COURSEPAGE-DUMP_AnalyticalInstrumentation_CHROMOTOGRAPHY.ppt

Chromatography

1. CHROMOTAGRAPHY
•History
•Separation
•Definition
•Types
•Paper
•Thin Layer
•Column
•Adsorption
•Ion-exchange
•Affinity
•Gel filtration
•Liquid-liquid
•Gas-liquid
•High Performance
Liquid Chromatography

2. History
•Chromatography (from Greek
chroma, color and grapheinto write)
•Primarily for the separation of plant
pigments such as chlorophyll and
Xanthophylls
•New forms of chromatography developed in the
1930s and 1940s
•First true chromatography
is usually attributed to
Russian botanist Michael
Tswett1906

3. Separation
•Differences Physical Characteristics
•Solvent
•Definition-Components of mixtures separated -
RATE –CARRIED THROUGH STATIONARY PHASE BY LIQUID OR
GASEOUS MOBILE PHASE.
•‘SOME’ components held firmly –stationary phase
•ADSORB –sheet, column
•AFFINITY
Sl.
No
Mol.
Property
Physical Property Separation Technique
1 Size Diffusion
Sedimentation
Gel Permeation, Dialysis
Ultracentrifugation
2 Shape Affinity Affinity Chromatography
3 Ionic Charge Ion exchange
4 Polarity Volatility
Solubility
Adsorptivity
Gas-Liquid
Liquid-liquid
Liquid-Solid

4. •Martin Consden and Gordon (AA)
•Filter Paper –Stationary Phase
•Mobile i. H
2O; solvent (n-butanol, acetic
acid, isopropanol, formamide, chloroform,
benzene, cyclohexane)
•Affinity –water –paper -solvent
•Solute –separated; Partition (i aqueous –
stationary; ii organic -mobile)
•Relative rate of Flow R
f
(Retardation Factor)
•R
f = Distance traveled by solute
Distance traveled by solvent
Paper Chromatography

5. •Sub-types
•Ascending
•Descending
•Dimensional -one/two
•Forces
•Propelling –capillary
•Solubility
•Retarding –gravitational / Partition
•Detection–color reaction
−Ninhydrin (AA)
−UV, Fluorescence, radioactivity
•Applications
•Fatty acids
•Carbohydrates
•Amino Acids

6. THIN LAYER CHROMATOGRAPHY
•Similar Paper, separation low mol. Wt.
•Adsorption (Alumina)/ Partition (Silica gel)
•Preparation
•Stationary phase –0.25/0.5mm glass (aluminium oxide,
magnesium oxide, silica gel; cellulose, polyamide,
polyethylene powders) –slurry-organic solvent 40
0
C
•Sample –micropipette, dried –ascending
Solvent system Stationary Compounds separated
Butanol: AceticAcid: Water(4:1:1)
Silica gelAmino acids
Chloroform: Methanol: Water
(65:25:4)
Silica gel Phospholipids
Petroleum ether: Propanol (99:1) Kieselghur Plant pigments

7. •Detection
•Advantages
−Short time, resolving power, wide choice of stationary
phase, selective principle, Easy isolation, low cost
•Uses
−Separation small molecules, drugs, contaminants and
adulterants, resolve plant extracts, metabolites,
biomolecules
Sl. No Compounds Detectant
1 CHO Anisaldehyde in H
2SO
4
2 AA Ninhydrin in alcohol
3 Lipids Rhodamine B
4 Steroids &
Terpenoids
Antimony trichloride
5 Hydrocarbons Potassium permanganate in
H
2SO
4
6 Nucleic acids Acridines

8. COLUMN CHROMATOGRAPHY
•CC defined as a separation process involving uniform
percolation of liquid through column packed with finely
divided material. The separation in the column is
EFFECTED BY DIRECT INTERACTION BETWEEN SOLUTE
COMPONENTS AND SURFACE OF STATIONARY PHASE OR
ADSORPTION OF SOLUTE BY STATIONARY PHASE
•CC involves ion exchange, molecular sieve, adsorption or
partition phenomenon
•Packing –column (2.5X100 cm), glass wool plug, slurry(+
solvent+heat) cool, even packing, elutent run.
•Matrix-mechanical stability good flow, chemically stable,
available different particle size, chemically inert.
•Cellulose-B1-4 D glucose –cross linked epichlorohydrin
•Dextran-A 1-6 D glucose -,,,, ,, (pH ~12)
•Agarose-3,6 anhydro galactose & D galactose, cross linked 2,3
dibromoproponal pH~3 -14
•Polyacrylamide-cross linked N-N-methylene bisacrylamide~ 2 -11
•Silica-orthosilicic acid (Si-OH)

9. •Sample application
•Remove mobile phase suction. Sample
allowed to run slowly –column-pipette slow
addition mobile 2-5 cm; connect reservoir –
constant flow.
•High density solution packing (1% sucrose,
sinks)
•Capillary/ syringe
•Factors
•Dimension
•Particle size
•Low viscosity
•Temperature
•Flow rate
•Packing

10. •Elution
•Isocratic
•Stepwise
•Gradient
•Retention Time
•Time taken each compound to emerge
•Volume mobile phase –elution volume
•Chromatogram
−Detector end ~ signals plotted –function
time or volume-peaks

11. ION-EXCHANGE CHROMATOGRAPHY
Process that allows the separation
ofionsbased on their charge. It can
be used for almost any kind of
charged molecule including
largeproteins, small nucleotides
and amino acids. The solution to be
injected is usually called asample
and the individually separated
components are calledanalytes. It
is often used in protein purification,
water analysis, and quality control
etc.

12. HISTORY
Ion methods have been in use since 1850, when H.
Thompson and J. T. Way , researchers in England, treated
various clays with ammonium sulfate or carbonate in
solution to extract the ammonia and release calcium .
In 1927, the first zeolitemineral column was used to
remove interfering calcium and magnesium ions from
solution to determine the sulfatecontent of water.
The modern version of IEC was developed during the
wartime.A technique was required to separate and
concentrate the radioactive elements needed to make the
atom bomb. Researchers chose adsorbentsthat would
attach onto chargedtransuranium elements , which could
then be differentially eluted.
In the early 1970s, ion exchange chromatography was
developed atDow Chemical Company as a novel method
of IEC usable in automated analysis .

13. PRINCIPLE
Ion exchange chromatography retains
analytemolecules on the column based on ionic
interactions. The stationary phase surface displays
ionic functional groups that interact with analyte ions
of opposite charge. This type of chromatography is
further subdivided into cation exchange
chromatography and anion exchange chromatography.
The ionic compound consisting of the cationic species
and the anionic species are retained by the stationary
phase.
Cation exchange chromatography retains positively
chargedcationbecause the stationary phase displays
a negatively charged functional group.
Anion exchange chromatography retains anions using
positively charged functional group.

14. ANION EXCHANGER
The resin in an anion exchanger is positively charged. Anion
exchangers are named so because of their affinity to anions.
Anion exchangers can be classified as: -
Weak:-Weak anion exchangers tend to lose their charge
as the pH changes (weak base).
Eg:-DEAE (diethylaminoethane).
Strong:-Strong anion exchangers are able to maintain
their positive charges across a variable pH range (strong
base). Eg:-Q (quaternary resin).
CATION EXCHANGERS
Cation exchangers are named for their ability to attract
cations or positively charged particles. In this case, the
resin of the chromatography system is negatively
charged.
Cation exchangers can be classified as :-
Weak-A weak cation exchanger is comprised of a weak
acid that gradually loses its charge as the pH decreases.
Eg.. Carboxymethal groups
Strong-A Strong cation exchanger is comprised of a
strong acid that is able to sustain its charge over a wide
pH range. Eg..Sulfopropyl groups

16. STAGES
EQUILIBRATION
In this step a buffer with the desired
conditions is applied, and all of the
charged group in the stationary phase
are associated with ions of the opposite
charge. When this process in complete,
equilibrium has been reached.
APPLICATION OF SAMPLE
In this step, the sample is applied to the
stationary phase. Only samples carrying
a charge opposite to the stationary
phase will bind to it while those with the
same charge or no charge will not bind.
These unbound particles will wash out
during this stage.

17. ELUTION
This step involves changing the buffer conditions to
particles that have bound to the stationary phase. Done
by several ways:-
A. By Changing the pH of the buffer solution. When the pI of a
protein is the same as the pH of a solution, the net charge of
the protein will be zero. Therefore, when the buffer
solution's pH reaches the pI of the protein, the protein's net
charge will be zero. The protein will no longer bind to the
stationary phase and will be released and washed out.
B. By Increasing the salt concentration. As the salt
concentration of the buffer increases, salt ions replace
the bound protein. Proteins with weaker ion interactions
will be released at lower salt concentrations. Proteins
with stronger charges will have a higher affinity for the
stationary phase and will remain bound to the column
longer.
REGENERATION
This step simply involves removing all bound protein from
the stationary phase so that it is ready for another
process

18. APPLICATIONS
The treatment of water for drinking purpose (commercial,
industrial, and residential), and wastewater treatment. Ion
exchangers can soften the water, deionize it, and even be
used in desalination.
Seperation of Protiens and Polysacharides, Nucleotides,
Amino acids, Vitamins
The recovery of valuable metals is also possible.
In food industry –wine making, sugar manufacture.
In the medical world, development and preparation of key
drugs and antibiotics, such as streptomycin and quinine.
Treatments for ulcers, kidneys etc.

20. ADSORPTION CHROMATOGRAPHY
•Principle
Certain solids -ABILITY TO HOLD MOLECULES -SURFACE –
INVOLVES WEAK ATTRACTIVE FORCES –VAN DER WAAL AND
HYDROGEN BONDING . Phenomena exploited separation.
Solute different components –interact differently –
stationary phase –some settle, others migrate –
separation achieved
WEAK INTERMEDIATE STRONG
Sucrose
Cellulose
Starch
Calcium Carbonate
Calcium sulphate
Calcium phosphate
Charcoal
Magnesium
Aluminium

21. •Preparation
•Column
•Packing
–Dry
–Wet
•Factors
–Adsorbent, solvent choice, flow rate, dimension,
temperature
•Removal separated components
–Extrusion
–Elution
•Applications
•Polycyclic aromatic compounds, phenols, amines etc
•Plasma corticol
•Aliphatic hydrocarbons from aromatic hydrocarbons
•Geometrical isomers
Adsorbent Substance separated
Activated carbon
Alumina
Calcium phosphate gel
Silica gel
Peptides, AA, CHO
Small organic molecules, proteins
Proteins, polynucleotides
Sterols, AA

23. GEL PERMEATION/ GEL FILTERATION
CHROMATOGRAPHY
•Principle
Molecular sieve/ Gel exclusion
chromatography. SEPARATION –PARTITION-
MOLECULAR SIZE . Column packed tiny inert
substance –small pores. Larger molecules
pass –eluted
•Gels –cross linked polymers, inert
•Dextran –Polysaccride, dry beads –Sephadex
•Polyacrylamide –Acrylamide with NN methylene -
bisacrylamide. Size regulated –Biogel
•Agarose –Linear polymer D-glucose and 3,6-
anhydro galactose -Sepharose

25. Name Fraction range for
protein (Daltons)
Dextran (Sephadex)
G –10
G –15
G –25
G –50
G –75
G –100
G –150
G –200
0-700
0-1500
1000-5000
1500 -30,000
3,000 -80,000
4,000-1,50,000
5,000-3,00,000
5,000-6,00,000
Poly-acrylamide (Bio-Gels)
P –2
P –4
P –6
P –100
P -300
100-1800
800-4000
1000-6000
5000-1,00,000
60,000-4,00,000
Agarose
Bio-gel A -0.5
Bio-gel A-1.5
Bio-gel A-150
10,000-5,00,000
10,000-15,00,000
1,00,000 –50,00,000

26. •Applications
•Separation low mol.wt compounds from
high mol.wt compounds
•Separtion of desired protein from mixer of
proteins
•Determination of molecular wt of protein

28. AFFINITY CHROMATOGRAPHY
•Principle
Make use –SPECIFIC AFFINITY
BETWEEN SUBSTANCE –ISOLATE
MOLECULE –SPECIFICALLY BIND –
LIGAND. Column prepared –
covalently coupling binding molecule
–matrix. Elution –changing
conditions.
•Structure –binding nature –pre-requisite
–Sephacryl S, Sepharose, Bio-gel, Bio-beads
•Spacer arm –1,6, diaminohexane, 6-aminohexanoic acid,
1,4-bis(2,3-epoxypropoxy)butane

30. Ligands used in affinity chromatography
LIGAND MACROMOLECULE
Tryptophan
Benzamidine
Heparin
Poly U
Lysine
Avidin
5’-AMP
2’,5’-ADP
Concanavalin A
Protein A & G
Soybean lectin
A-Chymotrypsin
Thrombin
Coagulation factor
Poly A messenger RNA
Ribosomal RNA
Biotin containing enzymes
NAD+ dependent dehydrogenase
NADP+ dependent dehydrogenase
Glycoprotein containing A-D-glucose & A-D-
mannose
Immunoglobins
Glycoproteins N-acetyl-galactopyranosyl
residues