Introduction to Chromatography

Chromatography
is 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.

Components:
mobile phase: a solvent that flows through the supporting medium

stationary phase: a layer or coating on the supporting medium that interacts

with the analytes

supporting medium: a solid surface on which the stationary phase is bound or

coated
 Chromatography separation in progress

 The analytes interacting

most strongly with the
stationary phase will take
longer to pass through the
system than those with
weaker interactions.

 These interactions are

usually chemical in nature,
but in some cases
physical interactions can
also be used.
Chromatographic techniques classified based on

1) the type of mobile phase used in the system

Mobil phase Type of Chromatography
Gas Gas chromatography (GC)
Liquid Liquid chromatography
2) the type of stationary phase used in the system
Gas Chromatography
Stationary phase Type of chromatography
Solid support Gas-solid chromatography
Liquid-coated support Gas-liquid chromatography

Liquid Chromatography
Stationary phase Type of Chromatograhy
solid, underivatized support Adsorption chromatography
liquid-coated or derivatized support Partition chromatography
support containing fixed charges Ion-exchange chromatography
porous support Size exclusion chromatography
support with immobilized ligand Affinity chromatography
3) the type of support material used in the system

 Packed bed (column) chromatography

 Liquid chromatography

 Open tubular (capillary) chromatography

 Gas chromatography

 Planar chromatography
 Paper chromatography
 Thine layer chromatography
Types of Chromatography:- chromatography can be classified based on the type of mobile
phase, stationary phase and support material
Theory of Chromatography

1) Typical response obtained by chromatography

Chromatogram - concentration versus elution time

Wh

Wb

Inject
Where:
tR = retention time tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
The separation of solutes in chromatography depends on two factors:

 a difference in the retention of solutes (i.e., a difference in their

time or volume of elution
 a sufficiently narrow width of the solute peaks (i.e, good
efficiency for the separation system)

Peak width & peak position

determine separation of peaks
2) Solute Retention:
A solute’s retention time or retention volume in chromatography is directly
related to the strength of the solute’s interaction with the mobile and stationary
phases.

Retention on a given column pertain to the particulars of that system depends

on the size of the column and the flow rate of the mobile phase

Capacity factor (k) more universal measure of retention, determined from tR.

k = (tR –tM)/tM

capacity factor is useful for comparing results obtained on different systems

since it is independent on column length and flow-rate.
Capacity factor (k)

The value of the capacity factor is useful in understanding the retention

mechanisms for a solute, since the fundamental definition of k is:
n Astationary phase
k =
n Amobile phase

k is directly related to the strength of the interaction between a solute with the
stationary and mobile phases.

nAstationary phase and nAmobile phase represents the amount of solute present in each
phase at equilibrium.

Equilibrium is achieved or approached at the center of a chromatographic peak.

When k' is < 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum

Load Eluent
(sample) (solvent)

k’ > 1000 k’ < 0.001

Compounds of
Compounds of
interest released
interest stick to
from sorbent
sorbent

Waste Extract
 Mobile phase Mobile phase
Load
Sample
(injection)
1 < k’ < 10
Compounds of Differential
interest in a interaction with
plug stationary phase
separates
compounds in
time and space.

Detector Detector
A simple example relating k to the interactions of a solute in a column is
illustrated for partition chromatography:

A (mobile phase) KD A (stationary phase)

where: KD = equilibrium constant for the distribution of A between the

mobile phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

As KD increases, interaction of the solute with the stationary phase becomes

more favorable and the solute’s retention (k) increases
 Separation between two solutes requires different KD’s for their interactions
with the mobile and stationary phases

since G = -RT ln KD

peak separation also represents different changes in free energy

3) Efficiency
Efficiency is experimentally related 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 theoretically related 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

Estimate s from peak widths, assuming

Gaussian shaped peak:
Wh
Wb = 4s

Wh = 2.354s

Dependent on the amount of time that a solute spends in the column (k’ or tR)
Number of theoretical plates (N)
compare efficiencies of a system for solutes that have different retention times

N = (tR/s)2

or for a Gaussian shaped peak

N = 16 (tR/Wb)2

N = 5.54 (tR/Wh)2

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
Plate height or height equivalent of a theoretical plate (H or HETP):
compare efficiencies of columns with different lengths:

H = L/N
where: L = column length
N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical
plate

H can be also used to relate various chromatographic parameters (e.g., flow rate,
particle size, etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?

 Eddy diffusion
 Mobile phase mass transfer
 Stagnant mobile phase mass transfer
 Stationary phase mass transfer
 Longitudinal diffusion
Eddy diffusion (A-term) – 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 column,

some arrive at the end sooner then others simply
due to the different path traveled around the
support particles in the column that result in
different travel distances.

Longer path arrives at end of column after (1).

Longitudinal diffusion (B-term) – band-broadening due to the
diffusion of the solute along the length of the column in the flowing
mobile phase.

The degree of band-broadening due to longitudinal diffusion depends

on the:
 diffusion of the solute
 flow-rate of the solute through the column
Mobile phase mass transfer (c-term) – 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 the:

 size of the packing material

 diffusion rate of the solute

Stagnant mobile phase mass transfer (C-term) – 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 the:
 size, shape and pore structure of the packing material
 diffusion and retention of the solute
 flow-rate of the solute through the column
Stationary phase mass transfer (C-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 the:
 retention and diffusion of the solute
 flow-rate of the solute through the column
 kinetics of interaction between the solute and the stationary phase
Van Deemter equation: relates flow-rate or linear velocity to H:
H = A + B/µ + Cµ
where:

 = linear velocity (flow-rate x Vm/L)

H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer

 One use of plate height (H) is to relate these kinetic process to band
broadening to a parameter of the chromatographic system (e.g.,
flow-rate).

 This relationship is used to predict what the resulting effect would

be of varying this parameter on the overall efficiency of the
chromatographic system.
Plot of van Deemter equation shows how H changes with the linear
velocity (flow-rate) of the mobile phase

µ optimum

Optimum linear velocity (µopt) - where H has a minimum value

and the point of maximum column efficiency:

µopt is easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
4) Measures of Solute Separation:
separation factor (a) – parameter used to describe how well two solutes are
separated by a chromatographic system:

 = k′2/k′1 k′ = (tR –tM)/tM

where: k′1 = the capacity factor of the first solute
k′2 = the capacity factor of the second solute, with k′2 ≥ k′1

A value of  ≈ 1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention.
Rs is preferred over a since both
retention (tr) and column efficiency
(Wb) are considered in defining peak
separation.

Rs ≥ 1.5 represents baseline

resolution, or complete separation
of two neighboring solutes  ideal
case.

Rs ≥ 1.0 considered adequate for

most separations.
Resolution
Describes how well 2 compounds are separated

selectivity
efficiency
retention

Example: Given the following data and a 24.7 cm column

Compound Retention Time (min) Peak Width(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 the resolution between species C and D. What column

length is required for a resolution of 1.5?