COLUMN CHROMATOGRAPHY

INTRODUCTION OF CHROMATOGRAPHY

What is chromatography?

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one which is stationary phase and other is mobile phase move in a definite direction.

Background of Chromatography

The term “chromatography’’ is derived from Greek, chroma meaning “color” and graphein meaning “to write”

Chromatography is a new technique which was first invested by Mikhail Tswett, in 1906 in Warsaw.

He was successful in doing the separation Of colored substances by percolating vegetable extract through a column of ca carbonate.

The Calcium carbonate act as adsorbent and different substances got absorbed to different extent and this give rise to color bands at different position on the column. Tswett termed this system of colored band as the chromatogram and the method as chromatography.

Theory of Chromatograph

y

Plate theory Rate theory

Classification of Chromatography

Classification of Chromatography

Adsorption chromatography

Partition chromatography

Ion exchange chromatography

Size exclusion chromatography

Affinity chromatography

1- Adsorption Chromatography Gas –solid chromatography Thin layer chromatography HPLC

2-Partition Chromatography Gas liquid chromatography Liquid -liquid chromatography Paper chromatography

3-Ion exchange Chromatography Ion exchange chromatography HPLC

4 - Size Exclusion Chromatography

5- Affinity Chromatography (DNA affinity chromatography)

Chromatography types are further Subdivided into

Column Chromatography

It was developed by the American petroleum Chemist D.T Day in 1900

M.S Tswett , the polish botanist in1906 used adsorption column in his investigation .

column chromatography is also known as adsorption chromatography .In which the solid stationary phase

is packed in a tubular column and mobile phase is allowed to flow through the solid .

the column in which the stationary phase is packed consist of glass or Teflon tube, typically 10 to 50mm

in diameter.

TYPES OF COLUMN CHROMATOGRAPHY

GENERAL TYPES OF COLUMN

CHROMATOGRAPHY

Adsorption chromatography Gel filtration chromatography Ion exchange chromatography Affinity chromatography Gas chromatography High performance liquid chromatography

TYPES OF COLUMN CHROMATOGRAPHY ON THE BASIS OF FLOW OF SOLVENT

Column chromatogra

phy

Gravity column

chromatography

Flash column chromatogra

phy

Gravity Column Chromatography

INTRODUCTION This is the method employed by Mikhail

Tswett in 1906. It is still used commonly in developing countries although the advent of faster more efficient variants has led to a decline in its use in developing countries.

DEFINITION It is the type of column chromatography in

which the mobile liquid is passed by gravity through the column of stationary phase.

ADVANTAGES OF GCC The advantages of this technique is that it

requires little in a way of special equipment and gives good results with a relative low level of experimental expertise. The amount of supervision required is much lesser compare to that from other techniques.

Flash Column Chromatography

INTRODUCTION This method is developed by W. C. Stills in 1978,

which involves application of positive pressure to the mobile phase solvent from the top of the column.

DEFINITION Flash column chromatography is a specialized

chromatography technique that uses compressed gas (such as nitrogen or air) or a pump to push solvent through the column.

APPLICATION OF FCC The main application of flash chromatography are:

Purification of synthetic products, Isolation of target compounds from natural products, The simplification of mixtures prior to high resolution preparative (usually) liquid chromatography The fractionation of complex mixtures into simpler group for analysis.

DIFFERENCE BETWEEN GRAVITY

AND FLASH COLUMN

CHROMATOGRAPHY

Gravity column chromatograph

y

Flash column chromatograph

yIt is the type of column chromatography in which the mobile liquid passed by gravity through the column of stationary phase

Flash column chromatography is a specialized chromatography technique that uses compressed gas (such as nitrogen or air) or a pump to push solvent through the column

The normal particle size range of silica gel in traditional gravity column chromatography is 63 – 200 micrometer.

The normal particle size range of silica gel in flash column chromatography is 40 – 63 micrometer

Gravity column chromatography

Flash column chromatography

The mesh number of gravity column chromatography is 70 - 230

The mesh number of flash column chromatography is 250 – 400.

Large solvent and adsorbent consumption

Less solvent and adsorbent consumption

It requires long separation time period

It requires short separation time period

PRINCIPLE OF

COLUMN CHROMATOGRAPHY

The basis of all forms of chromatography is the distribution or

partition coefficients (kd), which describes the way in which a

compound (analyte) distributes between two immiscible phases. In this

a solid stationary phase and a liquid mobile phase is used and the principle

of separation is adsorption.

It is basically based on the basic operations of adsorption, partition,

ion exchange, ion pairing and moleculer exclusion.

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 the stationary phase

(adsorbent) moves faster and hence it is eluted out of the column first. The one with greater affinity towards the stationary phase (adsorbent) moves

slower down the column and hence it’s eluted later. Thus, the compounds are

separated.

The type of interaction between the stationary phase (adsorbent) and the solute is reversible in nature; the rate of movement of a component (Rf) is given as follows: Rf = Rate of movement of a component Rate of movement of a mobile phase This equation can be simplified as follows: Rf = Distance moved by the solute Distance moved by the solvent

RETENTION TIME: The time between sample injection and an analyte peak reaching a detector at the end of the column is termed as Retention time (tR). Each analyte in a sample will have a different retention time. A term called the retention factor is often used to describe the migration rate of an analyte through a column.

When the mixture of analyte is applied and the mobile phase, commonly referred to as the eluent is passed through the column. As

the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge

individually in the eluate as it leaves the column.

INSTRUMENTATION OF COLUMN CHROMATOGRAPHY:

BASIC COMPONENTS:

FIVE MAJOR COMPONENTS:

PUMP INJECTOR INJECTION VALVE

COLUMN DETECTOR

1) PUMPS:

A device designed to deliver the mobile phase at acontrolled flow-rate to the separation system. Pumps are generally used in column chromatography. MAJOR TYPES:Syringe Pumps

Reciprocating Pumps

Pneumatic Pumps

SYRINGE PUMPS:

RECIPROCATING PUMPS:

PNEUMATIC PUMPS:

2) INJECTOR:

A device by which a liquid, solid or gaseoussample is introduced into the mobile phaseor the chromatographic bed. Main types are:

Direct injector

Bypass injector

On-column injector

3) INJECTION VALVE:

The injection valve allows a defined amount of sample to bepumped onto the column. The sample and the sample'sinjection into the system are the most critical factors in anyanalytical process. Therefore, the quality, reproducibility, andflexibility of the sample injection valve are important.

FUNCTION:

Injection valves are used for injecting liquid additives directlyinto the column.

4) COLUMN:

The column is where the actual separation takes place. It is usually a glass or metal tube of sufficient strength to withstand the pressures that may be applied across it. The column contains the stationary phase. The mobile phase runs through the column and is adsorbed onto the stationary phase. The columns available are simple glass tubes, varying in length and diameter . They usually have a stopcock attached to control the solvent flow, and may have a fritted plate to support the adsorbent.

MAJOR TYPES OF COLUMN:

1- Gravity Columns:

 2- Flash Columns (Air or nitrogen pressure):

4-Vacuum Columns [Vacuum liquid chromatography (VLC)]:

5-High pressure Columns (HPLC):

3-Low and Medium Pressure Columns (pumped):

COLUMN LENGTH AND DIAMETER:

As a general practice, 15m columns are used for fast screening, simple mixtures, or very high molecular weight compounds. The 30m length has become the most popular one for most analyses. Very long columns (50, 60 and 105m) are for extremely complex samples.

Increased diameter means more stationary phase, even with the same thickness, for greater sample capacity. When sample capacity is a major consideration, as with gases, very volatile samples, and purge and trap or headspace sampling, large id or even PLOT columns may be appropriate.

5) DETECTORS:

The UV detectors

The electrical conductivity detectors

The fluorescence detectors

The refractive index detectors

WORKING OF COLUMN

CHROMATOGRAPHY

GRAVITY COLUMN CHROMATOGRAPHY

Gravity Column Chromatography

It is a type of column chromatography which work under the force of gravity.

Used for large scale separation of components.

This method is a lot slower to run. They also are more difficult to pack with

adsorbent.

PROCEDURE

1. Packing of the column2. Sample loading3. Elution 4. Detection of components5. Isolation of compound

Steps involved

Packing of the column

Sample loading

Elution

Detection of components

Isolation of compound

1. Packing Of The Column

There are two common methods of packing a gravity column:

i. The slurry methodii. The dry pack method

i. The Slurry Method

• A slurry of adsorbent and solvent is made and poured into column.

• Place a flask under the column, open the pinch clamp, and allow the liquid to drain into it.

• Transfer the slurry to the column until all the stationary phase is added.

• Drain the excess solvent.• Close the pinch clamp. • The column is now packed and ready for

use.

ii. The Dry Pack Method

• Fill the column with solvent.• Using a funnel, sprinkle dry

stationary phase into the solvent, allow solvent to drain.

• Let the stationary phase settle and gently tap the column.

• Drain the excess solvent.• The column is now packed and

ready for use.

2. Sample Loading

The analyte sample is dissolved in a very small amount of solvent (1-3ml) and added to the top of the column.

After this, a small layer of white sand is added to the top of the column.

3. Elution

i. Isocratic Elution Technique

The same solvent composition or solvent of same polarity is used throughout the process of separation.

ii. Gradient Elution Technique

Solvent of gradually increasing polarity is used during the process of separation.

4. Detection of Components

a. For colored compounds, they can be seen visually to separate.

b. For colorless compounds, either Thin Layer Chromatography (TLC) or Gas Chromatography (GC) may be used to identify the compounds present in the different fractions.

5. ISOLATION OF DESIRED COMPOUND

The solvent of compound containing fraction is evaporated through Rotary evaporator and the compound is isolated.

Recrystallization of compound may be used to further purify the product.

FLASH COLUMN CHROMATOGRAPHY

Step 1: Preparation of Column

Cotton wool at bottom and little amount of sand

OR

Addition of dry silica gel by scooping

Pouring through beaker

Step 2: Pre-Elution of Column

When properly packed, the silica gel fills the column to

just below the indent on the pipette. This leaves a space of 4-5 cm on top of

the adsorbent for the addition of solvent. Clamp the filled column securely to any stand using a clamp

Pour a good amount of your elution solvent onto the silica gel.

The solvent flows slowly down the column.

Monitor the solvent level, both as it flows through the silica gel and the

level at the top.Make sure it does not go below the

top of the silica.

OR

The process can be speed up by

Pipette bulb Automated System

This helps in the quick travel of solvent through the column and save the time (it is the main importance of flash column

chromatography)

When the bottom solvent level is at the

bottom of the column, the pre-elution process

is completed and the column is ready to load.

Step 3: Sample Loading

WET LOADING

DRY LOADING

WET LOADING

Dissolve your sample in the minimum possible volume of pre-elution solvent and

apply it evenly to the surface of the silica.

Once the sample is in the column, fresh eluting solvent is

added to the top and you are ready to begin the elution

process.

DRY LOADING

Dissolve the sample to be analyzed in the

minimum amount of solvent and little

amount of silica gel.

Swirl the mixture until the solvent evaporates and only a dry powder

remains.

Transfer this dried powder onto the

top of the prepared column

Add fresh eluting solvent to the top. Now you are

ready to begin the elution process

Step 4: Eluting the Sample

Step 5: Detection of Sample

Step 6: Isolation of Separated or Desired Compound

PHASES USED IN COLUMN CHROMATOGRAPHY

STATIONARY PHASE USED IN COLUMN CHROMATOGRAPHY Most common stationary phase used are: Alumina may be acidic, neutral or basic Silica gel is slightly acidic.

SELECTION OF STATIONARY PHASE removal of impurities No. of components to be separated affinity differenced between components length of the column used quantity of the adsorbent used

PROPERTIES OF STATIONARY PHASE (ADSORBENT)

Most stationary phases influence the flow rate and resolution characteristics

The larger the particle faster the flow rate but smaller the particle the larger the surface area and potentially greater the resolving power.

For greatest effectiveness the particles of adsorbent should be of uniform size and large surface area (for instance, 150 mesh alumina has a surface area of 155 m2/g)

concentration of the mixture

The strength of adsorption depends upon the compounds involved. Since the adsorbents are polar, the more polar compounds are adsorbed more strongly. Thus, non-polar compounds are eluted first.

MOBILE PHASE USED IN COLUMN CHROMATOGRAPHY THE FUNCTIONS OF THE MOBILE PHASE

ARE: to introduce the mixture into the column as

a solvent to develop the zones of separation as  developing

agent to remove pure component out of the column as

an eluent.

CHARACTERISTICS OF MOBILE PHASE

Selection of solvents requires a balancing act between solvent and compound polarities.

For most separations, the solvent should be less polar than the compounds.

If the solvent is much more polar than the compounds, the compounds will remain in the mobile phase, and separation will not occur.

If the compounds are much more polar than the solvent, no compounds will elute since the solvent is unable to move compounds from the adsorbent sites.

order of polarity for silica gel and alumina is as follows: hexane < petroleum <ether < carbon tetrachloride < toluene <

dichloromethane < chloroform < diethyl ether < ethyl acetate < acetone < propanol < ethanol < methanol < acetic acid < water.

In complex separations, a series of increasingly polar solvents is used.

A large increase in polarity might cause all of the components to elute at once, as well as cause other problems with the column packing. Consequently, small polarity changes are accomplished by careful use of mixed solvents. For example, pure hexane may be used as the first solvent.

ADVANTAGES AND DISADVANTAGES OF

COLUMN CHROMATOGRAPHY

Column chromatography is used to determine the number of components of a mixture and used to separate and purify substantial quantities.

Column chromatography should be used to separate a mixture of liquids or solutes into its components individually. In fact, it is the most frequently used method of purifying mixtures of products in research laboratories.

Wider choice of mobile phase.

ADVANTAGES OF COLUMN CHROMATOGRAPHY

DISADVANTAGES OF COLUMN CHROMATOGRAPHY

Requires some technical skill and take some time. Column chromatography is less full proof than paper

chromatography and requires constant attention while the experiment is being performed.

This method is time consuming and tedious especially for large samples. If it is unnecessary to separate large quantities of sample, analytical methods such as paper chromatography may be more suitable and easier to perform.

Air bubbles can entrapped between stationary and mobile phase in case of dry packing due to which cracks appear in the adsorbent layer

FACTORS AFFECTING COLUMN EFFICIENCY

Dimension of the column

Particle size of adsorbent

Nature of solvent

Temperature of the column

Pressure

APPLICATIONS OFCOLUMN CHROMATOGRAPHY

Separation of mixtures of compounds

Removal of impurities or purification process

Isolation of active constituents

Isolation of metabolites from active fluids

In establishing the identity or non-identity of two substances

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