CHROMATOGRAPHY 1. vxssdllllllllllllllllllllllllllllllllll

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1. CHROMATOGRAPHY
Dr. Sobiya Afroz 1
st
year PG
Department of Physiology

3. Learning Objectives
•Definition
•History
•Introduction
•Types
•Principles
•Development
•Procedure
•Applications
•Advantages
•Disadvantages

4. •The term is derived from
the Greek word “chroma”,
meaning color.
•The method was first
employed by Tswett, a
botanist in 1903, for the
separation of plant
pigments using a column
of alumina.
•HPLC is used nowadays to
separate almost all
biological substances,
including proteins,
carbohydrates, lipids and
nucleic acids

6. CHROMATOGRAPHY
•Chromatography means colorwriting
•Chromatography is a method of analysis and separation
technique of organic and inorganic compounds
•Used for large and small quantities
•Quantitative and qualitative analysis

7. Introduction
•Proteins differ in their molecular size and charge .
•They can be separated, purified, isolated according to the
following properties
•Molecular weight
•Solubility
•Electric charge
•Adsorption properties
•Bioaffinity

8. Types of chromatography
•Planar chromatography
•Eg.thinlayer chromatography
•Column chromatography /
flash chromatography
•Gas chromatography
•Liquid chromatography
•High performance liquid
chromatography HPLC

10. CLASSIFICATION
CHROMATOGRAPHY
PARTITION
TLC
PAPER
CHROMATOGRAPHY
ION EXCHANGE
CATIONIC
ANIONIC
GEL FILTRATION GAS LIQUID
AFFINITY

11. Principle
•Chromatography is a technique used to separate a group of
similar substances on the basis of certain physical
characteristics.
•A continuous redistribution between two phases namely
stationary and moving (mobile) is done.
•The stationary phase selectivity retards the substances relative to
mobile phase by virtue of number of attractive forces such as van
der waalsinteractions , hydrogen bonds and dipole moments.

12. •ADSORPTION
•PARTITION
•MOLECULAR SIEVING
•ION EXCHANGE
•AFFINITY

13. ADSORPTION CHROMATOGRAPHY
•In this technique the separation is based on differences in adsorption at the
surface of a solid stationary medium.
•The common adsorbing substances used are alumina, silicates or silica gel.
•These are packed into columns and the mixture of proteins to be separated is
applied in a solvent on the top of the column.
•The components get adsorbed on the column of adsorbent with different
affinity.
•The fractions slowly move down; the most weakly held fraction moves
fastest; followed by others, according to the order of tightness in adsorption.
•The eluent from the column is collected as small equal fractions and the
concentration of each is measured, in each fraction

15. PARTITION CHROMATOGRAPHY
•Separation of mixture of amino acids and peptides
•Get separated depending on distribution ratios or partition
coefficient between two immiscible liquids
•Stationary phase : Inert solid support like filter paper or silica gel
•Mobile phase : flowing through that support
•Used in paper and thin layer chromatography

16. PAPER CHROMATOGRAPHY
•Sample is applied as a spot on the filter paper
•The edge of this paper is dipped in the solvent with ascending or
descending flow
•The constituents of the mixture get separated by their different
rates of migration on the paper
•Different spots are then identified by spraying the paper with a
suitable colour reagent

17. TYPES OF DEVELOPMENT
•ASCENDING CHROMATOGRAPHY
•DESCENDING CHROMATOGRAPHY
•RADIAL CHROMATOGRAPHY
•2D CHROMATOGRAPHY

19. •The fundamental measurement in chromatography is Rf value
which is a constant for a given compound in a particular solvent
system
•Mixture of compounds separated by this method is based on the
difference in their Rf value
Rf value = distance moved by solute
distance moved by solvent
Rf is Ratio of front
Rf is a constant for a particular solvent system at a given
temperature
It is always less than 1

20. Development
•AIM : To separate the given mixture of amino acids and compare
the Rf value with the standard by developing a chromatogram
•Apparatus: Whatmannfilter paper no. 1
chromatography chamber
solvent
capillary tubes
spraying apparatus
dryer

21. Preparation of sample
•1% of solution of amino acids in distilled water
•1gm each of amino acid in 100 ml of distilled water
•1Pinch each of amino acid 3ml of distilled water
•REAGENTS:
•SOLVENT SYSTEM :
•n Butanol: Acetic acid : water = 60:15:25
•SPRAYING REAGENT:
•1 % NINHYDRIN 10 mg in 10 ml acetone
•Paper size 12*27
•Standard amino acid solution : 1ml N HCl + pinch of AA powder

22. Procedure
•Keep aqueous solvent in the chamber for saturation
•Take Whatmannno.1 filter paper and draw horizontal line 2.5 cm
above the lower end of paper using a capillary tube
•Spot the test sample , standard amino acid solution at a distance
of 2.5 cmsfrom each other , 4 in one line
•Use capillary tubes to add solution
•Immediately dry it with a dryer
•Suspend the filter paper with lower end of paper dips into solvent
but the horizontal line lies above the surface of solvent

23. •After the solvent front has reached the desired level ( 2-3 hrs )
remove the paper and mark the solvent front with pencil
•Dry the paper
•Spray the ninhydrin solution uniformly with spraying apparatus on
backside of filter paper and dry thoroughly
•Observe the purple coloured spots and yellow coloured spots
•Mark the centre of the spots with the pencil
•Measure the distance of spots moved and the distance of solvent
front
•Calculate the Rf values
•Compare the results with Rf values of the standards

24. Calculation /Observation

25. THIN LAYER CHROMATOGRAPHY
•Paper is replaced by a thin layer of inert absorbent such as silica
gel , alumina or cellulose spread over a glass plate
•Better separation can be achieved by two dimensional
chromatography
•Separation in one direction first and then in a direction
perpendicular to it

27. Ion Exchange Chromatography
•Separation is based on the overall charge on the proteins
•Ion exchange resins are cross linked polymers containing ionic
groups as part of their structure
•Can be cationic or anionic exchangers
•The ion exchange resin is packed into a column in a suitable form
•Mixture to be separated is allowed to percolate through the
column and washed down with elution buffer.

30. GEL FILTRATION CHROMATOGRAPHY
•Separation of molecule takes place on the basis of molecular size
and shape
•Stationary phase : column of gel with openings and micro-
channels
•Smaller molecules retained , move slowly
•Larger molecules move out faster
•Removal by eluting
•Used to determine the molecular size

32. AFFINITY CHROMATOGRAPHY
•High affinity of specific chemical groups called ligands
•Mixture of proteins is passed through the column
•Unbound proteins are removed by washing with buffer
•Protein bound to ligand (specific)retained in column
•Elution : concentrated soluble ligand or buffer , competes with
immobilised ligand
•Used for purification of antigens, antibodies or enzymes

34. GAS LIQUID CHROMATOGRAPHY
•Stationary phase : liquid
•Supported by a column of an inert material as silica
•Mobile phase : gas
•Mixture separated by volatile at one end of column and vapours
are swept over the column by an inert gas such as argon or
nitrogen
•Particles are detected and quantified
•Used for separation of fatty acids

36. HPLC
•Stationary phase: usually is a bed of fine solid particles with narrow
size distribution, densely packed in a metal, glass or plastic tube –a
chromatographic column.
•The stationary phase may be either a bulk column packing or only a
part of it deposited on or, more frequently, chemically bonded to a
more or less inert support material.
•The mobile phase: (eluent) is a liquid, usually a mixture of two or more
solvents, often containing suitable additives, forced through the
column by applying elevated pressure.
•These features of HPLC are especially useful in pharmaceutical,
biomedical and clinical analysis.

37. •HPLC has become one of the most powerful tools in the
contemporary organic analysis for the separation and
determination of even very complex samples containing nonpolar,
moderately or strongly polar and ionic compounds, simple
species, high-molecular synthetic polymers or biopolymers

40. Medical Applications
•Inborn Errors Of Metabolism: detection of carbohydrates in urine,
serum , faeces
•Amino Aciduria: separation of amino acids
•Vitamins: A, D , E ,K Separation of vitamins
•Checking the purity of samples

41. •Applicationsofchromatography inthepharmaceutical
analysis:
•Qualitative and quantitative analysis of molecules.
•As chromatography is easy, precise, rapid, and accurate.
•It can be adopted successfully and efficiently in bulk and pharmaceutical
dosage form for routine quality control analysis of drugs.
•Chromatography plays a significant role in the development of a new
molecule, as it determines the structure, and monitors the synthesis or
reaction process.
•It is used to find out the amount of the drug from the pharmaceutical dosage
forms.
•analyze purified compounds for trace contaminants.
•It is used to separate the chiral compounds in pharmaceutical analysis.
•Chromatography is a robust and efficient quantitative tool for the diagnosis
and evaluation of diseases in drug and biomarker research.

42. •Applications of chromatography in the food industry:
•Chromatography is used in the processing of the food industry for
quality control, by isolating proteins, preservatives, vitamins, and
amino acids.
•The presence of chemical additives, the nutritional value of
processed foods such as junk food, fast food can be determined
with the help of a variety of chromatographic techniques.
•Many beverage industries use chromatography to confirm that
every batch of their product is the same.
•The chromatography is broadly used by food manufacturers in
determining the contents of processed food and other foodstuffs.
•It is used to determine food shelf-life, it is analyzed by which food
spoils.

43. •Applicationsofchromatography inthechemical
industry:
•The major application of chromatography in the chemical industry
is that it is used to monitor the reaction and synthesis of a
chemical and its products.
•It is used in pesticide and oil industries to detect different
contaminants present in the products.
•It is used in the chemical industry for determining the pollutants in
water, air, and chemicals.
•The chemical industries are used chromatography to monitor the
quality and purity of the samples.

44. •Applicationsofchromatography inforensicscience:
•The chromatography is used in forensic science to collect the evidence
and catch criminals.
•Chromatography is used in blood tests that can determine the amount
of alcohol, drugs, or toxic substances in the body after death.
•Chromatography is also to determine if their poison in the body.
•Gas chromatography is used to test samples of blood, urine, and
clothes, to recognize criminals and to help them bring justice.
•Since chromatography can reliably determine substances in the
bloodstream, it is used for screening of athletes for doping or
performance-enhancing drugs in sports.

45. •Applicationofchromatography intheenvironmentalanalysis:
•Chromatography is a widely used separation technique to determine the organic
trace components in environmental samples.
•Chromatography used for determining and identification environmentally ubiquitous
pollutants.
•Liquid chromatography with mass spectrometry (LC-MS) can be used to examine
many different pesticide residues.
•It is also used to determine the volatile and non-polar organic compounds (odors
and non-odorous) in the air.
•It is used to identify carcinogenic contaminants found in drinking or wastewater.
•Some other applications of chromatography includes, applications in the life
sciences, molecular biology, nanotechnology, petroleum industries, and diagnosis
of disease, etc

46. Advantages
•The advantages of chromatography include,
•it provides simple and rapid analysis with high resolution,
•it requires a very small amount of sample (Gram, PPM, and ng/ml)
•it has a broad range of mobile phase and stationary phase to
separate the components.

47. Disadvantages
•Chromatography equipment can only be operated by a trained
person.
•Chromatography instruments are expensive.
•An error occurs due to the overloading of the samples.
•Chromatography equipment must be handled with care because
of these parts are expensive and sensitive.
•Some of the chromatography techniques require more solvent to
separate the analytes.

48. Learning Objectives
•Definition
•History
•Introduction
•Types
•Principles
•Development
•Procedure
•Applications
•Advantages
•Disadvantages