Chromatography

Points Covered
• History
• Principle
• Related terms
• Performance parameters
• Classification
• Planar Chromatography
• Column Chromatography
• Gas Chromatography
• Liquid Chromatography
• Types of liquid chromatography
History
• First described by Mikhail Tswett
(1903)

• Separated plant pigments into

separate colored bands

• Coined term “chromatography”

• Derived from Greek words chroma

and graphein, which mean “color”
and “to write.”
 Principles of Chromatography

• Chromatography is a method in which the components of a mixture

are separated based on their differential interactions with two chemical
or physical phases: -
• a mobile phase
and
• a stationary phase
 Terms related to Chromatography
• Distribution / Partition coefficient (Kd ) :Pattern of distribution of a compound
(analyte) between two immiscible phases
Concentration in phase A
Kd
Concentration in phase B

• Stationary phase : Solid/gel/liquid mixture that is immobilized and held within

the system by support

• Mobile Phase : Liquid/gas which is passed over or through the stationary phase;
given after application of mixture on stationary phase
Cont…
• The mobile phase, commonly referred to as the eluent

• Elution volume : The volume of eluent required to cause elution

• Chromatogram : Pictorial record of detector response as a function

of elution volume or retention time which consists of peaks or bands
ideally symmetrical in shape , representing individual analytes
Cont…
• Chromatograph : Instrument used in chromatography

• Retention time (tr): Interval required for solute to pass from injector
through column to detector

• Dead time (tm): Time taken for a compound to pass through free
spaces between the matrix particles coated with stationary phase
Components of a chromatographic
system A Chromatogram
 Asymmetric peaks of chromatogram

FRONTING : Due to overloading of

the sample

TAILING : Retention of analytes by few

active sites on stationary phase

HOW TO SOLVE: chemically remove

the sites (capping)
 Performance parameter
• Adjusted Retention time (t'R): Difference between time spent by analyte in
stationary phase (tR) and time taken by analyte molecules to pass through the free
spaces (V0) between matrix particles coated with stationary phase (dead time / t M)
tR′ = tR − tM

• Capacity factor/ Capacity ratio/Retention factor (k'): Additional time taken by

analyte to elute out from column, relative to an unretained or excluded analyte not
inter-acting with stationary phase
Resolution (RS)

• Ratio of difference in retention

time (ΔtR) between two peaks (tRA
&tRB) to the mean (wav) of their
base widths (wbA & wbB)

• Capability of a system to resolve

one analyte peak from another

• Unresolved peaks referred as fused

peaks ( poor resolution)
Cont…

Selectivity factor or separation factor :

• Ratio of retention factor of two analytes - denoted as alpha

• Numerator is the retention factor for later eluting compound

α =KA/kB
Plate height (H)

• Length of column containing single theoretical plate

H = L/N * L= total length of column
* N= total no. of theoretical plates

• Significance : * Distribution of analyte throughout column

* Higher N and smaller H implies better efficiency
(sharper peaks)

• Affected by : * Longitudinal diffusion

* Multiple pathways
* Equilibration time between two phases
Relationship between the number of theoretical plates (N) and the shape of the
analyte peak.
 Van deemter equation

• Relationship between the factors (longitudinal diffusion, equilibration

time ,multiple pathways and flow rate of eluent) and plate height is
expressed by the Van deemter equation

• Contributes peak broadening •H - height equivalent to theoretical

plate
•μ - the flow rate of the eluent
•A, B,C - constants relating to multiple
paths, longitudinal diffusion,
equilibration time respectively
Optimum flow rate of column is the net result of the influence of flow
rate on longitudinal diffusion, equilibration time and
multiple pathways
 Chromatography

Planar Column

Paper Thin layer Gas Liquid

Adsorption Partition Ion- exchange Size -exclusion Affinity

 Sample preparation
• Solvent extraction : Most common method - to extract analyte from
aqueous mixture by organic solvent diethyl ether or dichloromethane

• Solid-phase extraction : More selective

Passage of solution through cartridge packed with appropriate stationary
phase selectively analyte of interest adsorbed and interfering substants
removed

• Sample derivatization : Facilitate better chromatographic separation and

detection by masking the functional groups of analyte , for example -OH,
-NH2, -COOH( sialyzation of OH grp, acylation of acid & alkylation)
 Planar chromatography
• Stationary phase coated or placed on a flat surface or plane on which
sample is added as a small spot or band.

• Support placed in a closed container with one edge in contact with the
mobile phase .

• Mobile phase travel across the plane by means of capillary action

• After given period of time , support removed from mobile phase and
dried prior to analysis
 Planar
Chromatography

Thin-layer
Paper
chromatography
chromatography
(TLC)
 Paper Chromatography
• Works on principle of partition chromatography based on differential distribution of
solutes between two immiscible liquids

• The analytes are separated according to their relative affinity towards the stationary
phase (water) or mobile phase (n-butanol)

• Support matrix : Paper (cellulose)

• Stationary Phase : Water molecules on the cellulose surface

• Mobile Phase : n-butanol

 Types of Paper Chromatography

• ASCENDING:
• Solvent placed at the base of the
container travel upward
direction across the paper.

• Paper base in contact with the

solvent at the base of the
container
Descending

• Solvent travel down the paper,

placed in solvent holder at the
top.

• The sample spot kept at the top

and solvent flows down the
paper from above
Radial

• Sample deposited at the center of a circular

filter paper.

• After drying the spot, the filter paper tied

horizontally on a Petri dish containing
solvent

• The wick of the paper dipped in the solvent

• The solvent rises through the wick and the

components are separated into concentric
ring
 Two dimensional
• Square or rectangular paper
used

• Sample applied to one of the

corners

• Development performed at a
right angle to the direction of
the first run

• Two different solvent used

 Thin Layer Chromatography
• Stationary phase (silica or alumina gel) attached to suitable matrix coated
thinly onto a glass, plastic or metal foil plate

• Sample applied as spot near edge (2 cm above) of coated plate

• Liquid mobile phase (ethanol/n-butanol/hexane) passed across plate held

horizontally/vertically, by capillary action

• Plate removed when mobile phase approaches 2 cm below the upper edge
of plate
Cont…
• Plate preparation –
Stationary phase applied to plate as slurry

Plate spreader used to impart uniform thickness to layer and

allowed to dry at room temperature

Required thickness : 0.25 mm (analytical)

2.0 mm (preparative)
General operation and system components of
thin-layer Chromatography
 Retardation factor Rf

Distance moved by solute front

Rf =

Distance moved by solvent front

 Ranges between 0 to 1
 Depends on retention by
stationary phase
 Analyte detection in Planar Chromatography
• Chemicals identified in planar chromatography based on the position of
their bands and by comparing their retention to reference compounds

• Separated components in planar chromatography detected by -

their natural color
their response to UV light (eg, through their fluorescence)
through visualization with chemical reagents that form colored
product
using radiolabels and autoradiography
Application of Planar Chromatography
Screening for
amino acid
metabolism
disorder

Amniotic fluid Detection of

Applications unknown organic
analysis
compounds

Drug screening
 Column Chromatography

Principle :
• Involves packing of stationary phase into glass or metal column

• Followed by application of analyte mixture on mobile phase

• It pass through column either under influence of gravity or by

application of gas pressure or by use of an efficient pumping system

• Separation of analytes occur on the basis of their distribution

coefficients and emerge individually out of the column
Column chromatographic techniques
ISOCRATIC ELUTION
If the composition of the mobile phase is
constant as in GC and some forms of
HPLC, the process is said to be isocratic
elution.

GRADIENT ELUTION
To facilitate separation when the composition of
the mobile phase may be gradually changed

Respect to pH, salt concentration or polarity this

is referred to as gradient elution. Isocratic elution (ie, constant mobile
phase composition) or gradient elution based
on solvent programming
(ie, varying mobile phase composition
 Stationary
phase

Column
Injector
Components

Fraction
collector Mobile
Detector and phase
chart
recorder
 Gas Chromatography

• Gaseous mobile phase used to pass a mixture of volatile solutes through

a column containing the stationary phase

• Mobile phase an inert gas such as nitrogen, helium, or argon or a low

mass gas such as hydrogen

• Solute separation based on differences in the vapor pressures of the

injected compounds and in the different interactions of these compounds
with the stationary phase.
• Components : * column (packed/capillary) housed in oven with
temperature regulation
* sample inlet point
* Carrier gas supply
* Detector, amplifier & data recorder system
Components of a GC system
Cont…

• Stationary phase : - polyethylene glycol (very polar)

- Methylphenyl silicone gum (medium polarity)
- Methyl vinyl silicone gum (non-polar)

• Application of sample : - low & non-polar samples applied dissolved in

organic solvent (acetone/heptane/methanol)

- Sample volume; 0.1-10 µl applied by injection;

“flash-vaporisation of samples

- Injection region preferably with temperature 20-

50oc higher than column [50-300oC ]
Cont…
• Mobile phase:
* nitrogen (packed columns)
* Helium/argon (capillary columns)
* Gas pre-purified by passing through molecular sieves to remove
oxygen , water vapor, hydrocarbons
* Constant flow rate of 40-80 ml/min (packed column) & 1-2 ml/min
(capillary column); maintained by gas flow controller

• Temperature control :
* programmed such that solutes with lower boiling point elute first
*Columns must be thermally conditioned
* Use of thermo-stable stationary phase
Cont…
• Detectors : - Flame ionization detector (FID)
- Photo ionization detector (PID)
- Electron capture detector (ECD)
- Mass spectrometer detector

• Applications : separation of volatile non-polar compounds (fatty acid

esters, fat soluble vitamins, pesticides, insecticides)
Classification of Column Chromatography
Column Chromatography

On basis of applied pressure

LPLC MPLC HPLC UPLC

(<5 bar) (5-60 bar) (>50 bar) (<1500 bar)
 High-pressure liquid chromatography

• Column:
* Conventional column :Made of stainless steel; withstand pressure
upto 50 Mpa
* Length : 3-50 cm; ID : 0.3-5 mm, flow rate: 1-3 ml/min
* Microbore columns: ID - 0.1-0.5 mm; having slower flow rates
(5-20 µl/min)
• Matrix and stationary phases:
Solid matrix preferred with spherical particles of uniform size (~5 µm); made of
silica or styrene/ divinylbenzene copolymers, following types:
- Microporous support
- Pellicular support (superficially porous)
- Bonded phases (stationary phase bonded chemically to inert support)
Cont…
• Application of sample:
*Usually by loop injector and sample flushed onto column by loop
outlet
*Guard column between injector and column to remove impurities
made of same material as column
*Retains all contaminants; replaced at regular intervals
• Mobile phase:
*Isocratic elution involves single pump to deliver single eluent
*Gradient elution uses separate pumps to deliver two eluents in
pre-determined proportions
*Purified eluent & degassed before use
*Degassing- warming, stirring vigorously with stirrer , vacuum
application
Cont…
• Pumps:

* Capable of output of at least 50 Mpa

* No pulses or cyclical variations in pressure
* Minimum flow capability : 10 ml/min
* Most commonly use-reciprocating pump ; maintain
constant flow rate through column
* Pumps inactivated automatically if pressure exceeds
safety limits
Cont…
• Detectors: high sensitivity required to detect vey low concentrations
* Variable wavelength detectors (uv-vis)
* Scanning wavelength detectors
* Fluorescence detectors
* Electrochemical detectors
* Mass spectrometer -detection of overlapping peaks
* NMR spectrometer –structural information
* Refractive index detector
* Evaporative light scattering detector (ESLD)
Components of HPLC
 Liquid
chromatography

Separation
mechanism

Molecular
Adsorption Partition Ion exchange Affinity
exclusion
 Adsorption Chromatography

• Principle : based on ability of solids (adsorbents) to hold molecules

at their surface by weak non-ionic forces

• Occur at adsorption sites

• Strength of interaction depending on functional groups present in

analyte
Cont…
• Stationary phase :
* Silica (Si-OH groups on surface); adsorb polar compounds;
slightly acidic reacting well with basic groups
* Alumina (more basic reacts well with acidic group)

• Mobile phase :
*With a polarity comparable to most polar analyte in mixture
* Alcohols (for –OH groups of analyte)
*Acetone/esters (for carbonyl groups)
* Hexane, heptane, toluene (non-polar analytes)
• Types : 1. Hydroxylapatite chromatography
2. Hydrophobic interaction chromatography (HIC)
3. Hydrophilic interaction liquid chromatography (HILIC)

• Applications : separation of non-ionic non-polar compounds

(triglycerides, vitamins, drugs)
 Hydroxylapatite Chromatography
• Principle : based on dipole-dipole & electrostatic interactions between calcium &
phosphate ions of crystalline hydroxylapatite [Ca10(PO4)6(OH)2] and functional
groups of proteins, nucleic acids

• Stationary phase : crystalline/spheroidal hydroxyl apatite (adsorbent) bonded to

agarose matrix

• Elution : with increasing strength of buffer (phosphate buffer 20mm to 500mm)

• Application : separation of ssDNA from dsDNA (dsDNA with higher affinity to

hydroxylapatite)
 Hydrophobic interaction Chromatography
(HIC)
• Principle : based on surface hydrophobicity of proteins due to presence of non-
polar amino acid residues, exposed to surface by strong salt solutions

• Stationary phase : alkyl (ethyl / octyl) or phenyl groups attached to polyamide

coated silica or agarose

• Elution : - use of eluent of decreasing ionic strength or increasing pH (increases

protein hydrophilicity)
- Non-ionic detergents e.g. Tween20, triton X-100

• Advantage : aqueous elution , minimises protein denaturation

 Hydrophilic interaction liquid
Chromatography (HILIC)
• Principle : based on separation of polar compounds forming H-bonding with
polar stationary phase
• Stationary phase :
* Silica
* Silica with bonded zwitterions
* -OH group rich compounds
• Mobile phase : relatively non-polar
• Elution : with solvents of increasing polarity
• Limitation : interference by highly polar contaminants in biological fluids
• Circumvented by : extraction of sample in organic solvent e.g. Acetonitrile
 Partition Chromatography
• Principle: based on differences in retention factor k and distribution coefficients Kd of
the analytes using liquid stationary and mobile phases.

• Stationary phase :

* liquid–liquid chromatography( liquid stationary phase attached to supporting matrix

by physical means)

* bonded-phase liquid chromatography(stationary phase covalently attached to the

matrix)

• TYPES:
* Normal phase LC
* Reverse phase LC
Cont…
• Advantage : • Disadvantage :
• Cheap • Gradual removal of stationary
phase
• High capacity
• Alteration of Chromatographic
condition
• Broad selectivity

• Can be overcome by using

bonded phase
 Normal-phase Liquid Chromatography
• Stationary phase - polar and mobile phase - relatively non-polar

• Most popular stationary phase - alkylamine bonded to silica

• Mobile phase - organic solvent e.g. hexane, heptane, dichloromethane or

ethyl acetate elutropic series

• Least polar eluted first and the most polar eluted last

• Application : To separate analytes having low water solubility

 Reversed-phase Liquid Chromatography
• Stationary phase - non-polar and mobile phase - polar, hence the name
reversed-phase.

• Most commonly used type - the bonded-phase form , alkylsilane groups are
chemically attached to silica

• Mobile phase - water or aqueous buffers, methanol, acetonitrile, tetrahydrofuran

• To analyze drugs and their metabolites, insecticide and pesticide residues, and
amino acids, peptide and proteins.
 Ion-Exchange Chromatography
• Principle : based on attraction between differently charged particles;
useful for separation of analytes having ionizable groups, carrying a
net positive/negative charge, which is in turn pH dependent
• Types:
- cation exchangers
- anion exchangers
• Application : separation & purification of :
* peptides & proteins
* Nucleic acids & polynucleotides
 Applications :
- Hemoglobinopathies
- Aminoacidopathies
- Water purification
• Materials: -
Matrices : polystyrene, cellulose, agarose, porous silica
• Functional groups :
* sulfonate & quaternary ammonium (strong)
* Carboxylate & diethyl ammonium (weak)

• Temperature: preferable at 60oC (decreased viscosity of mobile phase)

• Choice of exchanger : depends on :

* stability of analytes
* Effect of pH
• Choice of eluent :
* Chosen with pH at least one unit above or below pI of
analyte
* Cationic buffers for anion exchangers & anionic for cation
exchanger
* E.g. Tris (cationic), carbonate (anionic)
* Gradient elution preferred
 Size exclusion(gel filtration)
Chromatography
• Principle : separation based on molecular size & shape utilizing polymeric organic materials
possessing three dimensional network of pores conferring gel property to such materials

• Technique : * column of gel particles or porous glass granules in equilibrium with mobile
phase
* Large sized analytes completely excluded from pores (elute first)
* Smaller analytes distributed between mobile phase & gel particles
(elute later)
* Largest particles completely excluded from mobile phase (kd =0);
smallest gains complete distribution between two phases (kd =1)
* Remaining particles range from kd = 0 to 1
• Materials:
* Dextran, agarose, polyacrylamide, polystyrene
* Single mobile phase & isocratic elution
* UV spectrophotometric detector
* Long columns (more stationary phase; more pores)

• Applications :
* Purification of biological macromolecules e.g. Hormones,
antibodies, nucleic acid, microorganisms
* Determination of relative molecular mass (Mr) e.g.Globulins
* Desalting of substances having high Mr
Size-exclusion chromatography
 Affinity Chromatography
• Principle : utilizes unique property of extremely specific biological interactions to achieve
separation & purification.

• Material to be isolated binds reversibly to a specific ligand that is attached to an insoluble

matrix

• Materials : * Matrix – possess suitable and sufficient chemical groups allowing

stable covalent bonding with ligand
- Stable during binding & subsequent elution of
macromolecule
- Minimum non-specific interactions
- Exhibit good flow property
* Substances - uniform , rigid , spherical
- Cross-linked dextran, agarose, polyacrylamide, polystyrene,
cellulose, porous glass & silica
• Ligand - absolute specificity &
group selectivity

• Possessing group that attach ligand

to matrix

• Imparted by –NH2, -COOH, -SH & -

OH groups

• Spacer arm of 6-10 C between ligand

& matrix e.g. 1,6 diamino hexane, 6-
aminohexanoic acid
Cont…

• Procedure:-

- Ligand-treated matrix suspended in buffer, packed into column

- Sample containing desired macromolecule applied onto column
- After binding of macromolecule to ligand, column eluted with
more buffer to remove contaminants

- Purified compound recovered by non-specific or specific elution :

* Non-specific: pH alteration with dilute acetic acid or NH 4OH
* Specific: addition of high concentration of substance for which
purified compound has higher affinity than immobilized ligand
Cont…
• Application:
* Purification of enzymes, receptors, antibodies
* Separation of HBA1C from unmodified Hb by
phenylboronic acid
* Isolation of mRNA
* Isolation of proteins involved in nucleic acid
metabolism
Affinity Chromatography
 Metal
chelate

Dye
Ligand Covalent
Types of Affinity
Chromatography

Lectin
Affinity Immunoaffinity
 Lectin Affinity Metal Chelate

 Based on ability of lectin proteins (40-400

kda) to bind specific carbohydrate moieties  Binding of imidazole (histidine), thiol
(cysteine) or indole (tryptophan)
 Used in glycoprotein purification groups by metal ions like Ni2+, cu2+,
Zn2+
 Commonest source legumes
 Metal ions immobilized to agarose
 Elution carried out by :
* Affinity matrix by iminodiacetate or
tris(carboxymethyl)ethylene diamine
* pH manipulation
 Subsequently protein of interest eluted
* Addition of ethylene glycol by lowering pH or using EDTA
 Immunoaffinity Dye ligand

 Involves binding of proteins to triazine

 Monoclonal antibodies linked to agarose dyes may be by both ionic or
matrices by cyanogen bromide technique hydrophobic interaction
 Elution procedure include high salt  Most binding achieved at pH 7-8.5
concentration or urea or guanidine
hydrochloride cause protein denaturation  Advantages : 1. Cheap ,very stable
2. Readily coupled to
 Avoided by using chaotropic agents that conventional matrices
increase solubility or lowering the pH
to 3  Elution : salt gradient or affinity method
 Covalent Chromatography
Thiol (-SH) containing proteins bind to immobilized ligand containing –
S-S- group (e.g. Disulfide 2'-pyridyl group attached to agarose matrix )

Release of pyridine 2 thione; monitored spectrophotometrically at 343 nm

 Non-thiol contaminants eluted  Protein released by displacement

out using dithiothreitol, GSH, or
 Unreacted thiopyridyl groups cysteine
removed by mercaptoethanol  Matrix re-generated
Principle of purification of a protein (P-SH) by covalent chromatography
References:
• Wilson and Walker’s Principle and Techniques of Biochemistry and
Molecular Biology

• Tietz Textbook of Clinical Chemistry and Molecular Diagnostics