Chromatography

CHROMATOGRAPHY

The term Chromatography is a relatively new separation technique. In Greek, the term 'Khromatos' means ‘color’ and term 'graphos' means ‘writing’.

The Chromatography-technique was first discovered by Russian biologist Dr. Micheal Tswett in 1906, for the separation of color plant pigment on a column of alumina.

Nowadays various types of Chromatography are in use to separate almost any given mixture whether color or colorless into it’s as an analytical technique employed for the inorganic substance. Separation method is used for qualitative identification and quantitative determination.

Chromatography is a name given to technique by which members of a group of similar substance are separated by continuous redistribution between two phases. The first one is stationary phase and second is mobile phase.

Introduction Principle, Types & Uses

1.      Paper Chromatography

2.      Thin Layer Chromatography

3.      Column Chromatography

4.      Lon Exchange Chromatography

5.      Affinity Chromatography

6.      Gel Chromatography

Chromatographic Method	Basis
Adsorption Column Chromatography	Distribution of solute between solid and liquid phase on a column
Partition Column Chromatography	Distribution of solute between two liquids on a column
Paper Chromatography	Partition on a sheet of paper
Thin Layer Chromatography	Adsorption of partition on thin sheets
Ion Exchange Chromatography	Exchange of ion
Gel Permeation Chromatography	Size of solute
Gas Chromatography	Distribution of gaseous solutes between a gas, liquid/solid phase.
Electrophoretic  Chromatography	Separation on a sheet in the presence of an electric field

Chromatography – 2 classes

The two classes of Chromatography are,

1.      Gas

2.      Liquid Chromatography

a.      Adsorption

b.      Partition

c.       Ion Exchange

d.      Electrophoretic

Chromatography technique separates molecule on the basis of difference of size, shape, mass, charge, solubility, and adsorption properties. There are many different types of chromatography but they all involve interaction between three components.

a.      Mixture to be separated

b.      Stationary phase (solid phase which supports)

c.       Solvent system (mobile phase which moves over the stationary phase)

Basis of separation

            In chromatography, two immiscible phases (i.e. stationary and mobile phases) are brought into contact with each other. The mobile phase undergoes a series of interaction between stationary and mobile phases as it is being carried through the system by mobile phases. The difference in interaction governs the rate of migration of individual components under the influence of mobile phase. Under suitable condition, the resulting different rates of migration can bring about complete separation of the substance.

Solute property used for Chromatography separation

Technique                                                                  Solute Property

1. Adsorption                                                       a. Adsorption of particle
2. Partition                                                          b. Solubility
3. Ion exchange                                                   c. Ionization
4. Gel                                                                   d. Size and shape

Based on nature of stationary phase and mobile phase

                                                                                          Stationary      Mobile

1. Adsorption Chromatography                                      Solid                Liquid/gas
2. Partition Chromatography                                          Liquid              Liquid/gas
3. Gas Chromatography                                                  Solid/Liquid     Mixture of gas

Adsorption Chromatography

The technique in which stationary phase is solid and mobile phase is liquid/gas is adsorption Chromatography. The solid are absorbed in different parts of adsorbent column. Then adsorbent component are eluted by passing suitable solvent through column.

Partition Chromatography

The technique in which stationary phase is liquid, frequently water and mobile phase can be either liquid/gas.

Gas Chromatography

The stationary phase is solid/liquid and mobile phase is mixture of gas, inert gas, nitrogen, helium, argon.

Two types of GC are:

1. Gas – Solid Chromatography (Stationary Phase is Solid)
2. Gas – Liquid Chromatography (Stationary Phase is liquid)

Modes of Chromatography

Chromatographic separation may be achieved by using three modes

1. Column Chromatography
2. Thin layer Chromatography
3. Paper Chromatography

Column Chromatography

Column Chromatography is which stationary phase is attached to a suitable matrix is packed into a glass or metal column and the mobile phase is passed through the column either by gravity feed or by the use of pumping system or applied gas pressure. It is the most commonly used mode of Chromatography.

Thin Layer Chromatography

Thin Layer Chromatography in which the stationary phase attached to a suitable matrix which is coated thinly on to a glass or plastic plate. The mobile liquid phase passes across thin layer either horizontally or vertically.

Paper Chromatography

In Paper Chromatography, stationary liquid phase is supported by cellulose fiber of a paper sheet and the mobile phase passes along the gravity feed or capillary action over the stationary phase incorporated into a solid supporting material, cellulose.

PAPER CHROMATOGRAPHY

Paper Chromatography is an example of partition Chromatography.

Paper Chromatography was first introduced by Schonbein in 1961, but the technique became popular in 1963 through the work of R. Consden, A.H Gordon, A.J Martin and L.M Synege

This technique is a type of partition Chromatography in which the substance are distributed in two liquid phases i.e. one is stationary liquid usually water which is held in the fibers of paper and called the stationary phase. And the other one is mobilizing liquid or developing solvent and is called mobile phase. Organic solvent are commonly used as mobile phase.

In paper Chromatography, the separation of mixture of substance is mainly done by the flow of solvent on Whatmann Chromatography filter paper. The stationary phase is present in cellulose of filter paper and mobile phase is an organic solvent. Mobile phase i.e. organic solvent rises by capillary action and by absorption on the filter paper. Separation is achieved by the differential migration of different components which occurs due to difference in partition coefficient.

Procedure

In this technique, a drop of test solution is applied on small spot on the filter paper and the spot is dried. The paper is kept in closed chamber containing developing solvent and edge of filter paper is deepening into the solvent. Then the solvent is drawn out by capillary action and carries the mixture of the surface. The component of the mixture be separated migrate at different rates and appear as spots at different points of the paper. The substances are separated according to their relative solubility in the water and in organic solvent.

            The component having highest solubility will move rapidly along the strip of filter paper than the less soluble component.

(FIGURE)

R_f (Relative fraction) value:

The movement of the substance relative to the solvent is expressed in terms of R.f value i.e. migration parameter. Relative fraction is defined as the ratio of the distance travel by the substance and the distance travel by the solvent front.

Mathematically;

The identification of given compound may be made on the basis of its distance moved during development of relative to the distance moved by the solvent. For each compound the value of R_f is specific and R_f value is always less than one. Thus, an unknown compound can be identified by comparing its R_f value with its standard R_f value.

Figure 1: Determination of R_f value

Position coefficient or distribution coefficient (K­_d):

Basis of all form of Chromatography is partition coefficient. This describes a way in which the two compounds distribute in between two immiscible phases. For two such immiscible phases A and B, the value for the coefficient is constant at the given temperature and is given by the expression.

Partition coefficient can be defined as total amount of substance present in one phase divided by total amount of substance present in another phase.

Steps involved in Paper Chromatography

Various steps involved in paper chromatography are:

1. Choice of filter paper

The filter paper plays an important role in the success of paper Chromatography and various types of filter paper are available. Whatmann filter paper has a composition as a content 98.99 % of α-cellulose and rest is the mineral content.

            Components                          Percentage

·                     α-cellulose                  98.99%)

·                     β-cellulose                  0.3 - 1.0%)

·                     Pentosans                    0.4 - 0.8%)

·                     Ether soluble matter   0.015 – 0.02%

·                     Ash                              0.01 - 0.07%

Figure: Structure of cellulose with cross linking of glucose

In general paper is composed of randomly directed cellulose fiber. The cellulose itself is network of long chain of carbohydrate having mol. wt. > 10,000 and possessing hydrophilic character and cross linked by a stable hydrogen bond system. Water or other polar solvents is slightly held within the hydrophilic cellulose system.

Whatmann No. 1 filter paper is of great importance and is mostly used for the medium flow rate.

Table 1: Characteristics of Whatmann chromatographic papers

	Rate of Flow
	Fast	Medium	Slow
Thin papers	No. 4	No. 1	No. 2
	No. 5	No. 7	No. 20
	No. 54
	No. 540
Thick papers	No. 31	No. 3
	No. 17

Note: Generally used fast paper is No.4 and No.5

2. Choice of solvent

Paper chromatography is essential a partition chromatography and there are a wide variety of useful combination of stationary and mobile phase.

Stationary Phase: Stationary phase that can be used are classified as,

• Aqueous stationary phase: Water is rapidly held by paper. Aqueous stationary phase is stained by suspending a paper in a closed chamber whose atmosphere is saturated with water.
• Hydrophilic stationary phase: Ammonia, paraffin, phenol, methanol, glycerol are common hydrophilic solvent which are used as stationary phase. The organic solvent is dissolved in a very volatile diluents and paper is dipped into the solution. Hence, while drying in air, the volatile diluent evaporates, leaving the stationary phase i.e. liquid, uniformly distributed throughout the paper.
• Hydrophobic stationary phase: Solvent such as kerosene, aromatics and aliphatic hydrocarbon are used as hydrophobic stationary phase.

Mobile phase: Mixture of two or more solvent is used. Generally, a solvent or solvent mixture which gives an Rf value of 0.2 to 0.8 as the sample should be selected.

Some typical set of mobile phase

• Isopropanol – ammonia, water
• N butanol – acetic acid - water
• Water - phenol
• Formamide - chloroform
• Formamide – chloroform - benzene
• Formamide - benzene
• Formamide – benzene - cyclohexane
• Kerosene – 70 % isopropanol
• Dimethyl formamide – cyclohexane

3. Saturation of tank

The inner wall of tank is wrapped by filter paper before running the chromatography.

4. Preparation of sample

Sample that can be used in paper chromatography are of many types.

a)      Biological materials:

·         Serum

·         Extracts of plants and animal tissue.

·         Fermented broth

·         Culture

·         Food and food products

·         Vitamins

·         Antibiotics

If the sample is in solid form it may be dissolved in volatile solvent before loading the sample.

Sample should be in concentrated form. Some of the solid samples are dissolved in their specific solvents. E.g. Amino acids and sugars are prepared in 10 % isopropanol, lipids are prepared in chloroform.

5. Sample application / Sample loading

A horizontal line is drawn on the filter paper by pencil. This line is called origin line. On the origin line, cross marks are made with pencil in such a way that each cross is at least 2 cm away from each other.

Sample can be loaded by micropipette, Pasteur pipettes, microcap pipettes or by microsyringe. Platinum Pipette is generally used for qualitative purpose and it is also preferred for practical use because it can be used again and again by carefully washing and heating strongly after each application. If a loop is 0.4 mm diameter platinum wire and a cross section diameter is 1.5 mm a spot of about 10 ml is obtained.

Microsyringe: It can be used for both qualitative and quantitative purpose. Their capacity is 1-100 ml.

Microcap pipette: It is used for single use. They are filled by capillary action and bulb is used to expel the sample.

The sample volume of 10-20 ml is the ideal quantity to be spotted. With the help of micropipette, the sample is applied on X-marks. The spot are dried continuously by a stream of hot air. The spots are 0.5 cm to 1.5 cm in diameter.

6. Development of chromatogram

The loaded filter paper is dipped carefully into the solvent in such a way that it shouldn’t leave more than 1 cm and left undisturbed for at least 2 hrs until solvent front reaches upto 15 to 18 cm height from the original line.

The chromatogram can be developed by different methods:

1. Ascending technique: Movement of mobile phase is in upward direction and the technique is known as ascending technique. In this technique, mobile phase is placed in a suitable container at the bottom of chamber. Then, the lower end of paper containing spots is dipped into the solvent system without dipping the spots and then allowing it to rise up the paper by capillary action. The mobile phase i.e. solvent move in the upper direction against gravitational force.
2. Descending technique: In this technique, a flow of mobile phase is in downward direction. The solvent is kept in a trough at the top of the chamber and then allow floating down the paper. The movement of solvent is caused by capillary action or by the pull of gravitation force. The rate of flow of mobile phase is rapid as compared to ascending development so that chromatogram as developed in short time.
3. Radial paper chromatography: The technique is also known as solvent paper chromatography and this type of development is used in rare condition. A circular piece of paper is taken and a wick of about 2 cm is cut parallel to the radius from the edge of a center. The sample is deposited at the center of the paper at the upper end of the wick. The paper is then placed on the edge of a circular disc with the wick dipping into the solvent at the bottom of circular disc. As a result, the liquid ascends the wick and float radial through paper.

7. Drying the chromatogram

The wet chromatogram after development is dried in special drying cabinet which has been heated electrically with temperature control. The chromatogram can be died at 110°C for 5-10 minutes in hot air oven.

8. Visualization of the spot

Colored compounds are easily located on the paper but the compounds of biological origin are usually colorless and hence can’t be located on the chromatogram imply by visible inspection. The compounds on chromatogram can be located either by physical or biochemical methods.

a) Physical methods:

1. Fluorescence: Some colorless spots when held under a UV lamp fluorescence and reveal their existence i.e. if the compound are invisible under ordinary light, UV lamp can be used to locate the position of the spot. Although several compounds are relatively stable to UV light yet some compounds like steroids and vitamins they are destroyed when UV visualization technique is employed for identification.
2. Radioactive method: The location of substance may also be carried out by making use of radioactive substance where radioactivity can be detected by various counter devices.

b) Chemical Methods:

Chemical reagents are used to visualize spots. Such type of chemical are called chromogenic reagent or visualizing agent. The chemical reagent for locating spots can be gas, liquid or solid.

Gas:                 H₂S

Liquid:             Methyl, ethyl or butyl alcohols

Solid:               Potassium dichromate, aniline dyes, ninhydrin

Visualizing reagents are applied either by spraying or by dipping the chromatogram.

Spraying method: Chemical reagent is sprayed on the chromatogram uniformly by using a glass atomizer which is held in the position normally at the distance of 1 foot from the paper, and is moved slowly from top to bottom in a left to right direction.

Dipping method: In this method, a solvent is first taken in which substances are insoluble and then dipping is carried out in trough. Volatile solvent e.g. ethyl alcohol and methyl alcohol can be used because they can be easily evaporate from chromatogram.

9. Quantitative Estimation

The amount of material in each spot can be measured either by direct method or elution method.

Direct method:

i)        Comparison of visible spot: A rough quantitative measurement of a compound in a mixture can be carried out by comparing the intensity and size of spot with standard substance. For this purpose, accurately measured volumes of the mixture and known amount of component being investigated are applied separately on the origin line. Spots are dried, developed treated with a reagent which will react with the component to produce color. The size and intensity of colored spot produced by unknown component is the compared with that of known standard substance by visual inspection or under UV lamp.

ii)  Photo densitometry: A strip of paper containing the spot is cut and placed between two glass slides. Then the intensity of each spot is measured with a photoelectric device called photo densitometer.

Elution Method:

The spots from the developed chromatogram are eluted. The spots are cut off from the paper and then dissolve in a suitable solvent in a test tube. Generally ethanol is used. The eluate obtained from chromatogram can be estimated quantitatively by any physical or chemical methods.

Alternate method that can be used is spectrophotometry.

5 mg histidine – absorbance 0.08

X mg mixture – absorbance 0.03

Application

• Paper chromatography has been used for quantitative analysis of inorganic, organic and biochemical substance.
• Paper chromatography can be used for identification of compound in drugs, biochemical preparation and in natural products.
• It can be used for checking the purity of the sample or compound.
• It can also be used for separation and isolation of number of compounds such as acid, alcohol, vitamins, amino acids, antibiotics.
• It is also used to detect traces of pollutants in water, food or in soil. E.g. pesticides.
• It can be used for the separation of amino acids, sugars and many other compounds.
• It can be used to study several compounds of biological origin such as carbohydrates, sugars, lipids, acids, hormones, nucleotides.
• It can be used for the separation of inorganic ions.