Chromatography

Dr. Akepati S. Reddy

Associate Professor, Thapar University
Adjunct Scientist & Head, Dept. Analytical Services
TCIRD, Thapar Technology Campus
Patiala (PUNJAB) – 147 004
 Chromatography
• Science that deals with the separation of components from a mixture
based on the differences in their structure, size/shape and other properties
• Represent a broad range of physical methods of separation and
identification of components of mixtures
• The components to be separated are distributed between a stationary
phase and a mobile phase moving through the stationary phase in a
definite direction
• The separation occurs due to differences in the distribution constant of
individual components of the preparation (sample)
• Components of the preparation have different interactions with the
stationary phase and distribute themselves differently between the
stationary and mobile phases leading to the separation
• The components that display tighter interactions with the stationary phase
move more slowly, and those that spend most time in mobile phase move
along the mobile phase faster
 Applications
Can separate complex mixtures with great precision
Can purify basically any soluble or volatile substance if the right
adsorbent material, carrier fluid, and operating conditions are
employed
Can be used to separate delicate products since the conditions
under which it is performed are not typically severe
Well suited to a variety of uses in the field of biotechnology, such
as separating mixtures of proteins
 Chromatography: Classification
Based on the mobile phase
– Liquid chromatography
– Gas (liquid) chromatography
Based on the packing of the stationary phase
– Thin layer chromatography: stationary phase as a layer on glass, plastic or
aluminum plates
– Paper chromatography: thin film of liquid supported on an inert support
is stationary phase
– Column chromatography: stationary phase packed in a column
Based on the forces of separation
– Adsorption chromatography
– Partition chromatography
– Ion (exchange) chromatography
– Gel filtration chromatography
– Affinity chromatography
 Thin Layer Chromatography (TLC)
Used for identifying substances and testing purity of substances
Relatively quick method and needs small quantity of sample for
testing
Stationary phase is a thin layer of adsorbent (silica gel or alumina)
coated on a plate
Mobile phase is a shallow pool of solvent taken in a developing
chamber
Spot of sample is placed on the plate slightly above solvent level and
the plate is placed in the developing chamber
After allowing raising of solvent through the stationary, the plate is
removed from the chamber, trace of the solvent and outlines of the
spots (if seen) are outlined by pencil
 Thin Layer Chromatography (TLC)
For visualization of plots the plates can have appropriate substances
(dragendorff’s reagent for alkaloids, aniline phthalate for sugars,
ninhydrin for aminoacids, etc.) – UV lamp can be helpful
Retention factor (Rf) calculation for each of the spots and comparison
with that for known substances is used as basis for identification

Rf depends on temperature and on the solvent used

For better identification, better spot the known substance along the
unknown sample on the same plate
Impure substances develop two or more spots while pure substance
has a single spot
 Paper Chromatography
It is a method of partition chromatography: Separation is governed by the factor
of partition between two immiscible phases
Separation depends on the partition coefficient of the solute
c ( stationary )
K  C is concentration
c ( mobile )
Filter paper strips (cellulose fiber) are used as inert support for the stationary
phase (usually water) adsorption
Organic solvent immiscible with the stationary phase liquid is used as mobile
phase
Procedure includes
– Preparation of the paper with the stationary phase and assembly of the development
chamber
– Application of desalted sample on the paper for development
– Development of the chromatogram
– Detection and identification of substances
 Paper Chromatography
Development techniques can be
– Radial
– Ascending
– Descending
Multiple chromatography (repetition of the development after the first
development is completed) with the same or a different solvent
system (mobile phase) may also be practiced
• Can be one dimensional development (in the same direction)
• Can be two dimensional development (second one perpendicular to
the first one usually in a different solvent system
– Sample is applied on one corner of the filter paper, developed in the first
solvent system, filter paper is dried, rotated by 90 and developed in the
second direction
– The first solvent should be completed volatile
Radial development
 Column Chromatography
Stationary phase is packed into a column and mobile phase is a
moving liquid or gas forced either under pressure or under
gravity through the column
Definition Term
Mobile liquid phase with no affinity to the stationary phase (i.e.
Solvent
.inert towards it) & no effect on solutes
Any liquid with more affinity to the stationary phase than the
solvent but less than solutes and just capable to move the solutes Developer
.through the column
.Any liquid that passes out of the column Effluent
Any liquid that has lesser affinity to the stationary phase than
Eluent
.solutes but is capable to move them out of the column
.Fraction of eluent containing the required specific substance Eluate
Volume of mobile phase that passes out :)or retardation volume( Retention
.of the column, before elution of a specific substance volume (VR)
 Chromatography - Basic Operations
1. Feed Injection: Injected into the mobile phase flowing
through the system (with the help of a pump!)
2. Separation in the Column: As the sample flows through the
column, its different components will adsorb to the stationary
phase to varying degrees - those with strong attraction move
more slowly than those with weak attraction leading to the
separation of components
3. Elution from the Column: After the sample is flushed or
displaced from the stationary phase, the different
components will elute from the column at different times -
the components with the least affinity will elute first, while
those with the greatest affinity will elute last
4. Detection: Different components emerging out of the column
are collected, and a detector analyzes the emerging stream by
measuring a property which is related to the concentration
and characteristic of chemical composition
 Chromatography - The Chromatogram
• Chromatogram is an output from the detector
• Operational conditions such as pH and temperature have a
significant effect on the chromatogram
• Different peaks on the chromatogram correspond to different
components in the sample mixture
• Level of complexity of the sample is indicated by the number
of peaks
• Qualitative information about the sample composition is
obtained by comparing peak positions with those of standards
• Quantitative assessment of the relative concentrations of
components is obtained from peak area comparisons
• Column performance is indicated by comparison with
standards.
 Factors affecting solutes separation in CC
Effect Factor
Decreasing size improves separation (very small Particle size of solid
.particle size however needs high pressure) stationary phase
.Efficiency increases as ratio length / width increases Column dimensions
Non uniform packing results in irregular movement of
solutes through the column & less uniform zone Uniformity of packing
.formation (band broadening or tailing occurs)
Increase in column temperature results in speed of Column temperature
.elution but does not improve separation
Solvents should be of low viscosity (to give efficient
resolution) & high volatility (to get rapid recovery of Eluting solvent
.the substances)
.Uniform & low flow rate gives better resolution Solvent flow rate
Discontinuous flow disturbs resolution Continuity of flow
.Deactivation of adsorbent decreases separation Condition of adsorbent
.Substances of high concentration move slowly Concentration of solutes
 Different Types of Column Chromatography
Mechanism Mobile phase Stationary phase Mode or type
Solutes move at different rates according Liquid or gas Solid that adsorbs Adsorption
to the forces of attraction to the stationary the solutes Chromatography
.phase
Solutes equilibrate between the 2 phases Liquid or gas Thin film of liquid Partition
according to their partition coefficients formed on the Chromatography
surface of a solid
inert support
Solute ions of charge opposite to the fixed Liquid Solid resin that Ion Exchange
ions are attracted to the resin by containing carries fixed ions & Chromatography
electrostatic forces & replace the mobile electrolytes mobile counter ions
.counter ions of opposite charge
attached by covalent
bonds

Molecules separate according to their Liquid Porous gel with no Molecular Exclusion
:size attractive action on Chromatography
Smaller molecules enter the pores of the solute molecules
.gel, and need a larger volume of eluent
Larger molecules pass through the
.column at a faster rate

Special kind of solute molecules interact Liquid or gas Solid on which Affinity
with those immobilized on the stationary specific molecules Chromatography
phase are immobilized
 Traditional column chromatography
Has a stationary phase packed in a glass column and mobile phase is passed
through under atmospheric pressure
Chromatography includes
1. Packing the column: method depends on the density of the solid – wet,
dry and slurry methods can be followed – air bubble inclusion is avoided
2. Sample application: concentrate solution of the sample is evenly applied
to the top of the column without disturbing it
3. Elution: Can be isocratic elution (use of solvent of fixed composition) or
gradient elution (composition of the solvent, pH, polarity, ionic strength,
etc., change during elution)
– The change can be either continuous/linear or it can be stepwise/fractional
4. Detection: can be
– On column detection (for coloured and fluorescent substances)
– Through monitoring the eluted fraction by TLC/PC like techniques
– Through use of special detectors (refractive index, UV detector, etc.)
 Adsorption Chromatography
Oldest type (Father of Chromatography, Tswett, worked with it)
Stationary phase is an adsorbent (ideal adsorbent is insoluble in mobile
phase, inert to solutes, preferably colourless, and of suitable particle size)
• When liquid is used as mobile phase, it is called as Liquid-Solid
Chromatography (examples: TLC and HPLC)
• When gas is used as mobile phase, it is called Gas-Solid or Gas
Chromatography
Forces of attraction tending to adsorb solutes on the adsorbent are
– Dipole-dipole attraction (between adsorbent and polar solute)
– Hydrogen bonding (between hydrogen of the OH group of the adsorbent and
nitrogen/oxygen of the solute)
Forces tending to remove solutes from the adsorbent
– Elution (tendency of solutes to dissolve and move with mobile phase) - Strong
solvents like ether, hydrocarbons and carbonyl solvents can elute the solute
– Displacement – solvent molecules compete with solutes for the adsorption sites
of the stationary phase
 Commonly used adsorbents
Silica gel
• Prepared by acidification of sodium silicate with sulfuric acid, washing with water and
then drying (heated to 150-200°C its get rid of water)
• Active sites are hydroxyl groups attached to silicon atoms – these form hydrogen
bonds with solutes
• If silica gel contains water then it acts by mostly partition
Alumina
• Aluminum oxide –prepared by activation at 400°C overnight
• Strong positive fields of Al+++ and the basic sites easily polarize the compounds and
adsorb
• Separation of aromatics from olefins
• Three types of alumina (neutral alumina, 7-7.5 pH, acidic alumina, 4 pH and basic
alumina, 10 pH) – washing of aluminum oxide with HCl/NaOH.
Charcoal
• Non-polar charcoal: prepared by activation at 1000°C – acts by adsorption through
hydrogen bonds and electrostatic forces
• Polar charcoal: prepared at lower temperature and has water and so acts by partition
 Partition Chromatography
• It is liquid-liquid chromatography or gas-liquid
chromatography
• The two liquids must be immiscible
– They should have considerable difference between their solvent
strength parameters
– Pure water > Methanol > Ethanol > propanol > Acetone >
Ethylacetate > Ether > chloroform > Dichloromethane > Benzene
> Toluene > Carbon tetrachloride > Cyclohexane > Hexane >
Pentane
– The mobile phase is usually saturated with the stationary phase
(otherwise washout of stationary phase can occur)
 Partition Chromatography
• Stationary liquid is usually more polar in nature and present
as thin film on an inert solid support
– The support material should adsorb and retain the stationary
phase
– The inert support should be mechanically stable, easy to pack
and should not retard solvent flow
– Silica gel, diatomaceous earth and cellulose are example
support materials
• Separation occurs due to differences in partition coefficients
of solutes between the two liquids
• Good for the separation of more polar solutes that may not
be easily eluted from the system based on adsorption
• Paper chromatography is example
 Ion (Exchange) Chromatography
Ion chromatography, developed in 1940s during the war time Manhattan
project, is a type of liquid chromatography to separate and measure
ionic substances.
Two technologies, Ion Exchange Chromatography (IEC) and Ion
Chromatography (IC) are identified
• IEC: separates bio-molecules on the basis of their net surface
charges
• Ion exchange chromatography is commonly used in the
purification of biological materials
• Usually employed with HPLC
• IC: a general form of IEC – separates ions and polar molecules on the
basis of their charge properties
• Use weaker ionic resins for the stationary phase and a
neutralizing stripper column to remove background eluent ions
• Used primarily to investigate water quality (determining lower
concentrations of ions)
 Ion (Exchange) Chromatography
• Mobile phase is altered to suppress ionic nature of the analyte
• Stationary phase is incorporated with ions of opposite charge to
attract and retain analyte
• Glass column coated with a resin polymer (either positively
charged or negatively charged) is used
– Two types of columns: cation exchange columns (stationary phase
carries a negative charge) and anion exchange columns (stationary
phase carries a positive charge)
– Copolymerized styrenes with vinylic and aromatic functional groups
are used as the stationary phase
• Charged molecules in the liquid phase pass through the column
until a binding site in the stationary phase appears
• The molecule will not elute from the column until a solution of
varying pH or ionic strength is passed through it
 Ion (Exchange) Chromatography
Ion Chromatograph is composed of
• Pump to feed eluent
• Auto sampler
• Sample injector section
• Column related with separation of anions or cations
• Column Oven (?) to keep the column temperature constant
• Detector to registrate the separated substances
– The most common detector in Ion Chromatography is a
conductivity detector
 Stage 1. Water sample and
eluent are injected into
column -
- --
- -
-- -
-- - -
+ -+ -+ - - +
++ +-++-
- ++
Stage 2. Anions in the + -+ +
sample interact with cation
+ + ++ +- - -
exchange resin in the + --+-
+ + ++ -
+ +
column. The ‘blue’ anion +
+ -
interacts more strongly
+ + ++++ +
+ +
with the column and passes - -+
+ + + -+- + + +
through relatively slowly. + + + +
+ + ++ + + +
Ionic species Detection limits + ++ +
+ ++
F, Cl, NO2, Br, NO3 0.5 – 50.0 mg/l - +
SO4 1.0 – 100 mg/l -
-- ---
PO4 2.5 – 100 mg/l Li
0.05 – 10.0 mg/l
Na, NH4 0.25 / 0.5 – 25.0 / 50.0 mg/l * Stage 3. Anions leave the
K 0.5 / 1.0 – 50 / 100 mg/l * column. At a rate determined by
Mg 0.25 / 0.5 – 25.0 / 50.0 mg/l * the chemical properties of the
Ca 0.5 / 1.0 – 50.0 / 100 mg/l * anions, the resin and the eluent
 Molecular Exclusion Chromatography
Also known as Size Exclusion/Molecular Sieve/Gel Filtration/Gel
Chromatography.
• Stationary phase is porous beads (uncharged and inert polymeric
matrix also known as gel)
• Size of the pores in the beads is critical – larger size molecules are
excluded and eluted first and smaller ones reach interior of the
pores and elute last
Three types of gels are used
• Dextran (Sephadex - trade name): a homopolysaccharide of glucose
– dry beads are sowllen in water and used
• Polyacrylamide (bio-gel P – trade name): prepared by cross linking
of acrylamide with N,N-methylene bis acrylamide
• Agarose: a linear polymer of D-galactose and 3,6 anhydro-1-
galactose – on dissolution in hot water and cooling it forms gel,
which is held together by hydrogen bonds
 Molecular Exclusion Chromatography

• Separates molecules based on size and shape and capable of

separating larger molecules from smaller ones
• There is insignificant attractive interaction between the
stationary phase and the solute – hence no adsorption or
binding of the molecules to the support occurs
• The separation does not depend on temperature, pH, ionic
strength and buffer composition and hence can be carried out
under any conditions
• Commonly used in protein separation and purification
• The liquid or gaseous phase passes through a porous gel which
separates the molecules according to its size
• Extremely fast
Molecular Exclusion Chromatography

Layer of sample is placed on the column

Buffer is added to the column to wash the solute through the column
Material eluted is collected in fractions into separate tubes
 Affinity Chromatography
• Developed by Arne Wilhelm Tiselius in 1930s
• The most selective type of chromatography (a one step purification technique
for the target molecule)
– Can be used for the purification of any protein, provided the specific ligand is available
• The ligand (a molecule which recognizes the target molecule) is immobilized
on a support, packing material (affinity matrix), without affecting ligand’s
ability to bind with the target molecule
– Substrates for enzymes can be good ligands
• The affinity matrix (providing a structure for increasing surface area) must be
inert and should be easily modifiable
– Agarose (commonly used) and other polysaccharides
– Amino, hydroxyl, carbonyl and thio groups located on the matrix serve as ligand
binding sites
– The matrix should be able to withstand the decontamination process (may be with
caustic or urea)
• The ligands can be genetically engineered to possess a specific affinity
(antigens for antibodies!)
 Affinity Chromatography
• Sample is injected into the affinity chromatography column
– Only the target molecules will be retained in the column,
– Substances with no affinity will be eluted off
• Target molecules retained in the column are eluted off by
– Changing the pH or
– Changing the salt concentration or
– Changing organic solvent concentration, etc.
These cause desorption of the target molecules from the ligands
• Fouling of the matrix can occur when a large number of impurities
are present
Hence is usually implemented late in the processing
 Reverse phase chromatography
• A powerful analytical tool
• A hydrophobic, low polarity, stationary phase chemically
bonded to inert solid
– Silica esterified with C18 chain carboxylate as stationary phase
• Normal chromatography involves an organic mobile phase
and sample, and a polar stationary phase, while the Reverse
Phase Chromatography involves polar mobile phase and
organic stationary phase
• Separation is essentially an extraction operation
• Useful for separation of polar non-volatile organics
compounds which otherwise require derivatization in case of
normal chromatography
 Supercritical Fluid Chromatography
Relatively new analytical tool
Carrier here is a supercritical fluid - CO2, N2O,
NH3, SF6, N-propane, N-butane, Xe, CCl2F2
and CHF3 are used
Supercritical fluids are better described as a
phase equilibrium between liquid and gas
with no boundaries
The gas like and the liquid like characteristics
of supercritical fluids make them unique
for chemical separation
Works almost parallel to HPLC
Density, in addition to pressure and
temperature, can be modified for
optimized conditions of working
High Performance Liquid Chromatography (HPLC)
• A liquid chromatography under pressurized conditions is
known as High Performance Liquid Chromatography (HPLC)
• Involves use of Partition Chromatography or Adsorption
Chromatography or Ion Chromatography or Size Exclusion
Chromatography
• Mobile phase is a liquid of low viscosity flowing through the
stationary phase
• Stationary phase is
• an immiscible liquid coated onto a porous support
• a thin film of liquid phase bonded to the surface of a sorbent
• a sorbent of controlled pore size
• The column is shorter and has smaller diameter
• The process is conducted at high velocity and pressure drop
• Through controlling pressure, temperature and mobile phase
polarity, optimal results are obtained
 Gas Chromatography (GC)
Mobile phase is generally an inert gas (nitrogen, helium, argon and
carbon dioxide)
– Influenced by the type of detector used
– Carrier gas system may contain molecular sieve for the removal of water
and other impurities
Packed columns:
– Gas-solid (adsorption) chromatography: contain solid adsorbent (zeolite,
silica gel, activated alumina, etc.)
– Gas-liquid (partition) chromatography: Finely divided inert solid support
material (diatomaceous earth) is coated with liquid stationary phase
Capillary gas chromatography:
– Glass or fused silica capillary tubes coated with an absorbent or other
solvent is used as the stationary phase
– Wall coated open tubular: walls are coated with liquid stationary phase
– Support coated open tubular: walls are coated with thin layer of support
material and this adsorbs the stationary phase
Schematic of a GC
 Gas Chromatography (GC)
Sample injection
• Microsyringe is used for injecting the sample through rubber
septum into a flash vapourizer port
• Temperature of the sample port is >50°C higher than the boiling
point of the least volatile component of the sample
• Vapourization of injected sample forms mixture of carrier gas,
vapourized solvent and vapourized solute
Column temperature
• Depends on the boiling point of the sample
• Controlled within 1/10th °C by placing the column in a Column Oven
Detectors
• Non-selective detectors (independent of the compounds analyzed
but depend on the carrier gas), Selective detectors (for a group of
compounds) and Specific detectors (for a single compound)
• Concentration dependent and mass flow dependent detectors
• Detectors used are Thermal Conductivity Detectors (TCD), Flame
Ionization Detectors (FID), Electron Capture Detectors (ECD) and
Nitrogen-Phosphorus Detectors (NPD)
Thanks