Column chromatography

Objective:

• At the end of this lecture, student will be able to

• Explain the principle of Column chromatography

• Discuss the instrumentation of Column chromatography

• Discuss the application of Column chromatography in various fields

• Isolate and estimate phytoconstituents from natural source

Chromatography

• Laboratory technique for the Separation of mixtures

• Method of separating mixture of components in to individual components by: the mobile phase and the stationary phase

• Chroma -“color” and graphein – “to write”

• Colour bands – separation of individual compounds

• Measured or analyzed

Purpose of Chromatography

• Analytical

  Determine the purity of the substance, identify them

• Preparative

  Used to estimate the quantities of a substance

PRINCIPLES OF CHROMATOGRAPHY

Adsorption Principle

 Based on the affinity between stationary phases – components to be separated

 Mixture of components – mobile phase – moves – column of s.phase – components – more affinity towards stationary phase – moves slower – gets eluted later

• Components – less affinity towards stationary phase – moves faster – gets eluted first

• No two components have same affinity for the given set of stationary phase and mobile phase

Partition Principle

• Based on the solubility of the components – mobile phase

• Component – more soluble – mobile phase – travels faster – elutes first

• Component – more soluble – stationary phase – travels slower – elutes later

• No two components – same partition co-efficient – particular combination – stationary and mobile phase

Column chromatography

Column Chromatography – developed by American chemist D.T Day in 1900, M.S. Tswett, the Polish botanist, in 1906 used adsorption columns in his investigations of plant pigments

• Most useful methods for the separation and purification of both solids and liquids

• Solid – liquid technique – the stationary phase – solid & mobile phase – liquid

Principle – Adsorption

• Mixture of components dissolved in the M.P is introduced in to the column

• Components moves depending upon their relative affinities

• A compound attracted more strongly by the mobile phase will move rapidly through the column, and elute from, or come off, the column dissolved in the eluent

• In contrast, a compound more strongly attracted to the stationary phase will move slowly through the column

Adsorbents

• Silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc

• Alumina – less polar compounds

• Silica gel – good results with compounds containing polar functional groups

Adsorbent – criteria

• Particles should be spherical in shape & uniform in size

• Mechanical stability – high

• Shouldn’t react chemically

• It should be useful for separating for wide variety of compounds

• Freely available & inexpensive

• Particle size – 50 – 200 µm

Selection of Stationary Phase

Success of chromatography depends upon proper selection of Stationary phase

1. Removal of impurities

2. No. of components to be separated

3. Length of the column used

4. Affinity differences b/w components

5. Quantity of adsorbent used

Mobile Phase

• They act as solvent, developer & eluent

The function of a mobile phase are:

• As developing agent

• To introduce the mixture into the column – as solvent

• To developing agent

• To remove pure components out of the column – as eluent

• Choice of solvent – on the solubility characteristics – mixture

• Different mobile phases used: (in increasing order of polarity)

• Petroleum ether, carbon tetrachloride, cyclohexane, ether, acetone, benzene, toluene, esters, water, etc

• It can be used in either pure form or as mixture of solvents

Column Characters

• Support – stationary phase

• Material – column – good quality neutral glass – not be affected by solvents – ordinary burette – used as column for separation

• Column dimensions – length & diameter ratio (10:1,30:1 or 100:1)

• Various accessories are attached to the top and bottom of the column for maintenance of the elution process

Preparation of Column

• Glass tube – 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, so that the adsorbent layer is not disturbed during the introduction of sample or mobile phase

Packing Techniques

There are two types of preparing the column

• Dry packing / dry filling

• Wet packing / wet filling

Dry Packing Technique

• Adsorbent is packed in the column in dry form

• Fill the solvent, till equilibrium is reached

DEMERIT:

Air bubbles → cracks appear in the adsorbent layer.

• After filling tapping can be done to remove void spaces

Wet Packing Technique

• Ideal & common technique

• Material is slurried with solvent and generally added to the column in portions

• S.P settles uniformly & no crack in the column of adsorbent

• Solid settle down while the solvent remain upward – solvent is removed then again cotton plug is placed

Introduction of the Sample

• The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase

• The entire sample is introduced into the column at once and get adsorbed on the top portion of the column

• From this zone, individual sample can be separated by a process of elution

Elution

Development Technique (Elution)

• By elution technique – the individual components – separated out from the column

The two techniques are:

• Isocratic elution technique: Same solvent composition or solvent of same polarity – used – throughout the process of separation

• Example: chloroform only

• Gradient elution techniques: (gradient – gradually)

 Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of separation

 E.g. initially benzene, then chloroform, then ethyl acetate then chloroform

Detection of Components

• Compounds separated – column chromatography – colored, the progress of the separation can simply be monitored visually

• Compounds – isolated – column chromatography – colourless – small fractions – eluent – collected sequentially – labelled tubes and the composition – each fraction is analyzed by TLC

Analyzing the fractions:

• Analyze the fractions by thin-layer chromatography

Factors affecting Column Efficiency

1. Dimension of the column: column efficiency has been improved by increasing length/width ratio of the column

2. Particle size of column packing: separation to be improved by decreasing the particle size of the adsorbent

3. Activity of the adsorbent

4. Temperature of the column: The speed of the elution increases at higher temperatures

5. Packing of the column

6. Quality of solvents: solvents having low viscosities is giving better results

Applications

• Separation of mixture of compounds

• Purification process

• Isolation of active constituents

• Estimation of drugs in formulation

• Isolation of active constituents

• Determination of primary and secondary glycosides in digitalis leaf

• Separation of diastereomers

Advantages

• Any type of mixture can be separated

• Any quantity of mixture can be separated

• Wider choice of Mobile Phase

• Automation is possible

Disadvantages

• Time consuming

• More amount of Mobile Phase are required

• Automation makes the techniques more complicated & expensive

Summary

• Principle of column chromatography is adsorption

• Several adsorbents act as stationary phase

• Column material may be glass or stainless steel

• Column packing technique – dry and wet packing methods

• Elution technique – Gradient and isocratic elution technique

Frequently Asked Questions (FAQs)

1. What is the primary principle behind column chromatography?
   • Column chromatography relies on the differential adsorption of compounds on a solid stationary phase within a column.
2. How is the stationary phase chosen for a specific separation?
   • The choice of stationary phase depends on the chemical properties of the compounds to be separated.
3. What are the common applications of column chromatography?
   • Column chromatography is widely used in pharmaceuticals, chemical research, and other fields for compound separation and purification.
4. Can column chromatography be used on an industrial scale?
   • Yes, column chromatography can be scaled up for industrial applications.
5. Are there any limitations to column chromatography?
   • Column chromatography can be time-consuming and may not be suitable for separating very similar compounds.

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