Chromatography Methods Part 1

Chromatography
Introduction
Origin of Chromatography
The Russian botanist Mikhail Tswett first used
the term Chromatography (Latin for colored
drawing) in 1906, to describe the separation
that occurred when solutions of plant pigments
were passed through columns of calcium
carbonate or alumina, using petroleum ether.
Tswett in his work stated that:

chromatography is a method in which
components of a mixture are separated on
adsorbent column in a flowing system.
Chromatography now includes a number

of variations on the basic separation process, it
encompasses a wide range of techniques many
of which have their own terminology.
The IUPAC Definition of Chromatography

"Chromatography is a physical method of
separation, in which the components to be
separated are distributed between two phases,
one of which is stationary whilst the other
moves in a definite direction".
Solute migration and retention

The rate of migration of a solute through a stationary
phase is determined by its distribution ratio, K, which in
turn is determined by its relative affinity for the
two phases. In the context of chromatography, K is
defined as the ratio of the total solute concentration, CS
, in the stationary phase to that in the mobile phase,
CM
Thus, large values of K lead to slow solute migration, and

small values of K lead to rapid solute migration.
Solutes are eluted in order of increasing distribution ratio.
The larger the differences between the distribution ratios of
the solutes in a mixture, the more easily and quickly they
can be separated.
Because the interaction of solutes with the stationary phase
slows down their rate of migration relative to the velocity of
the mobile phase, the process is described as retardation or
retention.
Sorption processes
Sorption is the process whereby solute species are
transferred from the mobile to the stationary phase,
desorption being the reverse process. These processes
occur continually throughout a chromatographic
separation, and the system is therefore described as
being in a state of dynamic equilibrium. A solute is
repeatedly redistributed between the phases as the
mobile phase advances, in an attempt to maintain an
equilibrium corresponding to its distribution ratio, K.
Sorption mechanisms
There are four basic sorption mechanisms, and it is
common for two or more to be involved simultaneously
in a particular mode of chromatography.
Adsorption is a surface effect, not to be confused with

absorption, which is a bulk effect. Surface adsorption
involves electrostatic interactions such as hydrogenbonding, dipoledipole and dipole-induced dipole
attractions.
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.
Partition is a sorption process analogous to solvent

extraction, the liquid stationary phase being thinly
coated or chemically bonded onto an inert solid. Where
the liquid is bonded to the supporting solid, it is
debateable as to whether it behaves as a liquid and
whether the sorption process should be described as
modified partition, because adsorption may also be
involved. In true partition, solutes are distributed
according to their relative solubilities in the mobile and
stationary phases.
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. Both cationic and anionic ion-exchangers are
available.
Exclusion differs from the other sorption mechanisms in

that no specific interactions between solute species and
the stationary phase are necessary or desirable. The
stationary phase is a controlled-porosity silica or
polymer gel with a range of pore sizes, and solutes
remain in the mobile phase throughout the separation,
merely diffusing through the porous structure to
different extents depending on their size and shape.
Solutes whose size exceeds the diameter of the largest

pores are entirely excluded from the structure and
migrate at the same rate as the mobile phase. Solutes
smaller than the diameter of the smallest pores can
diffuse throughout the structure and have the slowest
rate of migration. Solutes of an intermediate size can
diffuse through some pores but not others, migrating at
rates between those of the largest and smallest species.
The chromatogram and its purpose

Chromatography is a dynamic system with the
separation process taking place continuously as the
solute moves over the stationary phase to attain the
equilibrium defined by the distribution ratio, K (Figure
1).The component can be considered to move through
the column or plate in a series of theoretical separation
steps with distribution between the two phases as
defined by K.
Therefore , in order to obtain efficient

separations, the mobile phase , stationary
phase and equilibrium conditions need to be
carefully selected to achieve the desired
separation as rapidly as is feasible but with
minimum dispersion or band broadening.
Figure 1
The eluted compounds are transported by the

mobile phase to the detector and recorded
as Gaussian (bell-shaped) curves.
The signals are known as peaks and the
whole entity is the chromatogram.
The chromatogram can be used to provide

information on separation efficiency:
Here w is the peak width at the baseline, t0 is the
dead time or retention time of an unretained solute,
i.e. the time required by the mobile phase to pass
through the column (also called the breakthrough
time). Hence the linear flow velocity, u, can
be calculated as:
Figure 9
u = L/t0
where L is the column length. A nonretained compound,
i.e. one that is not retained by the stationary phase,
appears at the end of the column at t0. tR is the
retention time; this is the period between sample
injection and recording of the peak maximum. Two
compounds can be separated if they have different
retention times. tR is the net retention time or adjusted
retention time.
tR = t0 + tR
t0 is identical for all eluted substances and represents
the mobile-phase residence time. tR is the stationary
phase residence time and is different for each
separated compound.
The longer a compound remains in the stationary
phase, the later it becomes eluted.
Retention time is a function of mobile phase flow

velocity and column length. If the mobile phase
is flowing slowly or if the column is long, then t0
is large and hence so is tR; tR is therefore not
suitable for characterizing a compound.
Therefore the retention factor or k value
(formerly known as the capacity factor, k) is
preferred:
Retention factors between 1 and 10 are preferred. If the

k values are too low, then the degree of separation may
be inadequate (if the compounds pass too rapidly
through the column, no stationary phase interaction
occurs and hence no chromatography).
High k values are accompanied by long analysis times.
Equation 1
k is independent of the column length and

mobile phase flow rate and represents the
molar ratio of the compound in the stationary
and the mobile phase, as mentioned earlier.
Problem 1
Calculate the k values of compounds 1 and 2
given:
t0=12.5 mm; tR1=33.1 mm; tR2=70.5 mm.
Two components in a mixture cannot be separated

unless they have different k values, the means of
assessment being provided by the separation factor, ,
formerly known as the relative retention.
Equation 2
with k2 > k1. If =1, then no separation takes

place as the retention times are identical. The
separation factor is a measure of the
chromatographic systems potential for
separating two compounds, i.e. its selectivity.
Selection of the stationary and mobile phases
can affect the value of .
Problem 2
Calculate the value of compounds 1 and 2.
(in problem 1)
The resolution, R, of two neighbouring peaks is defined

by the ratio of the distance between the two peak
maxima, i.e. the distance between the two retention
times, tR ,and the arithmetic mean of the two peak
widths, w:
Equation 3
where w1/2 is the peak width at half-height.
The peaks are not completely separated with a

resolution of 1, but two peaks can be seen. The
inflection tangents touch each other at the
baseline. For quantitative analysis a resolution
of 1.0 is too low in most cases. It is necessary to
obtain baseline resolution, e.g. R=1.5.
Resolution Values
Problem 3
Calculate the resolution of compounds 1 and 2.
given :
tR1=33.1 min; tR2=70.5 min; w1=17mm ;
and w2=29 mm
The chromatogram can also be used to calculate the

number of theoretical plates, N, in the column:
Equation 4
Equation 5
Problem 4
How many theoretical plates emerge from
calculations based on the last peak in Figure 9?
Given tR2 =70.5mm; w2 = 29 mm:
The height of a theoretical plate, H, is readily calculated

provided the length of the column is known:
Equation 7
where H is the distance over which chromatographic

equilibrium is achieved (see Figure 2) and is referred
to as the height equivalent to a theoretical plate
(HETP).
Problem 5
Using the chromatogram shown in Figure 10,
calculate:
(a) retention factors for peaks 15;
(b) separation factors for the best and worst
resolved pair of peaks;
(c) resolution of these two pairs of peaks;
(d) plate number for each of peaks 15.
Figure 10
Peak Symmetry
The normal dispersion of component molecules as
they move through the chromatographic system is
represented by a bell-shape Gaussian peak (Figure
8).However , if some molecules in the band are
more strongly retained on the stationary phase
,then these molecules will lag behind the main band
and will form a tail on the main peak(Figure 11).
Figure 11
Fronting occurs when some of the molecules move

ahead of the main band due to less than expected
retention by stationary phase .This may be due to too
large a sample being introduced onto the stationary
phase.
Peak symmetry can be described in various ways, see

Figure 12. Left: A line is laid at 10% of the peak height,
thereby defining the sections a0.1 and b0.1. The
first one describes the distance from the peak front to
the maximum, the second one the distance from
maximum to peak end:
Figure 12
Right: the definition according to United States

Pharmacopoeia (USP) uses a line at 5% of the
peak height. The first section is defined as f and
the peak width at 5% height as w0.05:
Generally a peak with T =1.0 is symmetrical
Problem 6
Calculate the tailing of the peak shown in Figure
12. with two methods

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Chromatography

Tswett in his work stated that:

Chromatography now includes a number

The IUPAC Definition of Chromatography

Solute migration and retention

Thus, large values of K lead to slow solute migration, and

Adsorption is a surface effect, not to be confused with

Partition is a sorption process analogous to solvent

Ion-exchange is a process whereby solute ions in the

Exclusion differs from the other sorption mechanisms in

Solutes whose size exceeds the diameter of the largest

The chromatogram and its purpose

Therefore , in order to obtain efficient

The eluted compounds are transported by the

The chromatogram can be used to provide

Retention time is a function of mobile phase flow

Retention factors between 1 and 10 are preferred. If the

k is independent of the column length and

Two components in a mixture cannot be separated

with k2 > k1. If =1, then no separation takes

The resolution, R, of two neighbouring peaks is defined

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

The peaks are not completely separated with a

The chromatogram can also be used to calculate the

The height of a theoretical plate, H, is readily calculated

where H is the distance over which chromatographic

Fronting occurs when some of the molecules move

Peak symmetry can be described in various ways, see

Right: the definition according to United States

Generally a peak with T =1.0 is symmetrical

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