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Chromatography Combination of laboratory techniques Used for separating…
Chromatography
Combination of laboratory techniques
Used for separating tye mixture compounds
General principle
Relative adsorption
Partition coefficient
Development of chromatogram
Frontal analysis
Elution analysis
Gradient analysis
Isocractic analysis
Displacement analysis
Column chromatography
Types
Adsorption column chromatography
Partition column chromatography
Ion exchange chromatography
Gel chromatography
Methodology
1.Selection of columns
1.gravity column
Mobile phase moves
through stationary phase
under influence of gravity
2.flash column
Mobile phase is pushed
By the stream of air or
Inertgas using adaptors
3.pumped column
Mobile phase moves by using
pumps generated at low
pressure or medium pressure
4.vacuum column
Mobile phase moved under
Influence of vacuum.
Hplc column
Mobile phase pushed
Through stainless steel columns
2.Selection of adsorbent and solvents
Adsorbent
Silica gek and alumina
Solvent
Petroleum egger
Cyclohexane
CCl4
Benzene
CHCl3
3.Preparation of column
Open tubular chromatography
4.Application of sample
5.Elution procedure
Isocratic elution
Gradient elution
Stepwise fractional elution
6.Detectors
Optical detector
Differential refractometers detector
Flame ionisation detector
Conductivity detector
Factors affecting
Nature of solvent
Dimension of column
Particle size of column packing
Pore diameter of column packing
Temperature of column
Advantages
1.used to separate any type of mixture
2.used to separate any quantity of mixture
3.wide range if mobile phases used
4.sample can be separated and reused
5.automation possible
Disadvantage
1.time consuming
2.expensive
3.conplicated
Application
Analytical uses
Separation of geometrical isomers
Separation of diastereomer
Separation of tautomeric mixtures
Separation on racemates
Thin layer chromatography
Principle
Normal phase tlc
Stationary phase - polar
Mobile phase - non polar
Reverse phase tlc
Stationary phase- non polar
Mobile phase - polar
Advantages
Simple equipments
Short time development
Wide choice of stationary phase
Early recovery of separated columns
Separation effects
Easy visulation of separated compounds
Sensitivity
Variable thickness of thin layers
Chemically inert stationary phase
Disadvantage
Separation length us insufficient
Difficult to reproduce
Only soluble cna be separated
Not automatic
Works in open system affected by temp and humidity
Methodology
1 Selection of coating material or absorbents
Inorganic adsorbents
Silica gel
Alumina gel
Diatomaceous earath
Magnesia
Others
Organic adsorbents
Cellulose and its acetylates
Charcoal and activated charcoal
2.Preparation of plates
Pouring
Dipping
Spraying
Spreading
Pre coated plates
Activation of adsorbent
Purification of silica gel G layers
Marking the plate and spotting of the sample
Preparation of the development tank
Selection of solvent system
Plate develooment
Detection of components Rf value
Evaluation of the chromatogram
Recovery of component
Application
Qualitative analysis
Quantitative analysis
Paper chromatography
Principle
Paper partition chromatography
Paper adsorption chromatography
Development techniques
Descending chromatography
Ascending chromatography
Ascending descending chromatography
Radial paper chromatography
Two dimensional chromatography
Methodology
Choice of proper chromatographic techniques
Choice of the filter paper
Choice of solvent
Preparation of sample
Spotting
Drying of chromatogram
Detection of spots
Calculation of Rf value
Advantages
Few quantitative material to carry a separation.
Less time
Requires small amount of sample
Economic method
Suitable for separation of both organic and inorganic compounds
Setup can be installed even in small olace
Convenient to use
Disadvantage
Cannot be used to separate volatile oil
Nit compatible with large amounts of sample
Not useful for quantitative analysis of the compounds
Cannot be used to separate complex mixture
Less accurate
Data cannot be saved for long period of time
Application
Separate plant pigments
Determine sequence in the DNA and RNA molecules.
Determine sequence of amino acid in protein
Detect forensic samples
Separate sugar moleculrs
Separate vitamins and antibiotics
Analyse drug metabolite in urine bloid
Detect unknown compounds
Determine insecticides in food component
Electrophoretic
Application
Used for DNA sequencing
Dna hybridisation
Determining biological fluid constituents
Analysis of antibiotics
Purification of vaccines
Chiiral analysis
Determining impurities
Assay of drugs
Diagnosis of genetic toxicity
Diagnosis of hereditary diaeasea
Analysis of carbohydrates
Used in field of forensic science
Molecular biology
Types
Zone electrophoresis
Moving boundary electrophoresis
Paper electrophoresis
Agarose gel electrophoresis
Capillary electrophoresis