What is Chromatography?

Chromatography is a technique for separating mixtures

into their components in order to analyze, identify,
purify, and/or quantify the mixture or components.

• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components
Illustration of Chromatography
Stationary Phase

Separation

Mobile Phase
Mixture Components
Affinity to Stationary Affinity to Mobile
Components
Phase Phase
Blue ---------------- Insoluble in Mobile Phase

Black  

Red  

Yellow          
 Types of Chromatography
• Liquid Chromatography – separates liquid samples with a liquid solvent (mobile
phase) and a column composed of solid beads (stationary phase)
• Gas Chromatography – separates vaporized samples with a carrier gas (mobile
phase) and a column composed of a liquid or of solid beads (stationary
phase)
• Paper Chromatography – separates dried liquid samples with a liquid solvent
(mobile phase) and a paper strip (stationary phase)
• Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent
(mobile phase) and a glass plate covered with a thin layer of alumina or silica
gel (stationary phase)
Principles of Paper Chromatography

• Capillary Action – the movement of liquid within the spaces of a

porous material due to the forces of adhesion, cohesion, and surface
tension. The liquid is able to move up the filter paper because its
attraction to itself is stronger than the force of gravity.

• Solubility – the degree to which a material (solute) dissolves into a

solvent. Solutes dissolve into solvents that have similar properties. (Like
dissolves like) This allows different solutes to be separated by different
combinations of solvents.

Separation of components depends on both their solubility in the mobile

phase and their differential affinity to the mobile phase and the
stationary phase.
 THIN LAYER CHROMATOGRAPHY Principle

 As the mobile phase rises up the TLC plate by capillary action, the
components dissolve in the solvent and move up the TLC plate.
 Individual components move up at different rates, depending on
intermolecular forces between the component and the silica gel stationary
phase and the component and the mobile phase.
 The stationary phase is SiO2 and is very “polar”.
 More polar analyte's interact more strongly with the stationary phase in
move very slowly up the TLC plate.
 By comparison, the mobile phase is relatively nonpolar and is capable of
interacting with analyte's by dipole-dipole and H-bonding.

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  This technique manipulates POLARITY
 More polar substances bind strongly to the adsorbent and
elute SLOWER
 Less polar substances bind weakly to the adsorbent and
elute FASTER
 The strength of interactions between the adsorbent
and eluting components vary approximately in this
order:
Salt formation > coordination > H-bonding >

More Polar dipole-dipole > van der Waals Less Polar

Polarity decreases
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 The Rf Value
A given compound will always travel a fixed distance relative to the
distance the solvent travels
This ratio is called the Rf value and is calculated in the following
manner:
. distance traveled by substance .

distance traveled by solvent front

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 THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front

Rf (B) = 3.0 cm = 0.60

5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf (C) = 0.8 cm = 0.16
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = 0.8 cm = 0.16
5.0 cm
5.0 cm
The Rf is defined as the distance the center of the spot moved divided
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by the distance the solvent front moved (both measured from the origin)
 Materials used in TLC

 Glass Plate
 Adsorbents
 Oven for activation of plate
 Developing chamber
 Mobile Phase
 A device for applying the adsorbent layer
 Storage facility for the prepared plate

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 Materials used in TLC

Hooper
A device for applying
Glass the adsorbent layer
Mobile
phase
Plate

Developing chamber

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 Stationery phase
Stationery phase Description Application

Silica gel G Silica gel with average Used in wide range

particle size 15µm pharmacopoeial test
containing ca 13%
calcium sulfate binding
agent
Silica gel G254 Silica gel G with Same application with Silica
fluorescence added gel G where visualization is to
be carried out under UV light.
Cellulose Cellulose powder of less Identification of tetracycline's
than 30µm particle size.

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 MOBILE PHASE
 TLC Solvents or Solvent Systems.
 A single solvent or mixture of two solvents can work
as mobile phase in TLC .Ex. petroleum ether, carbon
tetrachloride, chloroform, ethyl acetate, hexane can
used as mobile phase.
 The ability of mobile phase to move up is depend
on the polarity itself
 Volatile organic solvents is preferably used as
mobile phase.
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 MOBILE PHASE

SOLVENT POLARITY INDEX

Heksana 0

Butanol 3.9

Chloroform 4.1

Methanol 5.1

Ethanol 5.1

Acetonitrile 5.8

Air 9.0
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 TLC Plate Preparation


Methods used
to apply
adsorbent

Spreading Spraying Dipping

plate in
slurry

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  TLC Plate Preparation
 TLC plates are usually commercially available, with standard
particle size ranges to improve reproducibility.
 They are prepared by mixing the adsorbent, such as silica
gel , with a small amount of inert binder like calcium
sulphate (gypsum) and water. This mixture is spread as
thick slurry on an uncreative carrier sheet, usually glass,
thick aluminum foil, or plastic.
 The thickness of the adsorbent layer is typically around 0.1
– 0.25 mm for analytical purposes
 Around 0.5 – 2.0 mm for preparative TLC.
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 Experimental Procedure
 TLC Chamber Preparation
 Cut the filter paper so that it fits in the jar, touching the bottom
and reaching a height of about 1cm from the top of the jar
 To ensure that the filter paper will work, put it in the jar, and
then place an unused TLC plate in the jar. If the above criteria
are met and the plate doesn’t make any contact with the filter
paper, the setup should work
 Remove the TLC plate, and then completely saturate the filter
paper with the development solvent using a pasteur pipet.
 Fill the jar with development solvent to a depth no greater than
0.5cm
 Put the lid on the jar to preserve the saturated conditions
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 Application of sample
 Spotting the TLC Plate
 Dip the open end of a capillary tube into the solvent containing
the compound to be eluted
 Touch the end of the capillary tube lightly and very briefly to
the coated surface of the TLC plate
 Your spots should be made on the line drawn across the plate in
the correct lanes and shouldn’t have a diameter much larger
than the capillary tube
 After spotting the plate, place it in the saturated chamber and
close the lid
 Substances should be eluted until the solvent front reaches a height
of about 0.5cm from the top of the TLC plate
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 Development of Chromatogram

 Chromatoplate is kept in a tank at an angle 45°

 The bottom the tank is nearly covered up to 1 mm by
solvent.
 Three sides of tanks are lined with solvent saturate paper.
 The top of the tank is covered tightly.
 Solvent moves up and separation takes place in ascending
way with in few minutes.
 Plate is removed and dried.
 The separated components are located by either physical or
chemical method.

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 Applications of TLC
 Qualitative analysis: -
 If the separated components are colored then identification is very easy. All
the visualizing agents used in paper chromatography (Detecting agents or
indicators) can be used in TLC.
 From Rf value qualitative analysis can be performed.

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 Other Applications of TLC:-
 Analyzing the dye composition of fibers in forensic study
or Identifying compounds present in a given substance
 For Monitoring organic reactions.
 In clinical study to carry out qualitative and quantitative
analysis of biological and metabolic samples to detect
disease.
 Semi quantitative analysis can also performed by
extracting the spot in suitable solvent and it’s
determined by volumetric analysis or any other
instrumental technique.
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 Advantages of TLC over Paper Chromatography:

 Separation is sharper in TLC than paper

Chromatography.
 TLC is much more rapid
 The use of inorganic layer eliminates
background organic effects in spectroscopic
analysis.
 More reactive reagents like Sulphuric acid can
be used.
 The separated spots are more distinct hence
detection methods are more sensitive.
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High Performance Liquid
Chromatography (HPLC)
Stationary Phases
• Polar (“Normal” Phase):
• Silica, alumina

• Non-Polar (“Reversed Phase”)

• ODS Silica gel
• C18, C8
The Mobile Phase
• Normal chromatography
Hexane ; dichloromethane; isopropanol; methanol

Increasing strength

• Reverse phase chromatography

water ; methanol; acetonitrile; tetrahydrofuran (THF)

Increasing strength
Components of HPLC
1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detectors
6. Data Processing
7. Waste
Solvent Reservoir
• Mobile phase
• isocratic elution - single solvent separation teachnique
• gradient elution - 2 or more solvents, varied during separation
• To carry sample into the column
 Pumps
• To produce an appropriate pressure
to push solvent into the sample.

• A pump capable of pumping solvent

up to a pressure of 4000 psi and at
flows of up to 10 ml/min
Sample Injection System

• sample valve
• Syringe/injector

• Syringe :
• manual
• Autoinjector

• A fixed-volume loop of between 1 – 200 l

(20 l is often used as standard)
Columns
• straight, 15 to 150 cm in length; 2 to 3 mm i.d.
• packing - silica gel, alumina, Celite
HPLC Detectors

• UV/Vis
• Refractive index
• Fluorescence
• Evaporative light scattering (ELSD)
• MS
• Diode Array Detector (DAD)
Data Processing
• Using specific sowtare that is connected to HPLC machine
• Receive the information from HPLC machine and present
it as a graph
• The graph describes about qualitative data (Retention
time) and quantitative data (area under curve)
Application of HPLC
1. Pharmaceuticals industry
• To control the drug stability
• Quantity of drug determination from
pharmaceutical dosage forms, ex. Paracetamol
determination in panadol tablet
• Quantity of drug determination from biological
fluids, ex: blood glucose level

2. Analysis of natural contamination

- Phenol & Mercury from sea water

3. Forensic test
- Determination of steroid in blood, urine & sweat.
- Detection of psychotropic drug in plasma
Application of HPLC
4. Clinical test
- Monitoring of hepatic chirosis patient
through aquaporin 2 in the urine.

5. Food and essence manufacture

- sweetener analysis in the fruit juice
- preservative analysis in sausage.
The factors which influence the HPLC
performance
1. Internal diameter of column
- the smaller in diameter, the higher in
sensitivity
2. Pump pressure
- the higher in pressure, the higher in
separation
3. Sample size
4. The polarity sample, solvent and column
5. Temperature
- the higher in temperature, the higher in
separation
 advantages

1. Needs a small sample with a high accuracy and precis

2. Non-destructed sample during operation compared to
GC.
 Disadvantages
• Need a skill to run the instruments
• Solvents consuming
HPLC CHROMATOGRAM
Retention Time
• The retention time of a solute is taken as the elapsed time
between the time of injection of a solute and the time of
elution of the peak maximum of that solute.
 Affinity Chromatography
Introduction
A goal of biochemistry is to separate and identify chemical compounds.
chromatography is one of the most effective techniques for accomplishing
this.
In chromatography, substances are placed in a system consisting of two
physically distinguishable components -a mobile phase and a stationary
phase-and molecular species separate because they differ (many of them only
slightly) in their distribution between these two phase.
There are many kinds of chromatography:
Adsorption
Partition
Ion-Exchange
Molecular Sieve
Affinity
Column, paper, thin-layer and gas chromatography.
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What is Affinity Chromatography?
• A chromatographic technique for the separation of the
compound on the basis of a reversible interaction
between a compound and a specific ligand.
• Ideal for purification of compound from complex
mixtures several thousand folds in concentrated form.
• Originally developed for purification of enzymes
• Also purifies nucleotides, nucleic acids, immunoglobins,
membrane receptors, whole cells and cell fragments.
• High selectivity and high resolution
Principle
• When a complex mixture containing specific
compound is passed through a column containing
immobilized ligand, the specific compound will bind to
the ligand.
Ligand is immobilized to an insoluble solid matrix.
• A biological reversible interaction between a compound
and a specific ligand occurs as follows:
Compound (M) + Ligand (L)  ML complex
• This interaction occurs due to
• Electrostatic or hydrophobic interactions
• Van der Waals' forces or
• Hydrogen bonding.
Principle…

• Compounds not specifically bound are washed away

with the buffer.
• Compounds specifically bound are recovered from
ligand by reversing the interaction
• There are two methods of elution
Specific
Non-specific
Types of elution
Non-specific elution:
Change of pH or ionic strength or polarity.
• pH shift using dilute acetic acid or ammonium
hydroxide causes a change in the state of
ionization of groups in the ligand and the
compound.
• A change in ionic strength causes elution due to a
disruption of the ligand-compound interaction.
Specific elution:
Use of soluble competitive ligand.
Affinity Chromatography
The technique offers high selectivity, hence high
resolution, and usually high capacity for the proteins of
interest.
Purification that would otherwise be time-consuming,
difficult or even impossible using other techniques can
often be easily achieved with affinity chromatography.
The technique can be used to separate active biomolecules
from denatured or functionally different forms, to isolate
pure substances present at low concentration in large
volumes of crude sample and also to remove specific
contaminants.

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46
Affinity Chromatography
 Affinity chromatography separates
proteins on the basis of a reversible
interaction between a protein and a
specific ligand coupled to a
chromatography matrix.

 The kinds of Elution

 pH Elution
 Ionic Strength Elution
 Reduced Polarity of Eluent
 Competitive Elution
 Chemotropic Eluents

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Matrix/ Resins

• Most common matrices

Cross-linked dextrans, agarose, polymethylacrylate,
polyacrylamide, polystyrene, cellulose, porous glass and
silica.
• Matrix particles are uniform, spherical and rigid.
• An ideal matrix must have the following characteristics:
• Should possess chemical groups to which the ligand may be
covalently coupled.
• Should be stable during binding of the compound and its
subsequent elution
• Should interact only weakly with the compound to minimize
non-specific adsorption
• Should exhibit good flow properties
Ligand

• Ligand must specifically bind to one particular

compound.
• Ligand that display group selectivity selected as it will
bind to closely related groups of compounds that
posses similar chemical specificity.
• Ligand must possess a chemical group that should not
reversibly bind the ligand to the compound but should
attach the ligand covalently to the matrix.
• Most common chemical groups are
–NH2, -COOH, -SH and –OH.
Spacer arm
• Spacer arm interposed between ligand and matrix.
• It prevents ligand-matrix attachment that interfere with the
ability to bind the compound.
• Optimum length of the spacer arm is 6-10 carbon atoms.
• Their chemical nature is critical for separation.
• Some spacers hydrophobic - consists methylene (CH2) groups
• Others hydrophilic - consists carbonyl (CO) or imido (NH)
groups.
• Spacers important for small immobilized ligands and not for
macromolecular ligands.
• Matrix supports available with a variety of spacer arms and
ligands attached for immediate use.
Spacer arm
 Different group specific ligands and their affinity.
Ligand Affinity
5’AMP NAD+ dependent dehydrogenases, some kinases
2’5’-ADP NAPP+ dependent dehydrogenases
Calmodulin Calmodulin binding enzymes
Avidin Biotin containing enzymes
Fatty acids Fatty acids binding proteins
Heparin Lipoproteins, lipases, coagulation factors, DNA polymerases,
steroid receptor proteins, growth factors, serine protease
inhibitors
Proteins A and G Immunoglobulins
Concanavalin A Glycoproteins containing -D-mannopyranosyl and -D-
glucopyranosyl residues
Soybean lectin Glycoproteins containing N-acetyl -(or ) -D-
galactopyranosyl residues
Phenylboronate Glycoproteins
Poly (A) RNA containing poly (U) sequences, some RNA specific
proteins
Lysine rRNA
Cibacron Blue FF3G-A Nucleotide-requiring enzymes, coagulation factors
Purification steps
• Binding: buffer conditions are optimized to ensure that the target molecules interact
effectively with the ligand and are retained by the affinity medium as all other molecules
wash through the column.
• Elution: buffer conditions are changed to reverse (weaken) the interaction between the
target molecules and the ligand so that the target molecules can be eluted from the column.
• Wash: buffer conditions that wash unbound substances from the column without eluting
the target molecules or that re-equilibrate the column back to the starting conditions (in
most cases the binding buffer is used as a wash buffer).
• Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to
create an affinity medium.
• Pre-activated matrices: matrices which have been chemically modified to facilitate the
coupling of specific types of ligand.

54
 Some typical biological interactions
• Enzyme- substrate or inhibitor or cofactor
• Antibody- antigen or virus or cell
• Lectin – polysaccharide or glycoprotein or cell surface receptor,
cell
• Nucleic acid - complementary base sequence or histones or
nucleic acid polymerase or nucleic acid binding protein.
• Hormone/vitamin – receptor or carrier protein.
• Glutathione - glutathione-S-transferase or GST fusion proteins.
• Metal ions - Poly (His) fusion proteins or native proteins with
histidine, cysteine, tryptophan residues on their surfaces.
Applications
• Purification of enzymes, immunoglobulins and receptor
proteins
• Isolation of nucleic acids. eg. mRNA by selective
hybridization of poly (U) – Sepharose 4B by exploiting
its poly (U) tail.
• Isolation of complementary RNA and DNA using
immobilized single stranded DNA.
• Isolation of proteins involved in nucleic acid metabolism
by immobilized nucleotides