CHROMATOGRAPHY

 Chromatography is a physical method of separation in which the components to be

separated that distributed between two phases; stationary phase and mobile phase
moves in a definite direction.

 The terms that you should know in chromatography are:

 Mobile phase is solvent moving through the column

 Stationary phase is substance that stays fixed inside the column
 Eluent is fluid entering the column.
 Eluate is fluid exiting the column.
 The stationary phase usually in a column, but many take other forms, such as a
planar phase (flat sheet).

Column
Planar

 The two principal types of chromatography are :

 Gas chromatography (GC)

 Liquid chromatography (LC)
Gas chromatography (GC)

 Separates gaseous substances based on adsorption or on partitioning in a

stationary phase from a gas phase.

Liquid chromatography (LC)

 Includes techniques such as :

i. size exclusion (separation based on molecular size)

ii. ion exchange (separation based on charged)
iii. high performance liquid chromatography (HPLC – separation based on
adsorption or partitioning from a liquid phase)
iv. thin layer chromatography (TLC – a planar form of LC)
 Principles of chromatographic separation

 Process to separate compounds from mixture by passing the mixture through a

column that retains some compounds longer than others.

 The mechanisms of retention for various types of chromatography differ based

on establishment of an equilibrium between a stationary phase and a mobile
phase.
 The equilibrium distribution is described by the distribution constant:

[X]s
Kc =
[X]m

where

[X]S – Concentration of component X in stationary phase

[X]m – Concentration of component X in mobile phase
 In chromatography, when the mobile phase solvent is added to the column , it
is slowly emerge from the bottom of the column.

 The column normally is filled with solvent and chromatographic particles (e.g.
calcium carbonate, alumina, sucrose etc)

 The individual component of mobile phase interact with stationary phase at

different degrees.
 The equilibrium constant is governed by:

 temperature
 type of compound
 Stationary phase
 mobile phase

 In partition chromatography the distribution coefficient is called as partition

coefficient.

 KC ↑- solutes will be retained more strongly by the stationary phase, resulting

move along the column (be eluted) more rapidly.
 The true equilibrium between of two phases is not achieved because have some
lag of the analytes of molecules between the 2 phases that depend on:

i. The flow rate of the mobile phase.

ii. The degree of interaction mobile phase with the stationary phase

 Therefore, resulting in broadening band.

Distribution of two substances A and B,

along a chromatographic column in a
typical chromatographic separation.
 Classification of Chromatography Technique

 Can be classified according to the type of equilibration process involved,

which is governed by the type of stationary phase.

 Various bases of equilibration process are:

 Adsorption
 Partition
 Ion Exchange
 Pore penetration/Size Exclusion
 Adsorption Chromatography

 The stationary phase is a solid on which the sample

components are adsorbed.

 The mobile phase may be :

• liquid → liquid solid chromatography

• gas → gas solid chromatography

 The components distribute between the two phases

through a combination of sorption and desorption
process.
 Example :

Thin Layer Chromatography (TLC)

 The stationary phase is a plane, in the form

of a solid supported on an inert plate.

 The mobile phase is a liquid.

 Partition Chromatography

 The stationary phase is a liquid supported on an inert solid

 The mobile phase may be :

 liquid → liquid liquid partition chromatography

(LPC)
 gas → gas liquid chromatography (GLC)

 In the normal mode of operations of LPC:

 A polar stationary phase (e.g.: methanol on silica) is

used with a nonpolar mobile phase (e.g.: hexane).
 This favors retains of polar compounds and elution of nonpolar compounds
→ normal-phase chromatography

 If a nonpolar stationary phase is used, with a polar mobile phase, then

nonpolar solutes are retained more and polar solutes more readily eluted.
This is called → reversed phase chromatography and is actually the
most widely used.
 Ion Exchange Chromatography

 Ion exchange chromatography (IEC) uses

an ion exchange resin as the stationary
phase.

 The mechanism of separation is based

on ion exchange equilibrium.

http://www.separations.us.tosohbioscience.com/ServiceSuppo
rt/TechSupport/ResourceCenter/PrinciplesofChromatography/I
onExchange

http://www.youtube.com/watch?v=efUrl_djzQ0
 Size Exclusion Chromatography

 In size exclusion chromatography → solvated

molecules are separated according to their size by
their ability to penetrate a sieve like structure (the
stationary phase)

 Also known as gel permeation or gel filtration.

 The pores – small and exclude the larger solute

molecules, but allows smaller molecules to enter the
gel, causing them to flow through a larger volume

 This causes the larger molecules to pass through

the column at a faster rate than the smaller ones.
 Chromatography Nomenclature and Terms
SYMBOL OLD TERM SYMBOL NEW TERM
α Selectivity factor α Separation factor
HETP Height equivalent to a H Plate height
theoretical plate
k’ Capacity factor k Retention factor
n Number of theoretical N Efficiency, Number of
plates plates
neff Effective number of Neff Effective theoretical
theoretical plates plates, Effective plate
number
tm Mobile phase holdup time tM Mobile phase holdup time
tr Retention time tR Retention time
tr ’ Adjusted retention time tR’ Adjusted retention time
w Base peak width wb Bandwidth of peak
SYMBOL TERM
A eddy diffusion term = 2dp
 packing factor
dp average particle diameter
B longitudinal diffusion term = 2DM
 obstruction factor
DM diffusion coefficient
C interphase mass transfer term = dp2 / 6DM
Cm mobile phase mass transfer term
Cs stationary phase mass transfer term
SYMBOL TERM
L column length
u mobile phase linear velocity (cm/s)
ū average mobile phase linear velocity (cm/s)
 reduced velocity
h reduced plate height
Rs resolution
 Retention Time

 Retention time is time required for the sample to travel from the
injection port through the column to the detector.

c/d
e
f
 Theory of column efficiency in chromatography

 The separation efficiency of a column can be expressed in terms of the

number of theoretical plates in the column (movement of substance a
through a chromatographic column).

 A theoretical plate represents a single equilibrium step. The more

theoretical plates, the greater the resolving power (the greater number of
equilibrium steps).

 High efficiency , a large number of plates is necessary.

 The number of plates of efficiency can be obtained from chromatogram from
the expression:

tr 2 N = number of plates
N= 16( ) tr = retention time
wb wb = peak width

 The plate height is defined:

L N = number of plates
H= L = length of column
N H = height plate

 A theoretical plate represents a single equilibrium step. The more theoretical

plates, the greater resolving power (the greater number of equilibrium step)
 N = number of plates
tr = retention time
wb = peak width
b
The narrower the peak, the greater the number of plates.

N
 The effective plate number (Neff) calculated by using the adjusted retention time
instead of the absolute retention time is considered to be a better measure of
the efficiency of capillary columns.

t′r 2
Neff = 16( )
wb
t’r = tr - tm
Response
t’r = adjusted retention time
X 2
t’r2
tm = time required for the
t’r1 tr2 mobile phase traverse through
the column and it would take
X1 for an unretained solute to
tr1 appear.
tXm 0

Wb = peak width
1 3 6
Retention Time
 Retention factor in chromatography

 Retention or capacity factor k for sample peak can be defined by:

𝑡𝑟 −𝑡𝑚 𝑡 ′𝑟
k= =
𝑡𝑚 𝑡𝑚

 A large retention factor favours good separation. However, large

retention factors means increase elution time, so there is compromise
between separation efficiency and separation time.

 The retention factor can be increased by increasing the stationary phase

volume.
Example:
Calculate retention factor of tr = 36 and tm = 6.0
 The effective plate number is related to the retention factor and
effective plate number via:
 Resolution in chromatography

 The resolution of two chromatographic peaks is defined by:

2 (𝑡𝑟2−𝑡𝑟1) 2(𝑉2 −𝑉1)

Rs = Rs =
(𝑤𝑏1+𝑤𝑏2) (𝑤1+𝑤2)

 where tr1 and tr2 are the retention times of the two peaks (peak 1 eluted
first) and wb is the baselines width of the peaks
 The separation factor, α, is a thermodynamic quantity that is a measure of the
relative retention of analytes, and is given by:

𝑡′𝑟2 𝑘2
α= =
𝑡′𝑟1 𝑘1

 t’r1 and t’r2 (adjusted retention times) k1 and k2 (retention factors). This
describe how well the peaks are separated without taking peak width into
consideration. The resolution can, then, be written as:

√𝑁 α−1 𝑘2 𝑘1+𝑘2
Rs = ( )( ) kave =
4 α 𝑘𝑎𝑣𝑒+1 2
 The number of plates required for a given degree of resolution is given by:

2 𝑎 2 𝑘𝑎𝑣𝑒+1 2
Nreq= 16R ( )( )
𝑎−1 𝑘2

 The number of effective plates required is :

2 𝑎
Neff= 16R ( )2
𝑎−1
 Figure shows how the resolution increase
differently with increasing value N, k or α.

 N narrows the peak.

 k increases the retention time for both peak and

broadens them.

 It is more effective to try increasing the α or k by

varying the stationary and mobile phases.