Chapter 5 of 9

PRINCIPLE OF
CHROMATOGRAPHY

BMS481
BIOANALYTICAL CHEMISTRY
NURUL AILI ZAKARIA (PhD)
Course Learning Outcomes (CLOs)

At the end of the course, students should be able to:

1. Describe the fundamentals concepts of analytical techniques e.g. accuracy,
concentrations, molarity.
2. Illustrate the principles and applications of basic centrifugation,
chromatographic and spectroscopic analytical techniques in the isolation
and characterization of biological molecules.
3. Perform scientific experiments on biomolecules separation, characterization
and analysis.
Chapter Overview

Link to Practical
- Running a
Types & applications planar
- Column chromatography
Chromatography
Separation by -Planar
Chromatography Chromatography
- The main
components
Brief Intro -Principle
- What it is?
 Brief History

– The word Chromatography is derived from two Greek words-Chroma

means “color” and graphein to “write”
– Described by Mikhail Twsett in 1903 when separating plant pigments
using calcium carbonate
– Chromatography is a misnomer since it is no longer limited to the
separation of the colored substances.

Chromatography is a laboratory techniques for the separation of mixtures

 Link to

Introduction
Practical
Types &
- Running a
applications
planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle

- What it is?

What is it for?
Chromatography is a process whereby the:
❖ The components of a mixture may be separated
❖ Purity of a sample may be determined
❖ The separated components may be identified

Greek: chromos = color , graphy = write

 Link to

Introduction
Practical
Types &
- Running a
applications
planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle

- What it is?
How does it separates?

– A mixture can be separated using the differences in physical or chemical

properties of the individual components

➢ based on the states of matter of the two phases

➢ useful for separations of density and size
➢ useful chemical properties by which compounds can be separated
are: solubility, boiling point, and vapor pressure

Greek: chromos = color , graphy = write

 Link to

Terminologies
Practical
Types &
- Running a
applications
planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle

- What it is?

Chromatography Analyte Stationary phase Mobile phase

a physical method of • Substance which is to be • It is also called as • It is also known as
separation in which the purified or isolated adsorbent. solvent and eluent.
components to be during chromatography • May be a “solid or • May be a
separated are distributed liquid” supported on a “liquid/solvent/mixture
in h between two phases, solid (or) a gel of solvent/gas”
one of which is stationary, • Maybe ; 1) packed in a
while the other (mobile column 2) spread as a
phase) moves in a definite layer on a
direction glass/aluminium plate 3)
Distributed as a liquid
film
 Link to

Terminologies
Practical
Types &
- Running a
applications
planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle

- What it is?

Eluate Elution Chromatograph Chromatogram

• A machine that • Visual output of the
Is the separated Process of removing performs chromatograph
component the components chromatography by • Different peaks or
from the column gas or liquid patterns represent
separation different
components of the
separated mixture
 Separation by
Chromatograp
Types &
applications
- Column
Chromatograp
hy
Link to
Practical
- Running a
planar
chromatogra
phy: TLC
The Chromatogram
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle

- What it is?
 Introduction

➢ A sample that requires analysis is often a mixture of

many components in a complex matrix

➢ Often a standard containing either a single pure

compound or a mixture of known amounts of several
compounds will be used for comparison with the test
sample (by comparing the chromatograms).

Fig 1.
a) Chromatogram of an unknown mixture containing
five compounds as shown by five peaks
b) Chromatogram of a standard that contains caffeine
(1), aspirin (2) and salicylamide (5)
 Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography

Classification of Chromatography
-Planar Chromatography

Separation by * Answer questions in

i-learn
Chromatography

Brief Intro
- What it is?
- components
-Principle i. Based on physical state of mobile phase

Eg: column
Liquid-solid
adsorption TLC,
chromatography
HPLC
Liquid
Chromatography
Eg: column
Liquid-liquid partition
chromatography
Chromatography Paper partition

Gas Gas
Eg: GLC, GSC
Chromatography chromatography
 Link to Practical
- Running a planar
Types & applications chromatography:

Classification of Chromatography
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

i-learn

ii. Based on physical state of stationary phase

Chromatography
- components
Brief Intro
- What it is? -Principle

Chromatography
Stationary phase: solid Stationary phase: Liquid

Adsorption Partition
chromatography chromatography

Eg: column adsorption Eg: column partition

Ion exchange, size Paper partition
exclusion, affinity, TLC,
HPLC GLC
 Link to Practical
- Running a planar
Types & applications chromatography:

Classification of Chromatography
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

i-learn

iii. Based on polarity of stationary & mobile phase

Chromatography
- components
Brief Intro
- What it is? -Principle

Chromatography

Normal phase Reverse phase

chromatography chromatography

Stationary phase: polar Stationary phase: nonpolar

(silica gel) (Octadecylsilane or C18)
Mobile phase: nonpolar (n- Mobile phase: polar
hexane) (Acetonitrile, water)
 Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

Separation by chromatography
i-learn
Chromatography
- components
Brief Intro
- What it is? -Principle

– The basis of all forms of chromatography is the partition or distribution of a

compound (Kd), between two immiscible phases ; stationary phase and a mobile
phase.
– The value for Kd is a constant at a given temperature and is given by the expression:

Concentration of the compound in stationary phase

Kd =
Concentration of the compound in mobile phase

– If a mixture of compounds is applied to a chromatography column, they will

distribute between the two phases according to their Kd values
At equilibrium in a particular chromatographic system,
compound with a:

– Kd above 1 has a greater concentration in the stationary

phase than in the mobile phase
– Kd below 1 has a greater concentration in the mobile phase
than in the stationary phase
The Kd can also be use to describe the relative retention time of
a particular compound.
As Kd
increases, it
takes longer
for compounds
to separate
Requirements for column chromatography

➢ Column characteristics & selection

➢ Stationary phases
➢ Mobile phases
➢ Preparation of column
➢ Introduction of sample
➢ Development of column
➢ Detection of components
➢ Recovery of components
 Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

1. Column characteristics & selection

i-learn
Chromatography
- components
Brief Intro
- What it is? -Principle

Materials of construction Good quality neutral glass, plastic or nylon

Adsorbent (stationary phase)/ adsorbate 30:1
(mixture) weight ratio
Column length to diameter ratio (cm) 10-15: 1 or 30-100: 1
Multi component system is present Long column
Components with similar affinities for Long column
adsorbent are present
Components with different affinities for Short column
adsorbent are present

Weak adsorbent, few compounds ????

 Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

2. Stationary phases
i-learn
Chromatography
- components
Brief Intro
- What it is? -Principle

• Most commonly used adsorbent is silica gel (80-100 mesh or 100-200 mesh size)
• They adsorbs polar and unsaturated substances by formation of hydrogen bonds
with hydroxyl groups on the silicon atom

Type of mesh Size in microns

60/120 mesh 120-250 micron
100/200 mesh 75-150 micron
70/230 mesh *63-200 micron
230/400 mesh 37-63 micron
70/325 mesh 45-200 micron
 Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

2. Stationary phases
i-learn
Chromatography
- components
Brief Intro
- What it is? -Principle

Table 1 Adsorbents used in column chromatography

Weak activity Medium activity Strong activity

Sucrose Calcium carbonate Activate magnesium silicate

Starch Calcium phosphate Activated alumina

Inulin Magnesium carbonate Activated charcoal
Talc Magnesium oxide Activated magnesia
Sodium carbonate Calcium oxide Activated silica
 Link to Practical

Types & applications

- Column Chromatography
- Running a planar
chromatography:
TLC
It can also be your purification
-Planar Chromatography

Separation by * Answer questions in

BUFFERS
3. Mobile phase
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Chromatography
- components
Brief Intro
- What it is? -Principle

• Either gas or liquid which flows over/or through the stationary phase
either by use of a pumping system or applied gas pressure.

• Also referred as the eluent/solvent

• Act as a solvent to introduce the mixture into the column; as

developer to develop the zones for separation; and as an eluent to
remove the pure component out of the column

As the eluent flows through the column, the analytes separate on the basis of their
Kd and emerge individually in the eluate as it leaves the column
 Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

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Chromatography
- components
Brief Intro
- What it is? -Principle

3. Mobile phase
 Let's get to know more….

Stationary phase aka. Mobile phase

adsorbents
What does it do? Stays put! Flows around stationary phase

What is it? (physical form) Solid or liquid Liquid or gas

How does it effect sample Retains sample by surface Moves sample along
movement? interaction

Analogous to.. An obstacle course Motivating force

Example Silica gel Ethyl acetate
 4. Preparation (packing) of the column
Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

Chromatography
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a) Slurry packing (wet packing method)
- components
Brief Intro
- What it is? -Principle

The adsorbent is suspended in the mobile

phase and stirred very well to remove air
bubbles. The resulted slurry is then poured in
to the column.

At the bottom portion of the column, a piece

of glass wool or cotton/whatman filter paper
disc must be added before the slurry
application

After slurry application, the column must be

allowed to settle overnight. This is the ideal
method of column packing
https://www.youtube.com/watch?v=G4jyd8L0MWE
https://www.youtube.com/watch?v=8Ff1Xx-On-g
 4. Preparation (packing) of the column
Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

Chromatography
i-learn
a) Dry packing
- components
Brief Intro
- What it is? -Principle

In this method, the dry adsorbent is

poured to the column directly

Vibration is applied to get rid of air

bubbles then the mobile phase is
passed through the adsorbent.

The demerit with this method is that

air bubbles are entrapped between
the solvent and the stationary phase.
 4. Preparation (packing) of the column
Link to Practical
- Running a planar
Types & applications chromatography:
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

Chromatography
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a) Dry packing
- components
Brief Intro
- What it is? -Principle
5. Set up for the operation of a chromatography column
6. Detection and recovery of components

Each fraction is
Fractions are collected examined by TLC using
by elution analysis suitable experimental
conditions

From the column

Those fractions which
fraction, solvent is
give same Rf values in
evaporated, dried & the
TLC are added as a
material collected in
common fraction
eppendorf

After spectral analysis

(NMR, MS, XRD etc.) the
compound is identified
6. Detection and recovery of components
 Link to Practical

Applications of chromatography
- Running a planar
chromatography:
TLC
Types & applications

(in general)
- Column
Separation by Chromatography
Chromatography
- components -Planar Chromatography
-Principle
Brief Intro * Answer questions in
- What it is?
i-learn Inorganic ions Separation of copper, cobalt, nickel etc
Organic Purification of dyes (ie sudan red, methylene blue,
malachite green)
Separation of diastereomers
Separation of tautomers and racemates
Plant constituents Separation of geometrical isomers of cis and trans
isomers of bixin and crocetin dimethyl ether using
alumina
Purification od steroids, carotenoids, chlorophils and
xanthophylls
Separation of alkaloids and glycosides
Drugs and pharmaceuticals Purification of vitamin B12, Oxytetracyclin and
oleabdomycin
Separation of amino acids, antibody, proteins,
phospholipids and triglycerides.
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