SCH 2204 SEPARATION TECHNIQUES

Objective
The course is intended to empower learners with knowledge on the principles of
chromatographic and non- chromatographic techniques.

Expected Outcomes
At the end of the course a student is expected to:
1. Describe the theory in non-chromatographic techniques.
2. Describe instrumental components of chromatographic techniques.
3. Discuss the working principles of electrophoresis.
4. State the applications of separations techniques.

Course Description
Non-chromatographic techniques; distillation, crystallization, liquid-liquid extraction.
Sorption processes and theory of chromatography. Chromatographic techniques; paper and
thin layer, column; gas-liquid, high performance liquid chromatography and supercritical
fluid chromatography. Electrophoresis. Continuous flow measurements. Coupling of
instruments: GC-MS, GC-IR.
Practicals: Liquid-liquid extraction, planar chromatography and Column Chromatography.

Textbooks and References

1. Textbooks
a) Skoog, D.A. and Leary, J.J. (1992): Principles of instrumental Analysis (5 th Ed).
New York; Saunders college publishing.
b) Clifton, E.M. (1999): Chemical separations: principles, techniques and experiments.
USA; Wiley-Interscience.
c) Gurdeep, R.C. (2006): Analytical Chromatography, Himalaya Pub. House.

2. References for Introduction to Instrumentation

a) Harris, D.C. (1991): Quantitative Chemical Analysis. New York; W.H. Freeman and
company.
b) West, C.D. (1987): Essentials of quantitative analysis. McGraw-Hill book company
c) Hargis, G.H. (1988): Analytical Chemistry: Principles and Techniques, Prentice Hall
International, London.
d) Ian, D.W. Michael, C. and Poole, C.F. (2000): Encyclopaedia of separation science,
Vol. 1. Academia.
Course Journals
1. Journal of chromatography and separation techniques
2. Journal of separation science

Reference Journals
1. Journal of separation and purification technology
2. Journal of Chromatography A
THEORY OF CHROMATOGRAPHY
1.0 Introduction
Chromatography is a physical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary while the other moves in a definite
direction. In a chromatographic system, the sample is introduced in a small volume at the inlet of
a column or another carrier of the stationary phase. The mobile phase transports the sample
components in contact with the stationary phase throughout the column. Due to different
interactions between the sample components and the stationary phase, the sample components
migrate through the system at different speeds and elute from the column with different retention
times. The retention time is defined as the time between the sample introduction and the elution
from the column. At the end of the column, a detector provides a signal for all eluting
components (universal detection) or for a limited number (selective detection). In a sample with
many components, some compounds will coelute, partly or completely, depending on the
complexity of the sample and the peak capacity of the column.With mass spectrometric
detection, even coeluting components can be identified. Figure 1 shows ways in which
classification of chromatographic methods is done.
.

Figure 1. Classification of chromatographic methods

Chromatographic methods can be classified according to the separation principle, namely,
elution development, displacement development and frontal analysis. In practice only elution and
to a lesser extent, displacement development are commonly used.
Figure 3. Column chromatography

When the sample components are separated and detected by a detector connected to the outlet of
the column and the signals from the detector are visualized as a function of time, a
chromatogram is obtained.

Figure 3. Chromatogram of polymeric amines separated by gradient elution in HPLC

2.0 Theory of chromatography

Consider a sample mixture (consisting of components A B C) transported by a mobile phase over
a stationary phase. Each component or solute (A,B,C) is distributed between the two phases with
an equilibrium established defined by the distribution ratio (previously known as the partition
ratio); thus for component A.
where [As] is the concentration of A in a unit volume of the stationary phase, and [AM] is the
concentration of A in a unit volume of the mobile phase. The distribution ratio, KA, for A, is

Each component separated will have a different value for K, reflecting their relative affinities for
the stationary phase; the generalised form of the distribution equation for each component is

The larger the value of K the greater the affinity that component has for the
stationary phase, that is, Cs is larger and CM smaller.
Figure 4. Data symbols for components A B C

The resulting time the solute molecules spend attracted to the stationary phase constitutes a delay
with respect to the movement of the mobile phase. Consequently, a mixture of components
would be delayed by varying amounts due to their differing retention by the stationary phase.

Factors influencing retention

 Composition and properties of the mobile phase;
 type and properties of the stationary phase;
 the intermolecular forces between the component(s) and stationary and mobile phases;
and
 temperature.
Components being separated by chromatography may have polar character, that is, the molecule
contains polar functional groups or has non-polar alkane-like character. Like attracts like,
therefore solutes with polar groups have a stronger interaction with a polar stationary phase than
with a non-polar stationary phase and conversely non-polar groups are more attracted to a non-
polar stationary phase than a polar stationary phase. Hydrogen bonded solvents generally will
attract polar solute molecules but will exhibit varying degrees of repulsion to non-polar
molecules. The volume of mobile phase required to carry a band of component molecules
through the system to the detector is termed the retention volume, VR, and is measured from the
start of the chromatography to the peak maxima (Figure 4). In GC and HPLC a constant flow
rate of mobile phase is maintained and the time taken by a component band to pass through the
column is recorded as the retention time, t R. If the flow rate of the mobile phase through the
column is Fc then retention volume is calculated from

The average linear velocity of the mobile phase ū is calculated from the length of the column, L,
and the time the mobile phase takes to pass through the column, tM:

Retention volume and retention time are measured from the time the sample is introduced into
the chromatograph to when the component(s) are eluted from the column; no allowance is made
for the volume of mobile phase in the system nor the time the mobile phase takes to pass from
the injector to the detector. A correction therefore has to be made to obtain a more accurate
representation of the retention of a component by the stationary phase. V’R, the corrected
retention volume, accounts for the volume of mobile phase in the system

Where VM, tM are often referred to as the dead volume and dead time, respectively (Figure 4).
The greater the proportion of component or analyte in the mobile phase (CM), the faster it will
progress through the system; conversely the lower the proportion, the greater the retardation and
so retention time will increase. Retardation, RF, is therefore a function of the fraction of
component in the mobile phase:
Retention factor

Separation factor

The ability of a stationary phase to separate two components A and B, where B is the more
strongly retained component, is determined by their relative partition or distribution ratios and
hence their retention factors for a given stationary phase. The separation α is therefore a function
of the relative retention of each component by a stationary phase.
The separation factor for an adjacent pair of bands is a function of the type of stationary phase
used, the mobile phase and column temperature, and should be optimized for the most difficult
to separate early eluting pair of compounds. For separation to occur α should be greater than 1.0,
however in high performance chromatography such as capillary column GC and HPLC a is
almost 1.0 for closely eluting compounds having 'narrow' peaks.

2.1 Separating efficiency of a column

Figure 5. Peak heights of a Gaussian peak and width as a function of standard deviation.

The peak recorded in a chromatogram represents the distribution of molecules in a band as it

elutes from the column, the overall broadness being conveniently measured in terms of the width
of the peak. A number of independent factors such as sample-injector and detector
characteristics, temperature and column retention processes, contribute to the dispersion of
molecules in a band and band broadening. The cumulative effect of small variations in these
factors is described in statistical terms as the variance, in the elution process. Classical
chromatography theory considers that the separation process takes place by a succession of
equilibrium steps, the more steps in a column the greater the column efficiency with less band
broadening (variance) occurring. N, the theoretical number of separation steps (plates) in a
column determines its separating capabilities; N is therefore an indication of column efficiency.
σ, the standard deviation of the Gaussian peak, describes the spread of the molecules in a band.
Band broadening is also a function of time, the longer the band takes to elute the more time the
molecules have to spread out, therefore
2.2 Band Broadening
In all chromatographic systems, there is band broadening (Figure 1.2), caused by different
physical processes. In the columns, the following processes can occur:

 Eddy diffusion
 Longitudinal diffusion in the mobile phase
 Resistance to mass transfer: in the mobile phase, stationary phase, and stagnant
mobile phase
Eddy Diffusion
Eddy diffusion occurs due to the presence of multiple channels of different widths and lengths in
porous structures. Large inhomogeneous particles cause large contributions to band broadening
of eddy diffusion. In a packed column, the size of the eddy diffusion is proportional to the
particle size. A wide range of particle size also increases the eddy diffusion.
The main contribution of eddy diffusion to the plate height is

Longitudinal Diffusion
Longitudinal diffusion in the mobile phase is due to the natural tendency of a compound in a
concentrated band to diffuse into less concentrated zones. The contribution of longitudinal band
broadening is proportional to the diffusion constant.
Resistance to Mass Transfer

Resistance to mass transfer describes the band broadening caused by transporting the analytes by
diffusion and convection from one phase to the other. Resistance to mass transfer is, in general,
inversely proportional to the diffusion constants in either phase. In the mobile phase, there is an
additional link to eddy diffusion. The contribution to the plate height can be described by band
broadening taking place in the mobile phase, stagnant mobile phase, and stationary phase.
Combined Band Broadening in a Column

Figure 5. Van Deemter plot of plate height (H) as a function of linear flow

The van Deemter equation combines the different contributions in a simplified equation:

Where A is eddy diffusion, B is the longitudinal diffusion in the mobile phase, and C is the
resistance to mass transfer in the stationary phase (in GC) or in both the phases (LC). The
contribution of stagnant mobile phase is valid only with large particles and is therefore usually
neglected in the vanDeemter equation for high-performance systems. The contribution of
resistance to mass transfer in the mobile phase can be neglected in GC, but not in HPLC. The
van Deemter equation can be visualized by plotting the contributions to the plate height as a
function of the linear mobile phase velocity.