column chromatography

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