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CHAPTER 4
INTRODUCTION TO CHROMATOGRAPHIC
SEPARATIONS
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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

(sample) to distribute themselves between a stationary and a
mobile phase to varying degrees.

• The substances must interact with the stationary phase to be

retained and separated by it.
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• 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
quantitatively.
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• Anychromatography system is composed of three

components:

1. Stationary phase
2. Mobile phase
3. Mixture to be separated
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Chromatogram
If a detector that responds to solute concentration is
placed at the end of the column during elution & its
signal is plotted as a function of time a series of peak
obtained.

Such plot is called chromatogram.

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Peak positions used to identify components;

Peak areas used to determine the amounts of each
component/analyte.
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Classification of Chromatographic
Methods
1. Based upon physical means
The way stationary and mobile phase are brought into
contact

Column chromatography Planar chromatography

• Stationary phases is held in
narrow tube;
• mobile phase moves by pressure
or gravity
• Ex: - gas chromatography
(GC)
- high performance liquid
chromatography (HPLC)
• Stationary phase is supported on a
flat plate or in the interstices of a
paper;
• mobile phase moves through
capillary action or gravity
• Ex: – thin-layer chromatography
(TLC)
– paper chromatography
(PC)
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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

2. Based on the types of mobile phase

Either gas, liquid or supercritical fluid

Mobile Phase

i) Gas
iii) Supercritical fluid
Gas Chromatography
Supercritical-fluid Chromatography

ii) Liquid CO2 as mobile phase

Liquid Chromatography
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3. Based on the kinds of equilibria involved in the solute

transfer between the phases.
Interaction of analyte with stationary phase

a) Adsorption – The stationary is a solid on which the sample

components are adsorbed.

b) Ion Exchange – attraction of ions of opposite charges; for ionic

compounds anions or cations

c) 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

d) Size Exclusion
Separates molecules by size
Stationary phase is a porous matrix sieving
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Classification of Chromatographic
Methods
Chromatography

Partition Adsorption Ion- Size-

exchange exclusion

Liquid- Liquid-solid Liquid-solid Liquid-solid

liquid
Gas-liquid Gas-solid
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PARTITION
CHROMATOGRAPHY

Stationary phase is a liquid

supported on an inert solid
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• Partition chromatography
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.

 If the mobile phase is gas, the volatility (vapor pressure) and solubility in
stationary phase plays an important role.
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ADSORPTION
CHROMATOGRAPHY
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• Adsorption 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.
• The components distribute between the two phases through a combination of
sorption and desorption process.

• Stationary phase : solid

• Mobile phase : gas or liquid
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ION-EXCHANGE
CHROMATOGRAPHY
• Anion exchange – analyte is
anion; bonded phase has
positive charge.

• Cation exchange – analyte

is cation; bonded phase has
negative charge.
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• Ion-exchange chromatography
• Uses an ion exchange resin as stationary phase
• 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.

Sulfonic acids –SO3H (strong acid resin) Rz-(SO3H) + M+  Rz-(SO3M) + H+

Cation
Carboxylic acid –COOH (weak acid resin) Rz-COOH + M+  Rz-COOM + H+
exchanger
Quaternary ammonium group: strong base --H2N(CH3)3OH-
Rz-CH2N(CH3)3OH- + B-  Res-CH2N(CH3)3+B- + OH-

Amine group: weak base --NH(-R)2OH- Anion

Rz-NH(-R)2OH- + B-  Res-NH(-R)2+B- + OH- exchanger

Application:
Can be used for almost any kind of charged molecule including large proteins and amino acids.
• Protein purification
• Water analysis
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SIZE-EXCLUSION
CHROMATOGRAPHY

small molecules travel longer

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• Size-exclusion chromatography

• Also called gel permeation chromatography/gel filtration 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.

• Separation depends on the sizes of the pores relative to the sizes of the
molecules to be separated.

• The pores are normally small and exclude the larger solute molecule, but
allows smaller molecules to enter.
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• Small particles are retarded to a greater extent than large particles (some of which
may not enter the pores at all) & separation occurs.

• Molecules that are larger than the pore size of the packing are excluded.

• Applied to large molecules or macromolecular complexes such as proteins and

industrial polymers.
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TERMINOLOGIES IN
CHROMATOGRAPHY
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• 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:
• The time interval between its injection onto a column and the appearance of its
peak at the other end of the column.
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Migration Rates of Solutes

• Distribution constant, K

• Retention time, tR

• Capacity factor/retention factor, k’

• Selectivity factor, 
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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:

cS K is also called
Kc  stationary
mobile
partition coefficient/
partition ratio
cM
CS – Molar concentration in the stationary phase
CM – Molar concentration in the mobile phase
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Retention Time, tR

• Time required for the sample to travel from the injection port 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.
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Inject

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 a molecule of
the mobile phase to pass through the column.
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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?

tR -tM Describes the strength of the interaction

k’ = between a solute with the stationary and
tM
mobile phase
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
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Selectivity Factor, 

Describe the separation of 2 species (A & B) on

the column.

K
α  B
=
K A k’B
(t )  t =
α  R

(t )  t
B M
k’A
R A M

• A measure of the relative migration rates of species A and

B with a stationary phase material in chromatography.
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tR

tR
Response

tM

1 3 6

Retention time, min

Retention factor Selectivity factor

tR – tM tR(B) – tM
k’ =  =
tM tR(A) – tM
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Column Efficiency
• Efficiency is related experimentally to a solute’s peak
width.

• An efficient system will produce narrow peak.

• Two related terms widely used as quantitative measures

of chromatographic column efficiency:

i) Plate height, H
ii) Number of theoretical plates/plate count, N
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• The relationship between H and N is:

Larger value of N, the smaller

N  L /H
plate height, the narrower
chromatographic band, better
separation

where: L = length of column (usually in cm)

N = number of theoretical plates
H = 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
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• Experimentally, H and N can be approximated from the

width of the base of the chromatographic peak.

The equation:

N  number of plates
2
 tR 
N  16  
W 

 N can be calculated using tR and W

 To obtain H, the length of the column must be known
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• Another method for approximating N is to determine W½,

the width of the peak at half its maximum height.

N  number of plates
t 
2

N  16   R

W 
 t 
2

N  5.54  R

W 1/ 2 
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Resolution, Rs
• A measure of the separation of two chromatographic
peaks.
• Baseline resolution is achieved when Rs = 1.5 Complete
resolution

2[(t R ) B  (t R ) A ]
Rs 
WA  WB

Describes how well 2 compounds are separated

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Effect of Capacity Factor & Selectivity Factor on

Resolution
• Relationship between 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 k’
4  1 + k’

Simplified: Rs = √N
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Effect Resolution on Retention Time

• Relationship between the resolution of a column and
retention time:

2
tR = 16Rs2H  ( 1 + k’)3
u  - 1 (k’)2
Highest possible resolution in the shortest possible time

Simplified: tR = Rs2
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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
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17.63 min
Response
tR

16.40 min

tR

1.30 min
tM

1 3 6

Retention time , min

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i) Rs = 2[tR(B) – tR(A)]
WA + WB
Rs = 2(17.63 min – 16.40 min) column resolution, Rs
(1.11 min + 1.21 min)
= 1.06

2
ii) N = 16 tR
W

2
N = 16 16.40 min
1.11 min the average number of plates, N

= 3.49 x 103
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2
N = 16 17.63 min Therefore, calculate
1.21 min the N average
= 3.40 x 103
Nave = 3.44 x 103

the plate height, H

iii) H = L/N
= 30 cm / 3.44 x 103 = 8.7 x 10-3 cm
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LEARNING CHECK…
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LEARNING CHECK…
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BAND BROADENING
Efficient system produce narrow peak.

• 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.

The faster the mobile phase moves, the less time there is for equilibrium between
the phases and the mass transfer effect on peak broadening
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Multiple flow paths/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 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 the column after 1

Flow
Direction

1 2
Pathways of two molecules
during elution. Distance traveled
by molecule 1 is longer than
that traveled by molecule 2, thus
molecule 1 will take longer to
elute.
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Longitudinal diffusion – 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:

1) the diffusion of the solute

2) the flow-rate of the solute
through the column

Molecules diffuse from areas of high

concentration to areas of low concentration.

Flow

Over time….

Flow
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VARIABLES AFFECTING COLUMN

EFFICIENCY

• Mobile phase flow rate

• Particle size
• Diameter of column
• Film thickness
• Increase the length of the column-column efficiency
increase
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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.

Efficient column has small plate height

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EFFECT OF PARTICLE SIZE ON PLATE

HEIGHT

• The smaller the particle size, the more uniform the column
packing, then the more tolerant to the change in mobile-
phase velocity.

Efficient column has small plate height

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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.
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• Refer to table 26-3, the mobile phase mass-transfer

coefficient CM is known to be inversely proportional to the
diffusion coefficient of the analyte in the mobile phase DM
CM α 1/DM
• CM is proportional to the square of the particle diameter of
the packing material, d2p (packed column).
CM α d2p
• CM is proportional to the square of the column diameter, d2c
(capillary/open tubular column).
CM α d2c
• 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.
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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
d2f and inversely proportional to the diffusion coefficient
Ds of the solute in the film.
Cs α d2f α 1/Ds

• 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.
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Application of Chromatography
• Qualitative analysis
• Quantitative analysis
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Qualitative Analysis
• Based on retention time

• The sample produce the peak at the same retention time as a

standard under identical conditions.

• By comparison with known components, retention time is used for

identification of a component of a mixture.
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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.
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• 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.
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Internal Standard Method

• Uncertainties in sample injection can be overcome by use of an internal
standard.

• In this method, a measured quantity of an internal standard is added to both

standard and sample, and the ratio of analyte signal to internal standard is
recorded.

• Any inconsistency in injection of the sample will affect both the analyte and
internal standard.

• Properties of the internal standard should include:

a. The retention times of internal standard and analyte should be

different and the two peaks must be well separated

b. Internal standard should not react with the substance to be

examined;

c. It must be stable

• Using internal standards can significantly improve precision to better than 1%.
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• Area Normalization Method

• This technique can also overcome the uncertainties associated with

sample injections.
• In this method, complete elution of all components is necessary
• Areas of all eluted peaks are computed and calculated areas are
corrected for detector response.
• The concentration of the analyte is thus the ratio of its corrected peak
area to total corrected areas of all peaks.
• The method is not as versatile as the internal standard method.
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TAILING/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.

• Lead to poorer separations