Chromatography

Dr. Smily Pruthi ,
MD- Biochemistry
See video in mobile app –
tulip academy of medical
sciences

Greek chroma "color" and graphein "to write“
Physical method of separation in which the
components to be separated are distributed
between two phases – stationary ,and mobile
Website : www.tamsmed.com

 Used and named in the first
decade of 20th century for
separation of plant pigment
chlorophyll
 Russian botanist Mikhail
Tswett used columns of
CaCo3 for separating
chlorophyll

Ion exchange chromatography - Frank
Harold Spedding
Paper chrom. - Martin and Synge
GLC - Martin

 Mobile phase carries
sample through layer/
column containing
stationary phase
 Solutes distribute btwn
2 phases
 Lower aff. for stationary
phase – mobile phase
 Greater aff – stationary
phase

1.Analytical:To examine a mixture & its contents
2.Identification:To determine the identity of
components
3.Purification: To separate & isolate the
components
4. Quantification: To determine the amount of the
components

Chromatography
Planar Column
Paper Thin Layer Gas Liquid
(TLC)

Stat. Phase coated on sheet of paper or
bound to glass plate
Paper chrom.- Stat. phase is layer of water/
polar solvent coated on paper fibres
TLC – Thin layer of particles eg silica gel
spread evenly on glass plate/ plastic sheet
HPTLC – Thin layer consists of particles with
small dia. ( 4.5 μM)

Support particles on which stat. phase coated/
chemically bonded packed in tube, or stat.
phase coated on inner side of tube
Mobile phase – liquid/ gas
Instrument- gas/ liquid chromatograph

HPLC – Stat phase small dia. Particles
GC/ MS, LC/ MS – GC/ LC combined with mass
spectrometry

Graphical presentation of detector response
In analytical GC/ LC eluent exits from column &
passes through detector & produces series of
electric signals plotted as a func of time, dist/
volume

X-axis - Retention time - Time taken for a
particular analyte to pass through the system
(from the column inlet to the detector) under
set conditions
Y-axis - signal obtained by eg
spectrophotometer
 Optimal system - signal proportional to conc.
of analyte

WITH UNRESOLVED PEAKS WITH RESOLVED PEAKS

1. Adsorption chromatography
2. Ion exchange
3. Solvent partition
4. Gel filtration
5. Affinity
6. Steric exclusion

Based on electrostatic interaction btwn
charged biomol & opp charged groups on ion
exchange resins
Solutes sep. by difference in sign &
magnitude of ionic charge
Stat. phase – surface of plastic resin/ silica,
has func. groups with fixed cation/ anionic
charge

 Resins – cation / anion
exchanger
 Cationic exchange –
sulphonic / carboxylic
groups
 Anionic ex – Quart
Nitrogen containing
compounds eg Amberlite

Sep of amino acids, proteins, peptides, nucleic
acids
Separation & removal of inorganic ions from
aqueous mixtures. Eg Water purification beds
for preparing de ionized water

 Based on diff. distribution
of solutes btwn 2
immiscible liquids
 One liquid serves as stat
phase
 Thin film of liquid
adsorbed/ chem. Bonded
on surface of support
particles

Partition Chrom.
Gas liquid (GLC) Liquid liquid(LLC)
Normal Phase Reversed Phase
Polar liquid – stat phase Non polar – stat
MC

1.Ion Suppression
Ionic character of weakly acid/ basic analyte
suppressed through modificatn of mobile
phase pH
By neutralizing the ionic group, solute
interacts better with non polar stat phase

2. Ion – pair chrom.
 Counter ion added to mobile phase
 Forms ion pairs with analytes, & neutralizes
analyte ions
 Ion pairs sep. by RPC
 Used to separate therapeutic drugs & their
metabolites

Based on diff in adsorption of solute on solid
particle
Involves electrostatic, hydrogen bonds &
dispersive interactions among the molecule
and the adsorbent bed
Low reproducibility

Based on specific biological interaction btwn
analyte & ligand
Eg enz – substrate, hormone – receptor, ag –
ab etc.
Very selective

 Eg
 Carbo . With lectin
columns
 LDL & VLDL with
heparin col.
 Glycated Hb with phenyl
boronate columns

Gel filtration / molecular sieve /size
exclusion/ molecular exclusion
chromatography
Molecules separated on basis of size
Materials used for
stat phase - cross linked dextran, poly-
acrylamide, agarose.

 Beads of these
materials are porous
with pore sizes that
allow small molecules
to be trapped
 Larger molecules
remain in the mobile
phase & get eluted from
the column

Measure of successful chromatographic separation
Rs < 0.8 ⇒ Incomplete separation
Rs > 1.25 ⇒AcceptableWebsite : www.tamsmed.com

Improved by change in
1. Column retention factor ( distribution of
solute btwn stat & mobile phase)
2. Column efficiency ( Ease of physical
interaction btwn solute & column packing
material)
3. Column selectivity ( Chem. Interaction btwn
solutes & column packing)

Thin layer of sorbent, eg silica gel spread
uniformly over glass plate/ plastic sheet
Sample added as spot at edge
Plate placed in solvent tank with lower edge in
and sample band just over mobile phase
After mobile phase travels certain dist, plate
removed & dried

 Separation maybe achieved in descending/ radial
mode
 Separated components identified by diff
procedures eg UV illumination, spraying with colour
generating reagents
 HPTLC – small dia stat phase particles used →
↑ eff & reproducibility

Describes relative
migration of a
compound
Rf =Dist. From
application point to
solute center /
Dist. from application
point to solvent front

Technically easy
Relative low cost
Capacity to analyze multiple samples in
single run

Used for separating and analyzing
compounds that can be vaporized without
decomposition
Mobile phase is a inert gas like argon,
hydrogen, helium, nitrogen, called carrier gas
 Stat phase is microscopic layer of liquid on
inert solid support inside a glass or metal
column, eg methyl silicone polymers

More volatile solute elutes earlier than less
volatile one
Effluent from column carries separated
sample constituents to detector, which
produces a signal displayed as a series of
peaks
Volume and time at which unknown substance
gets eluted used to identify the substance by
comparing with ref. material values.

PACKED CAPILLARY
Filled with support
particles
Int dia 1 – 4 mm
Length – 1 m or
more
Glass / stainless
steel
Carrier gas -
Nitrogen
Inner wall of silica
tube coated with thin
film of liquid phase
ID 0.1 – 0.5 mm
Length 10 – 150 m
Hydrogen / Helium

Packed Col
Glass microsyringe injects 1 – 10 μL sample
through septum
Septum – interface btwn injector & chrom. sys
Analyte swept into col. By carrier gas
Problems
Septum leaks
Heat → septum decomp products in
column→ghost peaks in chromatogram

Capillary Col
Low sample capacity
Split mode – small portion of sample enters
column
Used when sample contains high conc. of
solute
Splitless mode – Most of sample enters col
Used when sample contains low sol. conc.
Temp control – Col. placed in oven

Flame ionization detector ( FID )
 MC used
 Col. Effluent mixed with H2 & air, & burned in flame
 Electron released detected by electrode
 Current generated used to identify & quantify
solute
Photo ionization detector ( PID)- UV light instead
Others – Thermal conductivity detector, mass
spectrometer

Sample Extraction
Eg To extract barbiturates from serum, serum
is acidified to convert barb. Into an organic
solvent
Sample Derivatization
To convert non volatile compounds into volatile
forms via chem. Modification eg esterification,
oximation, acylation
Also used to ↑ sens/ spec of some reacns

Analyte identification
Retention time at which unknown solute
elutes compared with that of ref compound
Appearance of rep peak at same time ⇒
similar constituents
Quantification
Electric signals from detector used to produce
quan. information

Parts
1. Chrom. Column
2. Solvent Reservoir
3. Pump
4. Injector
5. Detector ( spectrophotometer / fluorometer/
electrochem. Detector)
6. Computer

Columns
Stainless steel
ID 0.3 -0.5 mm
Length 50 – 250 mm
Column Packings
1. Bonded phase –
 Stat phase bonded chemically to silica
particles by silica ester/ silicone polymeric
linkage

Mech/ chem stable
Avail for ion exchange chromatography
Eg octa decylsilane bonded to silica
2. Polymeric packing eg polystyrene divinyl
benzene
3. Chiral packings – to separate enantiomers
4. Restricted access packing – outer layer of
particles coated with hydrophilic network,
pores coated with hydrophobic network

Sample concentration / purification – solid /
liquid phase extraction
Sample derivatization – eg labelling a. acids
with flourescent tags before / after chrom.
Solvent degassing
Dissolved gas bubbles generated while solvents
pass from reservoir into column
Create unstable electric signals
Removed by vaccuum degassing / Helium
purging Website : www.tamsmed.com

Chromatography

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