Chromatography is a Physico-Chemical separation method where the analyte is
contained within a liquid or gaseous mobile phase, which is passed through a
stationary phase. 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 extend. 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.
II.1 -Some important terminology

The mobile phase is called eluent, while when it emerge

from the end of the column it is called eluate

Eluent Column Eluate

in out

The process of passing mobile phase (liquid or gas)

through a chromatographic column is called Elution
Dead volume=The volume of mobile phase needed to move an un-retained solute
from the point of injection to the detector
II.2. Chromatographic theory

A- Plate theory

• The simplest way to think about chromatography is to imagine that a very large
number of "countercurrent distribution equilibrations" occur between the
mobile and stationary phases as the solute travel through the column.

• -It can be imagined that the column is divided into N segments, in each of which
equilibrium occurs of a solute between mobile and stationary phases. Each of
these segments is called a theoretical plate.
• If the total length of the column is L, the height equivalent to a theoretical plate
(H.E.T.P.) is

• By measuring the retention time (tR) or retention volume (VR) and bandwidth (w),
it is possible to measure the number of theoretical plate (N)
• Thus the width (w) is equal to four standard deviations of Gaussian
peak (4). Alternatively, if the width of the band is measured at
height equal to half of the peak height, then

• Note:
-N, the theoretical number of separation steps (plate) in a column
determine its separating capability; N is therefore an indication of
column efficiency.
-if N is constant, the width of chromatographic peak increases with
increasing retention time. That is, successive peaks on a
chromatogram should be increasingly broad.
• This theory was successful in:-

1-Give an equation that satisfactory describe the rate of solute migration

through the column.
2-Give approximate equation that account for the shape of elution band.

• But not successful in:-

1- Not illustrating effect of important variable such as flow rate and

packing characteristic on the width of the solute band or peak and
then upon H and N.
2- Ignore the diffusion of solute which is very important to H. Hence,
the plate to plate diffusion cause peak spreading which was not
mentioned in plate theory, this is called longitudinal diffusion.
B-Rate theory
This theory takes into account the finite rate at which solute can
equilibrated between the mobile phase and stationary phase.

• That is:
1-Equilibration is not infinitely fast (as plate theory assumed) and the
resulting band shape depends on rate of elution.

2-The band shape is also affected by diffusion of the solute along the
length of the column and by availability of different paths for different
solute molecules to follows as they travel between particles of the
stationary phase.
• All of the effects just mentioned depend on the rate, v, at which the mobile
phase passes through the column. Detailed consideration of the various
mechanisms by which the solute band is broadened leads to the "van
Deemeter equation" for plate height (H.E.T.P.)

• Where A, B, and C are constants characteristic of a given column (stationary

phase) and solvent (mobile phase) system. The equation says that there will be
an optimal velocity for the operation of any column, at which the plate height
(H.E.T.P.) reaches its minimum value with great efficiency.

• To understand the velocity-dependence of H.E.T.P we must explain each term

of “van Deemeter equation”
1) Term "A" (eddy diffusion and unequal pathway)
• This may arise from multiple paths of different length traveled by
solute molecules. The component molecules may therefore travel
different distances as they pass through a unit length of column,
packed with stationary phase particles, which was leads to
broadening of the solute zone, this phenomena is the result of Eddy
diffusion.

• The A term is independent of the mobile phase velocity, but is a

function of the size of stationary phase particles and the way they
are packed in the column.

• λ =a quantity characteristic of the column packing

• dp=is the average diameter of the stationary phase particles
 1

2 3

• Molecules finding relatively easy pathways including the rapidly moving stream
near the walls of the column, will move ahead of the main part of the band and
elute first (Path 3, in the figure).

• Whilst those following longer more erratic paths will take longer time to move
along the column and therefore lag behind (Path 1 and 2, in the figure).
2) Term "B" (longitudinal Diffusion)

• Longitudinal diffusion is used to describe diffusion that takes place along the axis
of the column and parallel to the movement of the mobile phase.

• Term "B" is related to the longitudinal diffusion in the column. Since the
concentration of the solute is lower at the edges of solute zone than at the center,
solute is always diffusing toward the edge of the zone.

• Longitudinal diffusion is time dependent since the longer a band takes to elute the
more time there will be for diffusion to take place; "B" is therefore inversely
proportional to the velocity of the mobile phase "v"
3) Term "C" (resistance to mass transfer)

• Transfer of component molecules between the mobile and stationary phase is

taking place continually during the elution process as the component molecules
try to attain the equilibrium defined by distribution ratio, KD.

-The term "C" account for finite time taken by solute for the mass transfer process
to occur by equilibration between mobile and stationary phases.

• -At high velocity, there is less time available for equilibration, and hence great
band broadening.
• Plot of the height of a theoretical plate (H.E.T.P.) as a function of
mobile-phase velocity (v) using the van Deemter equation. The
contributions to the terms A, B/v, and Cv also are shown
 Chromatographic resolution
The goal of chromatography is to separate a sample into a series of
chromatographic peaks, each representing a single component of the
sample. Resolution is a quantitative measure of the degree of separation
between two chromatographic peaks, A and B, and is defined as

• From this equation it is clear that resolution may be improved either by

increasing ∆tR or by decreasing wA or wB.
• -We can increase ∆tR by enhancing the interaction of the solutes with
the column or by increasing the column’s selectivity for one of the
solutes.
• -Peak width (w) is a kinetic effect associated with the solute’s
movement within and between the mobile phase and stationary phase.
The effect is governed by several factors that are collectively called
column efficiency ( ).
Two methods for improving chromatographic resolution:
(a) Original separation showing a pair of poorly resolved solutes
(b) Improvement in resolution due to an increase in column
efficiency
(c) Improvement in resolution due to a change in column
selectivity.