CHROMATOGRAPHY Dr. Smily Pruthi , MD- Biochemistry See video in mobile app – tulip academy of medical sciences
Definition 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
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History ď‚ž Used
and named in the first decade of 20th century for separation of plant pigment chlorophyll
ď‚ž Russian
botanist Mikhail Tswett used columns of CaCo3 for separating chlorophyll Website : www.tamsmed.com
Contd.. Ion
exchange chromatography - Frank Harold Spedding
Paper GLC
chrom. - Martin and Synge
- Martin
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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 Website : www.tamsmed.com
Uses 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 Website : www.tamsmed.com
Types Chromatography Planar
Paper
Column
Thin Layer (TLC)
Gas
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Liquid
Planar 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. ( 4.5Website μM) : www.tamsmed.com
Column chromatography 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
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HPLC
– Stat phase small dia. Particles
GC/
MS, LC/ MS – GC/ LC combined with mass spectrometry
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Chromatogram ď‚ž Graphical
presentation of detector response
ď‚ž In
analytical GC/ LC eluent exits from column & passes through detector & produces series of electric signals plotted as a func of time, dist/ volume
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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 Website : www.tamsmed.com
Chromatogram WITH UNRESOLVED PEAKS
WITH RESOLVED PEAKS
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Separation Mechanisms 1.
Adsorption chromatography
2.
Ion exchange
3.
Solvent partition
4.
Gel filtration
5.
Affinity
6.
Website : www.tamsmed.com Steric exclusion
Ion Exchange Chrom. 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 Website : www.tamsmed.com
Resins
– cation / anion exchanger
Cationic
exchange – sulphonic / carboxylic groups
Anionic
ex – Quart Nitrogen containing compounds eg Amberlite Website : www.tamsmed.com
Clinical App. ď‚ž Sep
of amino acids, proteins, peptides, nucleic acids
ď‚ž Separation
& removal of inorganic ions from aqueous mixtures. Eg Water purification beds for preparing de ionized water
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Partition Chromatography 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 Website : www.tamsmed.com
Partition Chrom. Gas liquid (GLC)
Liquid liquid(LLC)
Normal Phase
Reversed Phase
Polar liquid – stat phase
Non polar – stat MC
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Reversed phase chrom. 1.Ion Suppression ď‚ž Ionic
character of weakly acid/ basic analyte suppressed through modificatn of mobile phase pH
ď‚ž By
neutralizing the ionic group, solute interacts better with non polar stat phase Website : www.tamsmed.com
Contd.. 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 Website : www.tamsmed.com
Adsorption Chrom. 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 Website : www.tamsmed.com
Affinity Chrom. Based
on specific biological interaction btwn analyte & ligand
Eg
enz – substrate, hormone – receptor, ag – ab etc.
Very
selective
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Contd.. Eg Carbo
. With lectin columns
LDL
& VLDL with heparin col.
Glycated
Hb with phenyl boronate columns Website : www.tamsmed.com
Steric Exclusion Chrom. Gel
filtration / molecular sieve /size exclusion/ molecular exclusion chromatography
Molecules
separated on basis of size
Materials
used for stat phase - cross linked dextran, polyacrylamide, agarose. Website : www.tamsmed.com
ď‚ž Beads
of these materials are porous with pore sizes that allow small molecules to be trapped
ď‚ž Larger
molecules remain in the mobile phase & get eluted from the column Website : www.tamsmed.com
Resolution
Measure of successful chromatographic separation Rs < 0.8 ⇒ Incomplete separation Rs Website > 1.25 ⇒Acceptable : www.tamsmed.com
ď&#x201A;&#x17E; 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) Website : www.tamsmed.com
TLC 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 Website : www.tamsmed.com 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 Website : www.tamsmed.com
Retention Factor Describes relative migration of a compound Rf =Dist. From application point to solute center / Dist. from application point to solvent front Website : www.tamsmed.com
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Advantages of TLC Technically Relative
easy
low cost
Capacity
to analyze multiple samples in single run
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Gas Chrom. 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 Website : www.tamsmed.com
ď&#x201A;&#x17E; More
volatile solute elutes earlier than less volatile one
ď&#x201A;&#x17E; Effluent
from column carries separated sample constituents to detector, which produces a signal displayed as a series of peaks
ď&#x201A;&#x17E; Volume
and time at which unknown substance gets eluted used to identify the substance by comparing with ref. material values. Website : www.tamsmed.com
Gas Chrom. Parts
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Chromatographic column PACKED Filled
with support particles Int dia 1 – 4 mm Length – 1 m or more Glass / stainless steel Carrier gas Nitrogen
CAPILLARY Inner
wall of silica tube coated with thin film of liquid phase ID 0.1 – 0.5 mm Length 10 – 150 m Hydrogen / Helium
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Injector 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 Website : www.tamsmed.com
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 Website : www.tamsmed.com
Detectors 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 Website : www.tamsmed.com
Practical Considerations in GC 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 Website : www.tamsmed.com
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
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Liquid Chromatography Parts 1. Chrom. Column 2. Solvent Reservoir 3. Pump 4. Injector 5. Detector ( spectrophotometer / fluorometer/ electrochem. Detector) 6. Computer Website : www.tamsmed.com
Diag. Representation
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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 Website : www.tamsmed.com
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 Website : www.tamsmed.com
Practical Considerations 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
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