Introduction to

Chromatography
Introduction to Chromatography
Chromatography
 a separation technique based on the different interactions of compounds with
two phases, a mobile phase and a stationary phase, as the compounds travel
through a supporting medium
 technique in which the 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

Components:
mobile phase: a solvent that flows through the supporting medium (can be gas,
liquid or supercritical fluid)
stationary phase: a layer or coating on the supporting medium that interacts with
the analytes (fixed in place)
supporting medium: a solid surface on which the stationary phase is bound or
coated
Classification of Chromatographic Methods

A. Based on the type of support material

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

B. Based on the nature of the mobile phase (gas, liquid, supercritical fluid)
1. Gas Chromatography (GC)
o Mobile phase is gas
2. Liquid Chromatography (LC)
o mobile phase is liquid
3. Supercritical Fluid Chromatography (SFC)
o mobile phase is supercritical fluid
Classification of Chromatographic Methods

C. Based on the type of the stationary phase

Gas Chromatography
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support

Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized support
Partition chromatography liquid-coated or derivatized support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support
Affinity chromatography support with immobilized ligand
Elution in Chromatography

Elution
• a process in which solutes are washed through a
stationary phase by the movement of a mobile phase

Eluate
• mobile phase that exits the column is termed the eluate

Eluent
• a solvent used to carry the components of a mixture
through a stationary phase
Elution in Chromatography
At to: a solution of the sample containing a mixture of A and B in the mobile
phase is introduced at the head of the column

At t1 and t2 : first and further introduction of fresh mobile phase, the portion of
the sample contained in the mobile phase moves down the column, where
further partitioning between the mobile phase and the stationary phase occurs

• the average rate at which a solute migrates depends on the fraction of

time it spends in the mobile phase

• analytes interacting most strongly (B) with the stationary phase will take longer
to pass through the system than those with weaker interactions (A).

• resulting differences in rates cause the components in a mixture to

separate into bands, or zones, along the length of the column
t3 and t4: individual bands eluted from the column, where they can be
collected or detected
Chromatogram
• plot of concentration versus elution time or elution volume
• positions of the peak maxima on the time axis can be used to identify the components of the
sample
• peak areas provide a quantitative measure of the amount of each species
Effects of Relative Migration Rate and Band Broadening on Resolution

 A is more strongly retained by the SP than B, B

lags during the migration
 distance between A and B increases as they move
down the column
 broadening of both bands takes place also

Two methods of improving separation:

(1) increase the rate of band separation
(2) decrease the rate of band spreading
Variables that Influence Relative Migration Rate of Solute
• effectiveness of a chromatographic column in separating two solutes depends in part on the
relative rates at which the two species are eluted
• rates are determined by the ratios of the solute concentrations in each of the two phases

Distribution Constants/Partition Ratios

• chromatographic separations are based on differences in the extent to which solutes are
distributed between the mobile and the stationary phase

• ideally, distribution constant (Kc) is constant over a wide range of solute concentrations,
that is, cS is directly proportional to cM
Variables that Influence Relative Migration Rate of Solute

Retention Time

Where:
tR = retention time
tM = dead or void time
ts = time spent in SP

• tM is the time it takes for an unretained species to pass through

a chromatographic column

• Separations are based on the different times, tS, that

components spend in the stationary phase

• tR is the time required for the analyte peak to reach the

detector after sample injection
Retention Factor/Capacity Factor, k
• experimental parameter widely used to compare the migration rates of solutes on columns
• the amount of time a solute spends in the stationary phase relative to the time it spends in
mobile phase
• For solute A, the retention factor, kA

• kA < 1 solute emerges from the column at a

time near tM
• kA > 20 to 30 elution times become inordinately
long
• 1≤ kA ≥5 ideal separations
Selectivity Factor, α
• provides a measure on how well a column separates two analytes
• ratio of the partition ratio of a more strongly retained solute to the partition ratio of the less
strongly held solute KB : partition ratio of more strongly retained
species B
• For two species A and B, >1 KA: partition ratio of less strongly held or more
rapidly eluted species A

kB: retention factor for B

kA: retention factor for A

• To determine α from the chromatogram,

Band Broadening and Column Efficiency
• amount of band broadening that occurs as a solute passes through a chromatographic column
strongly affects the column efficiency

Rate Theory of Chromatography

• describes the shapes and breadths of elution bands in quantitative terms
• elution peaks look very much like the Gaussian or normal error curves
• as a single solute molecule undergoes many thousands of transfers between the stationary and the
mobile phases during elution, residence time in either phase is highly irregular
• transfer from one phase to the other requires energy, and the molecule must acquire this energy
from its surroundings
• residence time in a given phase may be very short after some transfers and relatively long after
others
• movement through the column can occur only while the molecule is in the mobile phase
• certain particles travel rapidly by virtue of their accidental inclusion in the mobile phase for a
majority of the time
• Other particles lag because they may be incorporated in the stationary phase for a greater-than-
average length of time results to a symmetric spread of velocities around the mean value
(represents the behavior of the average analyte molecule)
Band Broadening
• Some chromatographic peaks are nonideal and exhibit tailing or fronting
• Tailing and fronting are undesirable because they lead to poorer separations and less reproducible
elution times

Tailing can be due to:

• distribution constant that varies with
concentration

Fronting can be due to:

• distribution constant that varies with
concentration
• introduction of large amount of sample
onto a column
Quantitative Description of Column Efficiency
Efficiency is related experimentally to a solute’s peak width.
- an efficient system will produce narrow peaks
- narrow peaks  smaller difference in interactions in order to separate two solutes

Efficiency is related theoretically to the various kinetic processes that are involved in solute retention and
transport in the column
- determine the width or standard deviation (s) of peaks

Quantitative measures of chromatographic column efficiency:

(1) plate height, H (a.k.a. height equivalent of a theoretical plate, HETP)
N = L/H
(2) plate count or number of theoretical plates, N
L: length (cm) of the
column packing

• column efficiency increases = N increases = H becomes smaller

• differences in efficiencies are encountered in columns due to differences in column type, MP
and SP
Quantitative Description of Column Efficiency

• N can be determined from a chromatogram

• the retention time of a peak tR and the width of the peak at its base W (in units of time) are
measured:
The larger the value of N is for a column, the
better the column will be able to separate
two compounds.

• the better the ability to resolve

solutes that have small differences in
retention
• N is independent of solute retention
• N is dependent on the length of the
column
Effect of Mobile-Phase Flow Rate

 efficiency studies have been

carried out by determining H
as a function of mobile phase
velocity

• GC and LC show a minimum in H (or a maximum in efficiency) at low linear flow rates
• minimum for LC usually occurs at flow rates well below those for GC
• liquid chromatograms are obtained at lower linear flow rates than gas chromatograms
• LC columns are more smaller than those with GC columns
• it is impractical to use LC columns that are longer than about 25 to 50 cm because of high
pressure drops
• GC columns may be 50 cm or more in length
• the total number of plates and overall column efficiency are usually superior with GC columns
Longitudinal Diffusion Term
• results in the migration of a solute from the concentrated center of a band to the more dilute
regions on either side (that is, toward and opposed to the direction of flow)

• a common source of band broadening in GC than in LC where the rate at which molecules diffuse
is high

• contribution of longitudinal diffusion to plate height is inversely proportional to the linear velocity
of the eluent (analyte is in the column for a briefer period when the flow rate is high)
Stationary phase mass-transfer term
• band-broadening due to the movement of solute between the stagnant phase and the stationary phase

Since different solute molecules

spend different lengths of time in
the stationary phase, they also
spend different amounts of time
on the column, giving rise to
band-broadening.

The degree of band-broadening due to stationary phase mass

transfer depends on:

1) the retention and diffusion of the solute

2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and the
stationary phase
Stationary phase mass-transfer term
• When the stationary phase is an immobilized liquid:
 mass-transfer coefficient is directly proportional to df2 (square of the thickness of the film on
the support particles) and inversely proportional to DS (diffusion coefficient) of the solute in
the film
 df2 and DS reduce the average frequency at which analyte molecules reach the interface
where transfer to the mobile phase can occur

with thick films, molecules travel farther to reach the surface result is a slower rate of mass transfer
with smaller diffusion coefficients molecules travel slower and an increase in plate height

• When the stationary phase is a solid surface

 mass-transfer coefficient is directly proportional to the time required for a species to be
adsorbed or desorbed
Mobile phase mass-transfer term
• mobile-phase mass-transfer coefficient CM is inversely proportional to the diffusion coefficient of the analyte in the
mobile phase DM

a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence of multiple flow
paths through a packed column.

As solute molecules travel through the Different residence times in the

column, some arrive at the end sooner than column for molecules of the
others simply due to the different path same species vary.
traveled around the support particles in the
column that result in different travel
distances.
Longer path arrives at end of column after (1).
Mobile phase mass-transfer term

b.) Stagnant mobile phase mass transfer – band-broadening due to differences in the rate of diffusion of the
solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the
mobile phase within the pores of the support (stagnant mobile phase).

Since a solute does not travel

down the column when it is in
the stagnant mobile phase, it
spends a longer time in the
column than solute that remains
in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase

mass transfer depends on:

1) the size, shape and pore structure of the packing material

2) the diffusion and retention of the solute
3) the flow-rate of the solute through the column
c.) Mobile phase mass transfer – a process of peak broadening caused by the presence of different flow
profile within channels or between particles of the support in the
column.

A solute in the center of the

channel moves more quickly than
solute at the edges, it will tend to
reach the end of the channel first
leading to band-broadening

The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly
on:

1) the size of the packing material

2) the diffusion rate of the solute
 There is an optimum flow rate at which the plate height is a minimum and the separation
efficiency is a maximum
Summary of Methods for Reducing Band Broadening

1. Decrease particle size and column diameter

2. With gaseous mobile phases, the rate of longitudinal diffusion can be reduced
appreciably by lowering the temperature (lowers diffusion coefficient)
3. With liquid stationary phases, the thickness of the layer of adsorbed liquid should be
minimized
Column Resolution (Rs)
• resolution between two peaks is a second measure of how well two peaks are separated

Rs is preferred over a since

both retention (tr) and column
efficiency (Wb) are considered
in defining peak separation.

 Rs = 1.5 gives an essentially complete separation of A

and B
 Rs = 0.75 gives poor separation
 Rs = 1.0 zone A contains about 4% B, and zone B
contains about 4% A
 At Rs = 1.5, the overlap is about 0.3%
 Rs for a given stationary phase can be improved by
lengthening the column, thus increasing the number of
plates. The added plates, however, result in an increase in
the time required for separation
 2. Variation in the retention Factor
Optimization Techniques
• Increases in kB generally enhance resolution (but at
1. Variation in plate height
the expense of elution time)
• values of kB >10 provide little increase in resolution
but markedly increase the time required for
separations
• optimal value of kB lies in the range from 1 to 5
• For gaseous MP, k can often be improved by
• resolution of a column improves as the square temperature changes
root of the number of plates increases • For liquid mobile phases, changes in the solvent
• however, increasing the number of plates is composition optimizes k to yield better separations
expensive in terms of time unless the increase is
achieved by reducing the plate height and not
by increasing column length
• reduce the particle size of the packing material,
the diameter of the column, and the thickness
of the liquid film (ways to minimize plate height)
• optimize flow rate of the mobile phase
Optimization Techniques
3. Variation in selectivity factor

• optimizing k and increasing N are not sufficient to give a satisfactory separation of two solutes
in a reasonable time when α approaches 1

• increase α while maintaining k in the range of 1 to 10

• Options:
1. changing the composition of the mobile phase
2. changing the column temperature
3. changing the composition of the stationary phase (interchange columns)
4. using special chemical effects
General Elution Problem
chromatogram (a):
• conditions adjusted so that the retention factors for
components 1 and 2 (k1 and k2) are within the range of 1
to 5

• factors for the other components are far larger than the
optimum

• bands corresponding to components 5 and 6 are broad

and appear only after an inordinate length of time has
passed

chromatogram (b):
• changing conditions to optimize the separation of
components 5 and 6 bunches the peaks for the first four
components to the point where their resolution is
unsatisfactory
• the total elution time is ideal
General Elution Problem
Solution:

• change conditions stepwise that determine the values of

k as the separation proceeds

• For LC, variations in k are brought about by varying the

composition of the mobile phase during elution (gradient
elution or solvent programming)

isocratic elution (elution under conditions of constant

mobile-phase composition)

• For GC, the temperature can be changed in a known

fashion to bring about changes in k (temperature-
programming mode)
Applications of Chromatography
Qualitative Analysis:

• used for recognizing the presence or absence of components in mixtures that contain a limited
number of species whose identities are known based on retention time

• confirmation of identity would require spectral or chemical investigation of isolated components

• the chromatogram may not lead to positive identification of the species in the sample but it
often provides sure evidence of the absence of species

• failure of a sample to produce peak at the same retention time as a standard obtained under
identical conditions is strong evidence that the compound in question is absent (or present at a
concentration below the detection limit of the procedure)
Applications of Chromatography
Quantitative Analysis:

• based upon a comparison of either the peak height or the peak area of the analyte with that of
one or more standards
• analysis based on peak height can be used provided variations in column conditions do not alter
peak widths during recording of chromatograms; peaks should not be distorted or the column
overloaded
• analysis based on peak area is less sensitive to small changes in operating conditions;
independent of broadening effects

Calibration with Standards

1. External Standard Method

• involves the preparation of a series of standard solutions that approximate the composition of
the unknown

• the calibration is a plot of peak height (or peak area) vs. concentration of standard
Applications of Chromatography
Calibration with Standards

2. Internal Standard Method

• minimizes uncertainty in sample injection and variability due to instrument conditions

• a carefully measured quantity of an internal standard is introduced into each standard and
sample, and the ratio of analyte peak area (or peak height) to internal standard peak area
(or peak height) is obtained; the calibration curve is a plot of this ratio vs. concentration

• requirements for internal standard: must be completely resolved; must elute near peaks of
interest; similar in concentration to peaks of interest; chemically inert and absent in the
sample