INTRODUCTION TO
CHROMATOGRAPHIC
SEPARATIONS
What is chromatography?
 Chromatography is a powerful separation
method that is usually composed of mobile
phase and a stationary phase.
 This method is used to separate and identify the
components of complex mixtures.
 Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile phase to varying
degrees.
 Those components that are strongly retained by
the stationary phase move slowly with the flow
of mobile phase.
 In contrast, components that are weakly held by
the stationary phase travel rapidly.
 As a consequence of these differences in
mobility, sample components separate into
discrete bands that can be analyzed qualitatively
and/or quantitatively.
Classification of
Chromatographic Methods
 Can be categorized based on the followings:
1. Based on physical means
 The way stationary and mobile phase are brought
into contact
2. Based on the types of mobile phase
 Either gas, liquid or supercritical fluid
3. Based on the kinds of equilibria involved in
the solute transfer between the phases.
 Interaction of analyte between stationary and
mobile phases
Classification of Chromatographic
Methods
Column chromatography
 Stationary phases is held in
narrow tube;
mobile phase moves by
pressure or gravity
E.g. – gas chromatography
(GC)
– supercritical-fluid
chromatography (SFC)
Planar chromatography
 Stationary phase is
supported on a flat plate or in
the interstices of a paper;
mobile phase moves through
capillary action or gravity
E.g. – thin-layer
chromatography (TLC)
– paper chromatography
(PC)
* Based upon physical means
Column chromatography can be further
differentiated based on the types of mobile
phases and the kinds of equilibria involved in
solute transfer between the phases
Mobile Phase
i) Gas
iii) Supercritical fluid
Gas Chromatography
Supercritical-fluid
Chromatography
ii) Liquid
Liquid Chromatography
Types of Chromatography on The Basis of
interaction of The Analyte with Stationary
Phase
 Adsorption – for polar non-ionic compounds
 Ion Exchange – for ionic compounds
 Anion – analyte is anion; bonded phase has positive charge
 Cation – analyte is cation; bonded phase has negative
charge
 Partition – based on the relative solubility of analyte in mobile
and stationary phases
 Normal – stationary phase polar, the mobile phase nonpolar
 Reverse – stationary phase nonpolar, the mobile phase polar
• Size Exclusion – stationary phase is a porous matrix sieving
Classification of
Chromatographic Methods
Chromatography
Partition
Liquid-liquid
Adsorption
Liquid-solid
Gas-liquid
Gas-solid
Ion-exchange
Sizeexclusion
Liquid-solid
Liquid-solid
PARTITION CHROMATOGRAPHY
 Partition chromatography
 Accomplished by selective & continuous transfer
of the components of the mixture back & forth
between a liquid stationary phase and a liquid
mobile phase as the mobile phase liquid passes
through the stationary phase liquid
Stationary phase: liquid
Mobile phase: liquid or gas
 Partitioning
 distribution (by dissolving) of the components
between 2 immiscible phases:
 Relative solubilities of the components in the mobile
and stationary phase
 e.g. stationary phase – polar
 Polar components will retain longer than the non-polar
components.
 Non-polar components will move quickly through
stationary phase & will elute first before the polar
components, and vice-versa.
 Partition chromatography
 The stationary phase actually consists of a thin
film adsorbed (stuck) on or chemically bonded to
the surface of a finely divided solid particles.
 Partition chromatography
 If the mobile phase is gas, the volatility (vapor
pressure) and solubility in stationary phase plays
an important role.
ADSORPTION CHROMATOGRAPHY
 Adsorption (Affinity) Chromatography
 Components of the mixture selectively adsorb (stick)
on the surface of a finely divided solid stationary
phase.
 As mobile phase (gas/liquid) carries the mixture
through the stationary phase, the components of the
mixture stick to the surface of it with varying degrees
of strength & thus separate
 Stationary phase: solid
 Mobile phase: gas or liquid
ION-EXCHANGE CHROMATOGRAPHY
 Ion-exchange chromatography
 Method for separating mixture of ions
 Sample: aqueous solution of inorganic ions /
organic ions
 Stationary phase – small polymer resin “beads”
usually packed in a glass tube
 These beads have ionic bonding sites on their
surfaces which selectively exchange ions with
certain mobile phase compositions as the mobile
phase penetrates through it.
 Ion-exchange chromatography
 Ions that bond to the charged site on the resin
bead are separated from ions that do not repeated
changing of the mobile phase composition.
 The usual procedure is to initially use a mobile
phase with all the ions in the mixture bond & then
to change the mobile phase in a stepwise fashion
so that one kind of ion at a time is removed
 Done until complete separation achieved
SIZE-EXCLUSION
CHROMATOGRAPHY
 Size-exclusion chromatography
 Also called gel permeation chromatography
 Technique for separating dissolved species on the
basis of their size
 Stationary phase: porous polymer resin particles
(molecular sieves)
 The components to be separated enter the pores
of these particles & are slowed from progressing
through this stationary phase.
 Size-exclusion chroamtography
 Separation depends on the sizes of the pores
relative to the sizes of the molecules to be
separated
 Small particles are retarded to a greater extent
than large particles (some of which may not enter
the pores at all) & separation occurs.
TERMINOLOGIES IN
CHROMATOGRAPHY
 Terminologies in chromatography
 Elution:
a process in which species are washed
through a chromatographic column by
addition of fresh solvent
 Mobile phase: is one that moves over or through an
immobilized phase that is fixed in place in
a column or on the surface of flat plate
 Stationary phase: a solid or liquid that is fixed in place. A
mobile phase then passes over or
through the stationary phase
 Retention time: is the time interval btw its injection onto
a column and the appearance of its peak
at the other end of the column
Migration Rates of Solutes
 Distribution constant, K
 Retention time, tR
 Capacity factor,k’
 Selectivity factor, 
Distribution constant, K
 In chromatography, the distribution equilibrium
of analytes between the mobile and stationary
phases can often be described quite simple.
 Let say, we have analyte A. The distribution
equilibrium is written as:
A mobile  A stationary
 Therefore, the equilibrium constant K is called
distribution constant and is defined as:
cstationary
K = c
mobile
c – Molar concentration
K is also called
partition coefficient
or partition ratio
Retention Time, tR
Time required for the sample to travel from the
injection part through the column to the detector.
A typical chromatogram for a two-component mixture.
The small peak on the left represents a species that is not
retained on the column & so reaches the detector almost
immediately after elution is started.
tM
- time taken for the unretained
species to reach the detector.
- sometimes called dead time
- Rate of migration of the
unretained species is SAME as the
average rate of motion of mobile
phase molecules.
- So, tM can be expressed as the
time required for an average
molecule of the mobile phase to
pass through the column.
Retention Factor (Capacity factor), k’
 term used to measure the migration rates of
analytes on columns.
k’A = KA (VS / VM)
[unitless] for analyte A
How is k’A related to tR and tM?
k’A =
tR – tM
tM
When k’A is  1.0, separation is poor
When k’A is > 30, separation is slow
When k’A is 2-10, separation is optimum
Selectivity Factor, 
KB
 is defined as:  =
KA
distribution constants
k’
= B
k’A
capacity factors
= tR(B) – tM
retention times
tR(A) – tM
 A measure of the relative migration rates of
species A and B with a stationary phase material
in chromatography
Response
tR
tR
tM
1
3
6
Retention time , min
tR – tM
k’ =
tM
tR(B) – tM
 =
tR(A) – tM
Column Efficiency
 Two related terms widely used as quantitative
measures of chromatographic column
efficiency:
i) Plate height, H
ii) Number of theoretical plates, N
 The relationship between H and N is:
Column length
L
N =
H
Number of
theoretical plates
Plate height
 The efficiency of chromatographic columns
increases as the number of plates becomes
greater and plate height become smaller.
Efficient column has small plate height
 Experimentally, H and N can be
approximated from the width of the base of
the chromatographic peak.
The equation:
N = 16
tR
W
2
 N can be calculated using tR and W
 To obtain H, the length of the
column must be known
 Another method for approximating N is to
determine W½, the width of the peak at half
its maximum height.
N = 5.54
tR 2
W½
Resolution, Rs
 A measure of the separation of two
chromatographic peaks.
 Baseline resolution is achieved when Rs = 1.5
Rs =
2[tR(B) – tR(A)]
WA + WB
Effect of Capacity Factor & Selectivity
Factor on Resolution
 Relationship btw the resolution of a column
and the capacity factor k’, selectivity factor 
and the number of plates N is given by this
equation:
Rs = √N  - 1
4 
k’
1 + k’
Simplified: Rs = √N
Effect Resolution on Retention Time
 Relationship btw the resolution of a column
and retention time:
tR =
16Rs2H
u

-1
2
( 1 + k’)3
(k’)2
Simplified: tR = Rs2
Example
 Length of column: 30 cm
 Peak widths (at base) for A & B were 1.11 &
1.21 min respectively.
 Calculate:
i) column resolution, Rs
ii) the average number of plates, N
iii) the plate height, H
iv) length of column to achieve Rs 1.5
17.63 min
Response
tR
16.40 min
tR
tM
1.30 min
1
3
Retention time , min
6
i)
Rs =
2[tR(B) – tR(A)]
WA + WB
Rs = 2(17.63 min – 16.40 min)
(1.11 min + 1.21 min)
= 1.06
ii)
N = 16 tR
W
2
N = 16 16.40 min
1.11 min
= 3.49 x 103
2
N = 16 17.63 min
1.21 min
= 3.40 x 103
2
Therefore, calculate
the N average
Nave = 3.44 x 103
iii) H = L / N
= 30 cm / 3.44 x 103 = 8.7 x 10-3 cm
iv) (Rs)1
(Rs)2
√N1
=
√N2
1.06 = √ 3.44 x 103
1.5
√N2
N2 = 6.9 x 103
L = N x H
= 6.9 x 103 x 8.7 x 10-3
= 60 cm
BAND BROADENING
 Band broadening reflects a loss of column
efficiency.
 The slower the rate of mass-transfer processes
occuring while a solute migrates through a
column, the broader the band at the column exit.
 Some of the variables that affect mass-transfer
rates are controllable and can be exploited to
improve separations.
 Table 26.2 lists the variables that influence the
column efficiency.
 Their effect on column efficiency, as measured
by the plate height will be described in the
following slides
VARIABLES AFFECTING COLUMN
EFFICIENCY
VARIABLES AFFECTING COLUMN
EFFICIENCY
 Mobile phase flow rate
 Particle size
 Diameter of column
 Film thickness
EFFECT OF MOBILE PHASE FLOW RATE ON
PLATE HEIGHT
 From both the plots for LC and GC, we can see that
both show a minimum in H at low linear flow rates.
EFFECT OF PARTICLE SIZE ON
PLATE HEIGHT
 Refer to figure 26-11 , page 773
 The numbers to the right is the particle
diameters
 The smaller the particle size, the more
uniform the column packing, then the more
tolerant to the change in mobile-phase
velocity.
EFFECT OF DIAMETER OF THE
COLUMN ON PLATE HEIGHT
 For packed column, the most important
variables that affect column efficiency is the
diameter of the particles that making up the
packing.
 While for open tubular column, the diameter of
the column itself is an important variables.
 Refer to table 26-3, the mobile phase masstransfer coefficient CM is known to be inversely
proportional to the diffusion coefficient of the
analyte in the mobile phase DM.
 CM is proportional to the square of the
particle diameter of the packing material, d1p
(packed column).
 CM is proportional to the square of the
column diameter, d2p (open tubular column).
 As a conclusion, the bigger the column
diameter, the smaller the diffusion coefficient
DM. therefore, we can say that increase in
column diameter will increase the plate
height.
EFFECT OF FILM THICKNESS ON
PLATE HEIGHT
 When stationary phase is an immobilized
liquid, the mass-transfer coefficient Cs is
directly proportional to the square of the
thickness of the film on the support particles
d1l and inversely proportional to the diffusion
coefficient Ds of the solute in the film.
 With thick films and smaller diffusion
coefficient, analyte molecule travel slower. As
a result, slower rate of mass-transfer and an
increase in plate height.
Application of Chromatography
 Qualitative analysis
 Quantitative analysis
Qualitative Analysis
 Based on retention time
 Provided the sample produce the peak at the
same retention time as a standard under identical
conditions
Quantitative analysis
 Analysis based on Peak Height
 The height of chromatographic peak is obtained by
connecting the base lines on either side of the peak by
a straight line and measuring the perpendicular
distance from this line to the peak.
 Analysis based on Peak Area
 Peak areas are usually the preferred method of
quantitation since peak areas are independent of
broadening effects.
 Most modern chromatographic instruments are
equipped with computer or digital electronic
integrator that permit precise estimation of peak
areas.
 Calibration Method
(also known as external method)
- Involve preparation of series of standard
solutions that approximate the
composition of the unknown.
- The peak heights or areas are plotted as a
function of concentration.
- The concentration of the component(s) to
be analysed is determined by comparing
the response(s) peak(s) obtained with the
standard solutions.
 Internal Standard Method
-
-
-
Equal amounts of an internal standard
substance is introduced into each
standard and sample.
The internal standard should not react with the
substance to be examined; it must be stable
and must not contain impurities with a
retention time similar to that of the substance
to be examined.
The concentration of the substance to be
examined is determined by comparing the ratio
of the peak areas (or heights) due to the
substance to be examined and the internal
standard in the test solution with the ratio of the
peak areas (or heights) due to the substance to
be examined and the internal standard in the
standard solution.
 Area Normalization Method
 In the normalization method, the areas of all
eluted peaks
 The percentage content of one or more
components of the substance to be examined is
calculated by determining the area of the peak(s)
as a percentage of the total area of all the peaks,
excluding those due to solvents or any added
reagents and those below the disregard limit.
TAILING AND FRONTING OF
CHROMATOGRAPHIC PEAKS
 A common cause of tailing and fronting is a
distribution constant that varies with
concentration.
 Fronting also arises when the amount of
sample introduced onto a column is too large.
TYPES OF COLUMN
 There are two types of column:
 Packed Column
 Capillary Column
PACKED COLUMN
 Packed column
 Modern packed columns are fabricated from glass
or metal tubing.
 These tubes are densely packed with uniform,
finely divided packing material or solid support,
coated with a thin layer (0.05 to 1 µm) of the
stationary liquid phase.
 The tubes are usually formed as coils.
CAPILLARY COLUMN
 Capillary column
 Also known as open tubular column
 Early wall –coated open tubular (WCOT) column
were constructed of stainless steel, aluminium,
copper or plastic.later, they are made from glass.
 The most widely used capillary columns are
fused-silica wall-coated (FSWC) open tubular
columns.
• Column constructed of fused silica tubing.
• Polyimide coating gives it strength (outer layer). This
resulting columns are quite flexible and can be bent
into coils.
• These capillaries have much thinner walls compared to
glass columns.
• Liquid stationary phases coated or chemically bonded.
 Most applications utilize FSWC open tubular
column (replacing WCOT glass column).
 Recently, 530-µm capillaries, sometimes
called megabore column have appeared on
the market.
• These columns maintain the features of capillary
column.
• These columns tolerate sample sizes that are
similar to those for packed column.
• However, the resolution with these columns is
lower compared to capillary column (resolution is
higher with column of smaller inner diameter).
• Advantage of megabore column over packed
column is their lack of bleeding (loss of stationary
phase with time).