Column Chromatography PDF

COLUMN CHROMATOGRAPHY Dr. Talal Al Qaisi 1  CONTENTS  PRINCIPLE  PRACTICAL REQUIREMENTS  FACTORS AFFECTING COLUMN EFFICIENCY  APPLICATIONS  ADVANTAGES  DISADVANTAGES  PARTITION COLUMN CHROMATOGRAPHY 2  HISTORY  This chroma...

COLUMN CHROMATOGRAPHY Dr. Talal Al Qaisi 1  CONTENTS  PRINCIPLE  PRACTICAL REQUIREMENTS  FACTORS AFFECTING COLUMN EFFICIENCY  APPLICATIONS  ADVANTAGES  DISADVANTAGES  PARTITION COLUMN CHROMATOGRAPHY 2  HISTORY  This chromatography was first demonstrated by TSWETT in 1906.  In all the early applications, the procedure was limited to the separation of coloured substances, hence the name chromatography. 3  CHROMATOGRAPHY It is a technique employed for separation of components of mixture by continuous distribution of components between two phases i.e. one phase moves (mobile )over the other(stationary) in a continuous manner. when chromatography is carried out in column it is called column chromatography. It is otherwise known as gravity chromatography. 4  TYPES OF COLUMN CHROMATOGRAPHY  ADSORPTION CHROMATOGRAPHY  PARTITION CHROMATOGRAPHY  ION EXCHANGE CHROMATOGRAPHY  GEL CHROMATOGRAPHY 5  TERMINOLOGY  Stationary phase: It is also called as adsorbent. It is a solid.  Mobile phase: It is also known as solvent and eluent. It is a liquid.  Sample: It is also known as adsorbate which get adsorbed..  Elution: process of removing the components from column.  Eluate: separated component. 6  ADSORPTION CHROMATOGRAPHY It is a surface phenomenon. when the stationary phase is solid and mobile phase is liquid or gas it is known as adsorption chromatography. 7 8  PRINCIPLE when a mixture of components dissolved in the mobile phase is introduced into the column, the individual components move with different rates depending upon their relative affinities. The compound with lesser affinity towards stationary phase moves faster and it is eluted out of the column first. The one with greater affinity towards stationary phase moves slower down the column and hence it is eluted latter. Thus the compounds are separated. 9 10  STATIONARY PHASE Ideal properties:  Particles should have uniform size and spherical shape [60-200] microns.  Should have high mechanical stability, inert, insoluble in mobile phase used.  Colorless  Should allow free flow of mobile phase.  Freely available and in expensive. 11  TYPES OF ADSORBENTS WEAK MEDIUM STRONG Sucrose Calcium carbonate Activated silica gel Starch Calcium phosphate Activated alumina Inulin Magnesium carbonate Activated charcoal Talc Magnesium oxide Activated magnesia Sodium carbonate Calcium hydroxide Fuller’s earth 12  SELECTION OF ADSORBENT WEAK STRONG ADSORBENT ADSORBENT Few components More components Different affinities Similar affinities Longer column Shorter column For efficient separation – Adsorbate:Adsorbent=1:20 or 1:30 13  PREPARATION OF STATIONARY PHASE  Adsorbents require activation before use.  This can be achieved by heating, where adsorbent losses water and other adsorbed materials.  Generally there is an optimum temperature for activation.  Alumina -400o C silica gel- 100o C for 1hr.  Long time heating leads to loss of its activity. 14  MOBILE PHASE  To introduce the mixture into column as solvent.  To remove pure component out of the column as eluant. 15  Different mobile phases used: Ex; In increasing order of polarity or elution strength. cyclohexane < carbondisulphide < ether < Benzene < toluene < esters < alcohols < chloroform < acetone