2. CHROMATOGRAPHY
• Chromatography is an important biophysical technique
that enables the separation, identification, and
purification of the components of a mixture for
qualitative and quantitative analysis.
• The Russian botanist Mikhail Tswett coined the term
Chromatography in 1906.
3. Term Definition
Mobile phase or carrier solvent moving through the column
Stationary phase or adsorbent substance that stays fixed inside the column
Eluent fluid entering the column
Eluate fluid exiting the column (that is collected in flasks)
Elution
the process of washing out a compound through a
column using a suitable solvent
Analyte
mixture whose individual components have to be
separated and analysed
5. Application of chromatography
• Pharmaceutical sector: based on their molecular weight and element
composition
• Food Industry : In food spoilage and additive detection
• And determined the nutritional quality of food.
• Forensic science: crime scene testing like analyzing blood and hair
sample of crime place.
7. TYPES OF CHROMATOGRAPHY
• Paper chromatography
• Thin-layer chromatography
• Column chromatography
• Ion-exchange chromatography
• Affinity chromatography
• Gas liquid chromatography
• High-pressure liquid chromatography (HPLC)
8. Paper chromatography
First introduced by German scientist Christian Friedric Schondein in
1865.
In pc consists of a layer of cellulose highly saturated with water.
Paper chromatography is an analytical chemistry technique for
separating and identifying mixtures that are or can be colored,
especially pigments.
Based on their polarity of molecules.
Mobile phase – liquid and Stationary phase- paper strip
10. Application of PC
• To study the process of fermentation and ripening.
• To check the purity of pharmaceuticals.
• To inspect cosmetics.
• To detect the adulterants.
• To detect the contaminants in drinks and foods.
• To examine the reaction mixtures in biochemical laboratories.
• To determine dopes and drugs in humans and animals.
11. THIN LAYER OF CHROMATOGRAPHY
• Thin Layer Chromatography is a technique used to isolate
non-volatile mixtures. The experiment is conducted on a
sheet of aluminium foil, plastic, or glass which is coated with
a thin layer of adsorbent material. The material usually used
is aluminium oxide, cellulose, or silica gel.
• Each spot has a retention factor (Rf) expressed as:
• Rf = dist. travelled by sample / dist. travelled by solvent
12. APPLICATION OF TLC
• TLC is extremely useful in Biochemical analysis such as separation or
isolation of biochemical metabolites from its blood plasma, urine,
body fluids, serum, etc.
• It is widely used in separating multicomponent pharmaceutical
formulations.
• It is used in the food industry, to separate and identify colours,
sweetening agent, and preservatives
• It is used in the cosmetic industry.
• It is used to study if a reaction is complete.
• Analysing ceramides and fatty acids.
13. Coloumn chormatography
• Column chromatography is the technique used to separate the single
components of a mixture using a column of suitable adsorbent
packed in a glass tube.
• The mixture is placed on the top of the column, and an appropriate
eluant is made to flow down the column slowly.
15. Application of Column Chromatography
• Column Chromatography is used to isolate active ingredients.
• It is very helpful in separating compound mixtures.
• It is used to determine drug estimation from drug formulations.
• It is used to remove impurities.
• Used to isolate metabolites from biological fluids.
16. Ion Exchange Chromatography
• Ion exchange chromatography (or ion
chromatography) is a process that allows the
separation of ions and polar molecules based
on their affinity to ion exchangers.
• Any charge molecule like protein , nucleic
molecule are purified by IAC.
17. PRINCIPLE OF IEC
• Reversible exchange of ions between ions present in
solution and ion exchange resin.
• Cation exchange chromatography:
• Positively charged proteins are reversibly adsorbed to
immobilized negatively charged beads/polymers.
• Anion exchange chromatography:
• Negatively charged proteins are reversibly adsorbed to
immobilized positively charged beads/polymers.
• Con techonger
18. APPLICATION OF IEC
• Softening of hard water
• Demineralization of water
• To analyse base composition of nucleic acid
• To concentrate the metal ions in the sample
• To measure the additives in food and drug sample
• To separate protein mixtures
• Food beverage, industry , metal finishing , chemical pharmaceutical
tec. Sugar and sweetner production.
19. AFFINITY CHROMATOGRAPHY
• Affinity chromatography is a separation method based on a specific
binding interaction between an immobilized ligand and its binding
partner. Examples include antibody/antigen, enzyme/substrate, and
enzyme/inhibitor interactions
20. PRINCIPLE OF AC
• One component (ligand) is bonded to a support (in a similar way to a
phase chemically bonded to silica gel) and the other (sample) is
adsorbed from solution, the process being reversible
• Antigen enzyme
• Hormone antibody
• inhibitor carrier
22. APPLICATION OF AC
• It is used to purify a large variety of macromolecules such as enzymes,
immunoglobins, nucleic acids etc.
• It is also used to separate cells that include fat cells, T and B
lymphocytes, spleen cells, lymph node cells, oocytes and chick
embryo neural cells. Affinity Chromatography: Applications
• Large scale purification for proteins and enzymes
• Preparation of sample by isolation of cellular proteins
• Direct analysis of complex biological systems (boronate affinity
column to measure glycated haemoglobin)
• Use with HPLC to measure hormones, proteins, drugs, herbicides
23. High Pressure Liquid Chromatography (HPLC)
• High-performance liquid chromatography or commonly known as
HPLC, is an analytical technique used to separate, identify or quantify
each component in a mixture
• HPLC is thus basically a highly improved form of column liquid
chromatography. Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high pressures of up
to 400 atmospheres.
25. APPLICATION OF HPLC
• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
• Product purity and quality control of industrial products and fine chemicals
• Separation and purification of biopolymers such as enzymes or nucleic acids
• Water purification
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
26. GASE LIQUID CHROMATOGRAPHY
• In GLC the mobile phase is an unreactive gas, such
as nitrogen (Carrier gas).
• The stationery phase is a non volatile liquid held on
a finely divided inert solid support.
27. PRINCIPLE OF GLC
• • This is fast separation of complex mixtures and allows for
quantitative determination of each compound.
• 1. Analyte (sample) brought in by syringe into heated injection
chamber.
• 2. Nitrogen gas (inert) flows through carrying sample into the column.
• 3. Column has thin film of liquid
• 4. After separation the carried gas and separate mixture flows in
detector which activates a recorder
• 5.Traces a series of peaks on chromatogram.
30. GEL FILTRAION CHROMATOGRAPHY
• Gel filtration is a technique of partition chromatography in which the partitioning
is based on the molecular size of the substances to be separated.
• It is otherwise known as Molecular Exclusion Chromatography and
• Gel Permeation Chromatography
• Mobile phase-liquid
• Stationary phase - porous beads or material with a well defined range of pore
size.
• Gel filtration is well suited for:
• biomolecules that may be sensitive to changes in pH,
• concentration of metal ions or cofactors and
• hard environmental conditions.
31. PRINCIPLE OF GEL FILTRATION
• The basic principle of gel filtration is quite straightforward. Molecules
are partitioned between a mobile phase and a stationary phase
(comprising a porous matrix of defined porosity) as a function of their
relative sizes.
• Porous matrix spherical particles chemically and physically stable and
inert (no reactivity and adsorptive properties).
• The packed bed is equilibrated with buffer which fills the pores of the
matrix and the space in between the particles.
33. APPLICATION OF GEL FILRATION
CHROMATOGRAPHY
• Gel filtration plays a key role in the purification of enzymes,
polysaccharides, nucleic acids, proteins, and other biological
macromolecules.
• Gel filtration can also be used to facilitate the refolding of denatured
proteins by careful control of changing buffer conditions.
• It is used in protein fractionation experiments.
• Gel filtration technique is also used in molecular weight determination.
• Separation of sugar, proteins, peptides, rubbers, and others on the basis of
their size.
• Can be used to determine the quaternary structure of purified proteins.
