ii) Chromatographic separations

Chromatography was originally developed by the Russian botanist, Michael Tswett in 1903 for
the separation of coloured plant pigments by percolating petroleum ether extract through glass
column packed with powdered CaCO3. Coloured zones were produced by the various pigments
migrating through the column at different rates, the components being isolated by extrusion and
sectioning of the CaCO3 packing. Modern chromatographic techniques are more complex and are
used for a wide variety of separations frequently involving colourless substances to be separated
and quantified.

Basic principle

A chromatographic separation involves placing of a sample onto a liquid or solid stationary

phase and passing a liquid or gaseous mobile phase through or over it a process known as
elution. Sample components or solutes whose distribution ratios between the two phases differ
will migrate (be eluted) at different rates and this differential rate of migration will lead to their
separation over a period of time and distance.

Stationary phase is a phase that is fixed in place either in a column or on a planar surface.
Stationary phase is solid or liquid film coated on solid surface.

Mobile phase is a phase that moves over or through the stationary phase carrying with it the
analyte mixture. The mobile phase may be gas, liquid or supercritical fluid.

Chromatography is a technique I which components of a mixture are separated based on

differences in the rates at which they are carried through a fixed or stationary phase by a gaseous
or liquid mobile phase.

Elution is a process in which solutes are washed through a stationary phase by movement of
mobile phase.

Chromatographic mechanisms
During a chromatographic separation, solute molecules are continually moving back and forth
between the stationary and mobile phases. While they are in the mobile phase, they are carried
forward with it but remain virtually stationary during the time they spend in the stationary phase.
The rate of migration of each solute is therefore determined by the proportion of time it spends in
the mobile phase or in other words by its distribution ratio.

The process whereby a solute is transferred from a mobile to a stationary phase is called
sorption and the reverse is desorption. Chromatographic techniques are based on four different
sorption mechanisms, namely adsorption, partition, ion exchange and exclusion.

Adsorption is a surface effect involving electrostatic interactions e. g H – bonding, dipole –

dipole and dipole – induced dipole interactions. Solute species compete with the mobile phase
for a limited number of polar sites on the surface of the adsorbent of which silica gel is the most
widely used. Its surface comprises Si – O – Si and Si – OH (silanol) groups, the later being
slightly acidic as well as being polar which readily form H – bonds with slightly polar to very
polar solutes.

If a liquid is coated onto the surface of an inert solid support the sorption process is one of
partition and movement of the solute is determined solely by its relative solubility in the two
phases or by its volatility if the mobile phase is a gas.

Note: Both adsorption and partition may occur simultaneously and the contribution of each is
determined by the system parameters i.e the nature of the mobile and stationary phases, solid
support and solute. For example, a stationary phase of Al 2O3 is highly polar and normally
exhibits strong adsorptive properties. However, these may be modified by the presence of
adsorbed water which introduces a degree of partition into the overall sorption process by acting
as a liquid stationary phase. Conversely paper (cellulose) is relatively non polar and retains a
large amount of water which functions as a partition medium. Nevertheless, residual polar
groups in the structure of the paper can lead to adsorptive effects.

Ion exchange is a process whereby solute ions in the mobile phase can exchange with counter
ions carrying the same charge and associated with oppositely charged groups chemically bound
to the stationary phase. The stationary phase is a permeable polymeric solid such as an insoluble
organic resin or chemically modified silica, containing fixed charged groups and mobile counter
ions; which can exchange with the ions of a solute as the mobile phase carries them through the
structure. Both cationic and anionic ion-exchangers are available, the exchange processes being
represented as below;

Cation exchange nR-H+ + Xn+ (R-)nXn+ + nH+

Anion exchange nR+Cl- + Yn- (R+)nYn- + nCl-

R is a polymeric resin or silica

Xn+ and Yn- are solute cations and anions respectively of valency n.

Exclusion differs from other sorption mechanisms in that no specific interactions between solute
species and the stationary phase are necessary. The separating solutes remain in the mobile phase
throughout. Separations occur because of variations in the extent to which the solute molecules
can diffuse through an inert but porous stationary phase. This is normally a gel structure which
has a small pore size and into which small molecules upto a certain critical size can diffuse.
Molecules larger than the critical size are excluded from the gel and move unhindered through
the column or layer whilst smaller ones are retarded to an extent dependent on molecular size.

Note: In each chromatographic technique one of the four mechanisms predominates but it should
be emphasized that two or more may be involved simultaneously.

Characterization of solutes

As already described, the rate of movement of a solute is determined by its distribution ratio
defined as

C stationary phase
D=
C mobile phase

The larger the value of D, the slower will be the progress of the solute through the system and
the components of a mixture will therefore reach the end of a column or the edge of a surface
inorder of increasing value of D. in column methods, a solute is characterized by the volume of
mobile phase required to move it from one end of the column to the other. This is known as
retention volume, VR i.e the volume passing through the column between putting the sample on
top of the column and the emergence of the solute peak at the bottom.

VR = Vm + KVm

Vm is volume of mobile phase in the column (the dead or void volume)

K is retention factor which is directly proportional to D but takes account of the volume of each
phase.

Sometimes K is used to characterize a solute rather than V R. If K = 0, then VR = Vm and the

solute is eluted without being retarded or retained by the stationary phase. Large values of K,
which reflect large values of D result in very large retention volumes and hence long retention
times. At a constant rate of flow of mobile phase F, V R is related to retention time tR by the
equation

VR = FtR

If the flow of mobile phase is monitored by a detector and recorded such as is used in gas and
high performance liquid chromatography then tR can be used as a measure of VR.

Retention time, tR is the time between injection of a sample and appearance of solute peak at the
detector.

In paper and thin layer chromatography, the separation process is halted at a stage which leaves
the separated components in situ on the surface in form of spots. The rate at which a solute has
moved is then determined by its retardation factor, Rf which is defined as

distance travelled by the centre of the solute spot

Rf=
distance travelled by the ront of the mobile phase

Note: - Rf is inversely related to distribution ratio, D and can not be greater than 1. Distances are
measured from the point of application of the sample.

- As both tR and Rf are related to D they will depend on the conditions under which a
chromatogram is run.
A chromatogram is a display of separated components. In column chromatography,
chromatogram is obtained by plotting the detector signal against time to give a series of peaks
eah of which represents the distribution of an eluted component in the mobile phase. In planar
chromatography, chromatogram is a visualised display of the separated spots from a TLC plate
or paper chromatographic separation.

- To identify a component, comparison is made of their t R or Rf to those of the

standards.

Classifying chromatographic methods

a) Column chromatography
The stationary phase is held in a narrow tube and mobile phase is forced through the tube
under pressure or by gravity,
b) Planar chromatography
The stationary phase is supported on a flat plate or in the pores of a paper. The mobile
phase moves through the stationary phase by capillary action or under influence of
gravity.

Types of planar chromatography

i) Thin layer chromatography (TLC)

Separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate
covered with a thin layer of alumina or silica gel (stationary phase).
ii) Paper chromatography (PC)
Separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip
(stationary phase).

Thin layer chromatography

TLC may be either adsorptive or partition. In TLC any substance that can be finely divided and
formed into a uniform layer can be used as a stationary phase. Both organic and inorganic
substances can be used to form a uniform layer for RLC. Organic substances include: cellulose,
polyamide, polyethylene and inorganic: silica gel, aluminium oxide and magnesium silicate.
Stages in TLC

i. Sample application – capillary used to spot solution of each sample.

ii. Development – this is when the separation actually occurs.
iii. Visualization – viewed under UV light.
iv. Interpretation of results – comparison of retention factors.

Sample application (spotting) on TLC plates

- Before applying the sample, “draw guide lines” lightly with pencil 1 m away from the bottom
of TLC plate.

- Sample application is generally done by placing the solute mixture in a volatile solvent (weak
mobile phase).

- Use TLC capillary to transfer and spot dissolved samples.

Development of TLC plates

TLC developing chamber (just glass jar with solvent) is use.

- Place the spotted TLC plate in a developing chamber.

- Developing solution is drawn up the plate by capillary action.

- Remove TLC plate when the solvent reaches top line.

Note: During this approximately 20 minutes developing stage, compounds in the original spots
are being pulled through the silica gel.

Visualization of TLC results

This can be done by direct measurement or chemical derivatization.

 Direct measurement

- Allow the solvent to evaporate from surface of TLC plate.

- View results under UV light, look for grayish spots on the fluorescent green background.

- Mark spots with pencil while viewing under UV.

 Chemical derivatization

For components that do not fluoresce, a chemical substance is added to the separated component
spots that aid visualization under UV. This can be done either by iodine adsorption or oxidation
using sulphuric acid.

Interpretation of TLC results

- Determine retention factors (Rf) for each spot detected. The solvent front is marked and a ruler
is held next to the plate to allow calculation of Rf value.

distance spot has moved

Rf=
distance solvent has moved

- Use Rf of reference spots to identify the other components. The spots are identified by
comparison of their Rf values with those of standard components.

Advantages of TLC

- Easy separation and visualization of compounds.

- Capacity to analyse multiple samples in a single run.
- Relative low cost.

Applications

- Separation of carbohydrates
- Separation of lipid into different classes
- Separation of triacylglycerols

Paper chromatography
It was first introduced by a German scientist Christian Friedrich Schonbein (1865). It is
considered to be the simplest and most widely used of the chromatographic techniques because
of its applicability to isolation, identification and quantitative determination of organic and
inorganic compounds. It is carried out mainly by the flow of solvents on specially designed
paper.

PC is a variant of partition chromatography procedure in which the cellulose support is in the

form of a sheet or paper. Cellulose contain a large amount of bound water even when extensively
dry. Partitioning occurs between the bound water and the developing solvent.

In PC the mixture to be separated is spotted onto the paper and dried. Then the solvent flows
along the sheet either by gravity (descending chromatography) or capillary attraction (ascending
chromatography).

Procedure

- Place 25 mL of solvent in a 60 mL beaker. Cover the beaker and set it aside.

- Obtain a piece of chromatography paper and draw a line 1 cm from the bottom with a pencil.

- Place a small spot of each indicator (standard) on the line.

- Spot and label each of the indicators and the unknown.

- The spots should be about 2 cm apart.

-When the spots have dried, form the paper into a cylinder with the spots facing out. Staple the
edged together being careful to keep them straight and not allowing them to touch.

- Place the cylinder into 600 mL beaker and replace the cover. Be sure the cylinder is not
touching the sides of the beaker.

- Let the chromatogram develop until the solvent is 2 cm from top of paper.

- Remove chromatogram from beaker and immediately mark the solvent front with a pencil.

- Allow chromatogram to dry.

Take chromatogram to the hood and lightly mist it with water. Place it in the ammonia chamber.

- Remove the cylinder from ammonia chamber and unroll it. Immediately circle the coloured
regions with a pencil.

- Determine Rf values for each coloured spot in the knowns and unknowns.

- Use computed Rf values to identify the components unknown.

Applications

- Separation of amino acids.

- Separaion of mixtures of drugs.

- Identification of drugs.

- Analysis of metabolites of drugs in blood, urine.

- Separation of carbohydrates, vitamins, antibiotics, proteins e.t.c

Chromatographic performance

The ideal chromatographic process is one in which the components of a mixture form narrow
bands which are completely resolved from one another in as short a time as possible. The
performance of a particular chromatographic system can be assessed in the following ways.

a) Efficiency and resolution

The width of a band or peak is a measure of efficiency of the process. Resolution is the
ability to separate adjacent peaks of components with similar t R or Rf values. For column
chromatography, a plate number, N is used as a measure of efficiency. Assuming a
Gaussian peak profile, N is defined in terms of solute retention time, tR and peak width as
given by the standard deviation.
N=¿
In practice it is easier to measure baseline width of a peak, W b or the width at half height,
W½ and two alternative expressions derived from the above equation.
 N=16 ¿ …………………………….. (a)

Or N=5.54 ¿ ………………………… (b)

Note: Some laboratories favour the use of equation (a) but some favour (b) on the grounds that
peak width at half height can be measured with greater accuracy than the base width. To make
valid comparisons of efficiencies, the same formula should be used always as the computed
values of N using each of the above formulae may differ considerably.

Measurement of chromatographic efficiency from a Gaussian peak

An alternative measure of efficiency which is independent of the length of a
chromatographic column is the plate height, H (or Height Equivalent of a Theoretical
Plate, HETP) and given by
L
H=
N
Where L is the length of the column expressed in mm or cm.
Note: N is a dimensionless number so to ensure correct computation the units of t R and
W b or W h /2 must be the same.
Resolution, Rs is defined as difference between retention times of two adjacent solute
peaks, ∆t R divided by their average base widths, (W1 + W2)/2
2 ∆ tR
R s=
W 1+W 2
Measurement of the resolution of two adjacent Gaussian peaks
Note: Because of the Gaussian profile of the peaks, a 100 % separation is never attainable
but values of Rs approaching or exceeding 1.5 which is defined as baseline resolution are
satisfactory.
Question
1. A gas chromatographic method for the separation of a mixture of cyclohexane, t-
butanol and benzene on a 10 m capillary column gave the following data:

Parameter Cyclohexane t-butanol Benzene

tR 3 m 20 s 3 m 30 s 3 m 45 s

Wb 8s 9s 11 s

W h /2 4.6 s 5.1 s 6.2 s

Calculate
i) The plate numbers, using both plate number formulae for each solute
Plate numbers N=16 ¿ Or N=5.54 ¿
Cyclohexane N=16 ¿ N=5.54 ¿
t-butanol N=16 ¿ N=5.54 ¿
benzene N=16 ¿ N=5.54 ¿
ii) The plate heights
10000
Plate height H=
N
Cyclohexane H = 1.0 mm H = 0.95 mm
t-butanol H = 1.15 mm H = 1.06 mm
benzene H = 1.49 mm H = 1.37 mm
iii) Resolution between adjacent pairs of solutes
2∆tR
Resolution R s=
W 1+W 2
2(30−20)
Cyclohexane / t-butanol R s= =1.2
8+9
2( 45−30)
t-butanol / benzene R s= =1.5
9+ 11
Note: - The plate numbers are slightly higher when half height peak widths are used to
calculate them due to peak tailing increasing Wb hence comparison of efficiencies are
valid only if the same formula is used throughout.
- The cyclohexane and t-butanol peaks are not fully resolved (R s = 1.2) but the t-butanol
and benzene peaks have baseline resolution (Rs = 1.5)

2. The following data are a liquid chromatographic column

Length of packing 24.7 cm

Flow rate 0.313 mL / min

Vm 1.37 mL

Vs 0.164 mL
 A chromatogram of a mixture of species A, B, C, D provided the following data

Retention time (min) Width of peak base(Wb) (min)

Non retained 3.1

A 5.4 0.41

B 13.3 1.07

C 14.1 1.16

D 21.6 1.72

Calculate
i) The number of pates from each peak. Ans A= 2775.49, B = 2472.04, C =
2363.97, D = 2523.3
ii) The mean and standard deviation for N. Ans mean = 2533.7 SD = 0.2 x 103
iii) The plate height for the column. Ans 9.748 x 10-3
iv) The resolution for species B and C. Ans 0.717

b) Peak asymmetry (peak skew)

The concentration profile of a migrating solute is fundamentally symmetrical (Gaussian)
only if the solute distribution ratio, D remains constant over the concentration range of
the entire peak as shown by a linear sorption isotherm which is a plot of solute
concentration in stationary phase, Cs against that in mobile phase, Cm. However, curved
isotherms resulting from changes in the solute distribution ratio at higher concentration
levels lead to two types of peak asymmetry or skew described as tailing and fronting.
Both tailing and fronting are undesirable as closely eluting peaks will be less well
separated and retention data less reproducible. Where either occur, reducing the amount
 of solute chromatographed will generally improve the peak shape but slow desorption
may still cause some tailing.

Sorption isotherms and the resulting peak profiles

c) Kinetic effects
The ultimate width of a peak is determined by the total amount of diffusion occurring
during movement of the solute through the system and on the rate of mass transfer
between two phases. Both diffusion and mass transfer effects are interdependent and
complex being made up of a number of contributions from different sources. Because
they are kinetic effects, their influence on efficiency is determined by the rate at which
the mobile phase travels through the system.
Effects of diffusion and mass transfer on peak width. (a) Concentration profiles of a
solute at the beginning of a separation. (b) Concentration profiles of a solute after passing
some distance through the system.

Qualitative and quantitative analysis

There are three approaches to qualitative chromatographic analysis.
- Comparison of retention data for unknown solutes with corresponding data for
standards (known substances) obtained under identical conditions. For planar
chromatography (PC and TLC) retardation factors, R f values for standards and unknowns
are compared by chromatographing them simultaneously so as to eliminate variations in
laboratory materials and conditions. For column separations, retention times, t R or
volumes, VR are compared by chromatographing standards and unknowns sequentially
under stable conditions with as little time between runs as possible.
- Spiking samples with known solutes
For column separations where samples are known to contain certain solutes, a
comparison is made between two or more chromatograms run under identical conditions.
The first is of original sample and subsequent ones are obtained after adding a spike of
one of the known solutes. Any peak in the original chromatogram that is of increased size
in a subsequent one can then be identified as the corresponding spiked solute.
- Interfacing the chromatograph with a spectrometer
For column separations, this provides spectral information for each separated solute in
addition to retention data. Spectra of unknown solutes can be compared with those in
computerized library databases or interpreted manually even when pure standards are not
available.

For quantitative chromatographic analysis, in addition to ensuring stable and reproducible

conditions for sample preparation and chromatography, further specific requirements
must be met, namely;
- the analyte (solute) must be identified and completely separated from other components
in the chromatogram.
- standards of known purity must be available.
- a recognized calibration procedure must be used.
For planar chromatography, solute spot areas or densities can be measured insitu, or the
solute spots can be removed, dissolved and measurements made by another analytical
technique such as UV spectrometry.
For column chromatographic separations, quantitation can be by peak area, peak height
or peak height x retention time. Integrated peak areas are directly proportional to the
amount of analyte chromatographed when working within the linear range of the detector
and are the most reliable. They can be measured by computing integrators by
triangulation ( ½ base x height of the triangle that approximates to the Gaussian peak
profile) or by cutting out and weighing peaks drawn by a chart recorder.
Detector responses for an analyte are established by the preparation of a calibration graph
using chromatographed standards with or without the addition of an internal standard by
standard addition or by internal normalization.

Internal standardization
A calibration procedure whereby a constant amount of a selected substance, the internal
standard is added to all samples and analyte standards alike compensates for variations in
sample size and other parameters. The ratio of the detector response for the analyte in
each standard to the corresponding response for the added internal standard is plotted on
the y-axis of a calibration graph against the mass or concentration of the analyte on x-
axis. Response ratios for the analyte and added internal standard in the samples can be
used to determine amount of analyte in the samples by interpolation of the graph. If only
one or two analyte standards are prepared the amount of analyte in a sample can be
calculated by simple proportion.
analyte∈sample response ratio for sample
=
analyte∈ standard response ratio for standard

Internal normalization
For some purposes, only the relative amounts of the analytes in a multicomponent
mixture are required. These are normalized to 100 or 1 by expressing each as a
percentage or fraction of the total. Internal normalization is of particular value in
quantitative chromatography where several components of a sample can be determined
simultaneously and absolute levels are not of interest. The relative composition is
calculated from the instrument response, peak area in the case of a chromatographic
analysis, for each component in the mixture using the formula
Ax
% X i= × 100
ε Ai
Xi is one of n components
A is measured area or response
Example
The measured peak areas (using electronic integration with a computing-integrator,
computer and chromatography data processing software or geometric construction such
as triangulation, ½ base x height) and percentages by internal normalization, which must
total 100 percent are given in table below.
Peak areas and percentage composition by internal normalization for a 5-component
mixture

Component Measure peak areas Relative percent

(arbitrary units)

1 167.8 35.9

2 31.63 6.8

3 108.5 23.2

4 80.63 17.3

5 78.38 16.8

Totals 466.94 100.0

167.8
e.g for component 1, relative percent = ×100=35.9
466.94