Chromatography

Chromatography
 Chromatography is a laboratory technique that separates components within
a mixture by using the differential affinities of the components for a mobile 2
medium and for a stationary adsorbing medium through which they pass.

 Separation of components in a sample by distribution of the components in

two phases:

 Stationary Phase (either solid or liquid fixed on a column, capillary tube,

plate or sheet)

 Mobile Phase (either liquid or gas)

• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components
 Uses of Chromatography

 Analyze – examine a mixture, its components, and their relations to one 3

another

 Identify – determine the identity of a mixture or components based on

known components

 Purify – separate components in order to isolate one of interest for

further study

 Quantify – determine the amount of the a mixture and/or the

components present in the sample
 Applications of Chromatography
 Pharmaceutical Company – determine amount of each chemical found
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in new product

 Hospital – detect blood or alcohol levels in a patient’s blood stream

 Law Enforcement – to compare a sample found at a crime scene to

samples from suspects

 Environmental Agency – determine the level of pollutants in the

water supply

 Manufacturing Plant – to purify a chemical needed to make a product

 Chromatography Explained
 The Stationary phase is usually in a column but take other forms
such as planar phase (Flat sheet)
 Types of Chromatography:
1. Gas chromatography: Separates gaseous substances based
on adsorption on or partitioning in a stationary phase from
gas phase.
2. Liquid chromatography: It includes techniques such as;
a. Size Exclusion (separation based on molecular
size)
b. Ion exchange (separation based on charge)
c. High performance liquid chromatography (HPLC
is based on adsorption or partitioning from a
liquid phase) along with Thin Layer (TLC) a
planar form of LC
d. Electrophoresis (separation in an electrical
gradient based on the sign and magnitude of
solute charge).
HPLC relies on pumps to pass a pressurized liquid and a sample mixture
through a column filled with adsorbent, leading to the separation of the
sample components. The active component of the column, the adsorbent,
is typically a granular material made of solid particles (e.g., silica,
polymers, etc.), 2–50 μm in size. The components of the sample mixture
are separated from each other due to their different degrees of interaction5
with the adsorbent particles. The pressurized liquid is typically a mixture
of solvents (e.g., water, acetonitrile and/or methanol) and is referred to as
a "mobile phase". Its composition and temperature play a major role in
the separation process by influencing the interactions taking place
between sample components and adsorbent. These interactions are
physical in nature, such as hydrophobic (dispersive), dipole–dipole and
ionic, most often a combination. HPLC is distinguished from traditional
("low pressure") liquid chromatography because  operational pressures
are significantly higher (50–350 bar), while ordinary liquid
chromatography typically relies on the force of gravity to pass the mobile
phase through the column. Due to the small sample amount separated in
analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter,
and 30–250 mm length. Also HPLC columns are made with smaller
adsorbent particles (2–50 μm in average particle size). This gives HPLC
superior resolving power (the ability to distinguish between compounds)
when separating mixtures, which makes it a popular chromatographic
technique.
Thin-layer chromatography (TLC) is a chromatography technique used to
separate non-volatile mixtures. Thin-layer chromatography is performed
on a sheet of an inert substrate such as glass, plastic, or aluminium foil,
which is coated with a thin layer of adsorbent material, usually silica gel,
aluminium oxide (alumina), or cellulose. This layer of adsorbent is
known as the stationary phase.
 Chromatography Explained

(A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the
differential affinities among the mixture components for the stationary phase.
Principles and Theory

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Principles and Theory

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Principles and Theory

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Principles and Theory

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Principles and Theory

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Principles and Theory

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 Ion Exchange Chromatography (IEC)

 The most frequently used techniques for purification 13

 Used for separation of biomolecules
 Separates the molecules according to their net charge, like proteins, and
nucleic acids etc.
 Principle of IEC
 The forces of attraction between stationary phase ---- the mobile phase
 The ion exchanger consists: 14
 An inert support medium covalently bound to negative or positive
functional groups contained by resins
 Oppositely charged ions are attached to these functional groups
which are picked to be replaced by counter ions in the sample
THE BASIC COMPONENTS OF AN ION EXCHANGER

01

02

(Cation exchanger and Anion exchanger)

 Material Used for Exchange Resin
 Two types of material is used:
 Polystyrene 15
 Cellulose
 They differ from each other in:
 Flow properties
 Accessibility to ions
 Chemical and mechanical stability

01

02

(Cation exchanger and Anion exchanger)

 Material Used for Exchange Resin

 Two types of material is used: 16

 Polystyrene
 Cellulose

 Polystyrene:

 Polymerization of Styrene with Divinylbenzene cross linkages

 Useful for the separation of compounds with low MW

 Inadequate for separating macromolecules

 Material Used for Exchange Resin

 Two types of material is used: 17

 Polystyrene
 Cellulose

 Cellulose:

 High molecular weight compound

 Available in high quality pure state

 Best for separation of macromolecules

Exchange Medium

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01

02

(Cation exchanger and Anion exchanger)

Exchange Medium

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 Working

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 Elution can be done in two ways:

 By elution buffer of high magnitude of charge

 By changing the pH of the elution buffer

Procedure

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Procedure

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Procedure

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Procedure

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 Procedure

STRONG EXCHANGER WEAK EXCHANGER

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• Ion exchangers with functional • Ion exchangers with functional ion

ion groups that are totally ionized groups that are ionized at only a
at all working pH values. narrow range pH values

• Sulphonates (-SO3-) • Corboxylate (-COO-)

• Quaternary ammonium (-N+R3) • Diethylammonium (-NH+(CH2CH3)2)
Effect of pH on protein purification

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 Choosing Ion Exchanger

Protein Stability 27

If the protein is stable below its pI ----- Cation Exchanger

 If the protein is stable above its pI ----- Anion Exchanger

Molecular weight (MW) of proteins

<10,000 Da ----- Matrix with small pore size

 10,000-100,000 Da ----- Sepharose Column
 Factors affecting Ion Exchange

1. Ion Exchange Resin 28

The cross linking is

important for the effective
separation
2. Sample
The concentration and charge
of ions

3. Buffer
The pH of the buffer should
impart the same charge to
the sample ions as present in
the column.
 Advantages of IEC

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 Reusable

 Easily collectable

 Cost-effective and Quick separation

 Efficient technique

Low maintenance costs

 Applications

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 Softening of hard water
Deionization of water
To concentrate the metal ions in the sample
To separate protein mixture
To separate nucleic acids
 Size Exclusion Chromatography (SEC)

 SEC is a non-absorption technique for purifying proteins 31

 It uses porous particles to separate molecules of different sizes

 It is generally used to separate biological molecules, and to determine

molecular weights and molecular weight distributions of polymers

 It is usually applied to large molecules or macromolecular complexes such

as proteins and industrial polymers.

 The separation of molecules is called fractionation.

 Size of pores in beads determines (what goes through the beads and what
goes around the beads).
 • IEC
Types of SEC) • SEC

Gel permeation chromatography (GPC) Gel filtration chromatography (GFC)

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When an organic solvent is used as a When an aqueous solution is used to
mobile phase, the technique is known as Gel transport the sample through the column,

permeation chromatography the technique is known as Gel filtration

chromatography 
Uses a hydrophobic column packing
material and a non-aqueous mobile phase Uses a hydrophilic packing material and
(organic solvent) to measure the molecular an aqueous mobile phase to separate,

weight distribution of synthetic polymers fractionate, or measure the molecular

weight distribution of molecules soluble in

water, such as polysaccharides and

proteins.
 • IEC
Principle • SEC

 A mixture of molecules dissolved in liquid (the mobile phase) is applied to 33

a chromatography column which contains a solid support in the form of
microscopic spheres, or “beads” (the stationary phase).

 The mass of beads within the column is often referred to as the column
bed.

 The beads act as “traps” or “sieves” and function to filter small molecules
which become temporarily trapped within the pores.
 • IEC
Principle • SEC

• Larger molecules pass around or are “excluded” from the beads. 34

• Large sample molecules cannot or can only partially penetrate the pores,
whereas smaller molecules can access most or all pores.

• Thus, large molecules elute first, smaller molecules elute later, while
molecules that can access all the pores elute last from the column.

• Particles of different sizes will elute (filter) through a stationary phase at

different rates.
• IEC
• SEC

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 • IEC
Components • SEC

1. Stationary Phase 36

2. The Mobile Phase

3. The Columns

4. The Pump

5. Detectors
 • IEC
1. Components – Stationary Phase • SEC

 Stationary phase consists of semi-permeable, porous beads with well 37

defined range of pore sizes.

 Beads are crosslinked polymers

 Degree of crosslinking is controlled carefully to yield different pore sizes.

 Smaller pore sizes are used for rapid desalting of proteins or for protein
purification.

 Intermediate pore sizes are used to separate relatively small proteins.

 • IEC
1. Components – Stationary Phase • SEC

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• Very large pore sizes are used for purification of biological complexes.

• Stationary phase used for gel exclusion chromatography include dextran

(Sephadex™), polyacrylamide and dextran polyacrylamide (Seshadri™).

• Each is available with a variety of different ranges of pore size in the

beads, permitting separation of macromolecules of different size

 • IEC
1. Components – Stationary Phase • SEC

A good stationary phase should have following properties: 39

 It should be chemically inert.

 It should be inexpensive.

 It should not react with component to be separated.

 It should not react with eluent.

 It should be colorless, uniform in size and shape.

 It should be mechanically stable.

 • IEC
Gel categories and applications • SEC

1. Soft gel e.g. Dextran (Sephadex), Polyacrylamide gels 40

 Separation of proteins.

2. Semi-rigid gel e.g. bio beads

 Separation of non-polar polymers in non-polar solvents.

3. Highly rigid gels and glasses

Separation of polar systems.

 • IEC
Gel categories and applications • SEC

Dextran 41

 A homopolysaccharide of glucose residues. It’s prepared

Sephadex is a gel
with various degrees of cross-linking to control pore size. filtration medium
prepared by
crosslinking dextran
 Procured as dry beads, the beads swell when water is with epichlorohydrin

added.

 The trade name is Sephadex.

 It’s mainly used for separation of small peptides and

globular proteins with small to average molecular mass.
 • IEC
Gel categories and applications • SEC

Polyacrylamide 42

 These gels are prepared by cross linking acrylamide with N,N-

methylene bis acrylamide.

 The pore size is determined by the degree of cross-linking.

 The separation properties of polyacrylamide gels are mainly the same

as those of dextran's.

 They are sold as bio-gel P. They are available in wide range of pore
sizes.
 • IEC
Gel categories and applications • SEC

Agarose 43

 Linear polymers of D-galactose and 3,6 anhydro-1 galactose.

 It forms a gel that’s held together with H bonds. It’s dissolved in boiling
water and forms a gel when it’s cold.

 The concentration of the material in the gel determines the pore size.

 The pores of agarose gel are much larger than those of sephadex or bio-
gel p.

 It’s useful for analysis or separation of large globular proteins or long

linear molecules such as DNA
 • IEC
2. Components – The Mobile Phase • SEC

 The liquid used to dissolve the biomolecules to make the mobile 44

phase is usually called a buffer.

 The mixture of biomolecules dissolved in the buffer is called the

sample.

 The most common eluents for polymers that dissolve at room

temperature. e.g. Tetrahydrofuran, Chloroform, Dimethyl

formamide.
 • IEC
3. Components – The Columns • SEC

 Increasing the column length will enhance the resolution, and increasing
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the column diameter increases the capacity of the column.

 Columns with different porosity or mixed-bed columns, provide a better

separation.

 Proper column packing is important to maximize resolution:

 An over packed column can collapse the pores in the beads, resulting
in a loss of resolution.

 An under packed column can reduce the relative surface area of the
stationary phase accessible to smaller species, resulting in those
species spending less time trapped in pores.
 • IEC
3. Components – The Columns • SEC

 Shorter columns save time and solvent. 46

 Small particles (typically 5 mm) provide a better resolution.

 On the other hand, 5 mm (or even 3 mm) packings are more sensitive
towards contamination by samples containing impurities.

 Isocratic: A column set in SEC should always run in the same

(constant concentration) mobile phase.

 Gradient: Refers to maintaining a varying concentration in the

mobile phase. 
 • IEC
• SEC

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Preparative column vs
Analytical column
 • IEC
4. Components – The Pump • SEC

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 A highly constant flow rate has to be maintained during the entire
chromatogram. This is very important in SEC.

 A change of the flow rate of only 0.1% can cause an error in molar
mass of up to 10%.
 • IEC
5. Components – Detector • SEC

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 Concentration sensitive detection (e.g., UV absorption)

 Molecular mass sensitive detectors (e.g., viscosity detectors)

 Others (e.g., a Fourier Transform Infrared (FTIR) Spectrometer)

Thank you

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