column chromatography
DESCRIPTION
Chromatographic MethodTRANSCRIPT
COLUMN CHROMATOGRAPHY
byMr. Shaise Jacob
Faculty, Nirmala College of PharmacyMuvattupuzhaKerala, India
COLUMN CHROMATOGRAPHY
Column of stationary phase is used» Solid – S.P» Liquid – M.P
PRINCIPLE ◊ AdsorptionMixture of components dissolved in the M.P is introduced in to the column.Components moves depending upon their relative affinities.
PRACTICAL REQUIREMENTS 1.STATIONARY PHASE
Adsorbent in C.C should meet following criteria ◘ Spherical in shape ◘ Mechanical stability must be high ◘ They shouldn’t react chemically ◘ It should be useful for separating for wide variety of compounds ◘ It should be freely available & inexpensive
SELECTION OF S.P
Success of chromatography → proper selection of S.P, it depends on the following.
1. Removal of impurities
2. No. of components to be separated
3. Length of the column used
4. Affinity differences b/w components
5. Quality of adsorbent used
◊ MOBILE PHASE
They act as
▫ Solvent
▫ Developer
▫ Eluent
◊ COLUMN CHARACTERISTICSColumn - Neutral glass
Column dimensions - length & diameter ratio (10:1,30:1 or 100:1)
PREPARATION OF THE COLUMN
» Bottom portion of the column – packed with glass wool/cotton wool or may contain asbestos pad,
» Above which adsorbent is packed
» After packing a paper disc kept on the top
Two types of packing techniques are there.
1.Dry packing
2.Wet packing
DRY PACKING
Dry Packing Technique Adsorbent is packed in the column in dry form Fill the solvent, till equilibrium is reachedDEMERIT: Air bubbles are entrapped b/w M.P & S.P→
cracks appear in the adsorbent layer.
Wet Packing Technique
» ideal & common techniqueAdsorbent + M.P in a beaker & poured in to
column
◊ S.P settles uniformly & no crack in the column of adsorbent
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INTRODUCTION OF THE SAMPLE
Samples dissolved in M.P & introduced into the column at once → eluted
ELUTION PROCEDURES
Two techniques
1)Isocratic elution techniques
2)Gradient elution techniques
Isocratic elution techniques
(Iso means – same)
Same solvent composition or solvent of same polarity used throughout the process of separation
Gradient elution techniques
( gradient – gradually) Solvents of gradually ↑ polarity or ↑ elution
strength are used during the process of seperation.
E.g. initially benzene, then chloroform, then ethyl acetate then chloroform
DETECTION OF COMPONENTS
Colored components-Visually
Colorless components- Different properties which can be used are – uv / visible detector, flourescence detector, RI detector, Flame ionization detector…
RECOVERY OF COMPONENTS
◘ ELUTION
FACTORS AFFECTING COLUMN EFFICIENCY
1) Nature of solvents
2) Dimension of columns
3) Particle size of column packing
4)Temperature of the column
APPLICATIONS►Seperation of mixture of components
►Purification process
►Isolation of metabolites
►Estimation of drugs in formulations
Advantages of C.C» Any type of mix. can be separated
» Any quantity of mix. can be separated
» Wider choice of M.P
» Automation is possible
Disadvantages of C.C» Time consuming
» ↑ amounts of M.P required
» Automation makes the techniques more complicated & expensive
THANK YOU