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Index

INTRODUCTION & PHASE SEPRATION COMPOUMD OF MIXTURE TYPES OF CROMETOGRAPHY

PROCEDURE CHROMETOGRAPHY
GENERAL FACTORS INCREASING RESOLUTION

Introduction
Chromatography, firstly introduced by the Russian botanist Micheal Iswett (1906).
Chromatography is a Technique which can be used to isolate the various components of a mixture.

Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibriumdistribution) of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase. 1. Mobile phase: phase which sample is dissolved in may be gas, liquid, or supercritical fluid. 2. Stationary phase: phase which mobile phase is forced through.

Separates components in mixture:


Based on - polarity (-),(+) - boiling point - ionic strength - size

Types of chromatography

Partition Chromatography Column Chromatography Paper chrometography Thin layer chromatography Gas liquid Chromatography Ion exchange Chromatography

Partition Chromatography

Used in GC & LC Use of silica gel And cellulose. Molecules will partition into the stationary phase based upon affinity for stationary phase & eventually partition into mobile phase again

Column Chromatography

Its also called adsorption Chromatography Very similar to partition chromatography Adsorption just on surface, partition into thin layer Use of silica And Aluminum

Paper Chromatography
The paper chromatography is a simple method for the separation of smaller molecules from one an other.

The molecules to be separated are applied to sheets of suitable. Use for separation of amino acid, Nucleotides and other lower molecular weiught solutes.

Thin layer chromatography

In the complete this technique less time to

paper chromatography.

The surface of the plate consists of a very thin layer of silica gel on a plastic or Aluminium backing.. This method are used in sepration of Non poler molecules. Use for the rapid sepration of unsaturared fatty acid, trigyycerides, phospholipids, etc.

Gas liquid Chromatography

Separation based on size. Small molecules get trapped in pores & take longer to get out. A Involatele are thin layer. For the separation of ratio of lipid and amino acids.

Ion exchange Chromatography

Separtion based on relative strength of ionic bond An ion exchange has cations on surface Used for separation of proteins, RNA & DNA

Procedure of chromatoghaphy
1. Sample collection and preparation

2. Separation of chromatography

3. Detection

GENERAL FACTORS INCREASING RESOLUTION


1. 2. 3. 4. 5. 6. Increase column length Decrease column diameter Decrease flow-rate Decrease sample size Use proper pressure Use gradient elution

Thanks..

Nitesh kumar Sinha

Rinky sinha
Vikash choube

Bimlesh pandey
Sem. 1st (M.Sc.Biotechnology.)

Surguja university Ambikapur(c.g.)