COLUMN CHROMATOGRAPHY

COLUMN CHROMATOGRAPHY

It is also called adsorption chromatography.

·         Stationary phase                      solid

·         Mobile phase                           liquid

The idea of column chromatography was first coined by American Chemist D.T. Davy in 1897 for the study of difference in chemical composition of different natural petroleum products. But it was Russian Botanist Michael Tswett in 1906 who had successfully separated green plant pigments by column techniques. It is the technique in which stationary phase e.g. silica gel, alumina, cellulose and mobile phase is either gas or liquid. The separation takes place when components of mixture are most strongly adsorbed by solid stationary phase.

Principle:

It is known that role of adsorption varies with the given adsorbent for different materials. Thus the principle of selective adsorption is used in column chromatography. In this method, mixtures to be separated are dissolved in a suitable solvent and allow passing through a column containing the adsorbent. The compound which has greater adsorbent power is adsorbed in the upper portion of the column.  The next in the lower portion of the column is compound having lesser adsorbing than other and this process is continuous as a result the compounds are separated in adsorbed in various part of the column. Then, the column may be washed with suitable solvent and each component is separated and analyzed.

A typical column chromatography system consists of the glass column, a mobile phase reservoir at the top and bottom of column contains fraction collector. The column is usually of glass of constant cross sectional area and can be of any size and length. The column is commonly made up of pyrex glass which is thermostable and chemostable. Generally long column give good resolution of components but wide columns are better for dealing with large quantities. The length: width ratios of 10:1 to 100:1 have been found most satisfactory. The glass column used should have a means of supporting the stationary phase to the base of column as possible.

Commercial column possess either a porous glass plate fused on to base of column or suitable device for supporting or replaceable nylonate which support the stationary phase. The commercial column support is to use a small plug of glass wool together with a minimum amount of quartz sand and glass beads.

Adsorbent used in column chromatography

Common adsorbent for column chromatography are sucrose, cellulose, starch, calcium carbonate, calcium phosphate, calcium oxide, silica gel, charcoal, magnesium and aluminium oxide. In order to obtain columns with good filtration properties, the adsorbent should have following properties

1.   The adsorbent must not react with substance to be analyzed i.e. chemically inert.

2.   The adsorbent should be insoluble in the solvent to be used.

3.   The color of absorbent should be such that it doesn’t interfere with chromatogram i.e. adsorbent used should be colorless. But this is not a rigid rule.

4.   The physical and chemical properties of adsorbent should not changed under experimental condition i.e. should be stable.

5.   The particle size should be uniform i.e. it should be fine enough to hold the substance and give the sharp bands. Column efficiency can be increased by smaller particles 100-200 mesh

Among the various adsorbent, alumina and magnesium have been found to give satisfactory separation with most of compound. In general, alumina seems to have higher selective power give sharper and narrow band. Sucrose and starch have power to be very successful for the separation of chlorophyll.

Classification of adsorbents:

Adsorbents have been classified into 3 groups depending on the strength according to force with which they hold solutes.

a. Week adsorbents e.g. sucrose, starch

b. Intermediate adsorbents e.g. CaCO₃, Ca-phosphate

c. Strong adsorbents e.g. alumina, charcoal

Step in Column Chromatography:

1. Preparation of column / Packing of column

The glass wool or cotton plug is used as the support for the column. It is first kept in vertical position. The chromatography column is packed with adsorbent material by filling it about 1/3^rd full solvent and slowly adding slurry of material in solvent. This is carefully poured down with glass rod to stop air buddle being trapped into the column. The suspension is allowed to settle and excess solvents are allowed to run off. This process is repeated until the column of required height is obtained (5 cm of height). Column is packed with dry power but wet packing is more common. In the dry packing, the dry powered adsorbent is introduced through the open end. Then, vaccum is created at bottom and column is tapped with light object until no more setting takes place.

2. Solvent system

In case of column chromatography, a single solvent is used. For better separation, it is found that the solution of mixture should be prepared in non polar solvent such as Petroleum ether, benzene, carbon tetrachloride, ethylacetate, acetone, cyclohexane, ether, chloroform etc.

Solvent in column chromatography functions in the following ways

a. Solvent brings about the separation of mixture into different zones/bands.

b. Solvent puts the mixture on the column

c. Solvent also elutes the components of each separated bands

For all this processes, sometimes only a single solvent is used but it is advantage to use different solvents for different functions.

Polar solvents like water, alcohol etc hold the solute fast and separation of the solute from the solvent is more difficult. The more polar is adsorbent solute, the more polar the mobile phase should be used. This is reason why adsorption is generally not useful for polar compounds. Thus, the transfer of mixture to the adsorbent column is usually carried out by making use of non polar solvents whereas elution of solute from the column can be carried out by using strongly polar solvents.

3. Application of Samples

Several methods are available for the application of sample to the top of the prepared column.

1.   Simple way is to remove most of the mobile phase from above the column by substrate and just to drain remainder solvent into the column bed. Then, sample is carefully applied by pipette and allowed just to run in the column. A small volume of mobile phase is applied to wash final traces of sample into column bed. Mobile phase is then carefully added to column to the height of 2-5 cm. Then, column is connected to a suitable reservoir that contains more mobile phase so that the height of mobile phase can be maintained at 2-5 cm.

2.   An alternative procedure which avoids the necessity to drain the column to the surface of the bed is to increase the density of the sample by the addition of sucrose to a concentration of about 1 % when the solution is layered into the liquid above the column bed, it will automatically sink to the surface of column and hence can be quickly passed into the column.

3.   A third method involves the use of capillary tubing or use of roller pumps to pass the sample directly to the column surface.

In all cases, care must be taken to avoid overloading the column with sample, otherwise irregular separation will occur.

4. Column development and sample elution

In column chromatography, the term development implies the irrigation of the adsorbed mixture and resolution of component into distinct bands. After sample application, the next step is to remove the material from the column with appropriate solvent. Development may be carried out in several ways.

A. Frontal development: A large volume of mixture to be separated is passed through a column of adsorbent. Pure solvent appears first followed by least strongly held substance then a mixture of least and then least strongly held and so on. Only the strongly held substances appear in pure form.

B. Displacement development: A solvent is used which have the higher affinity for the stationary phase than any of the components of mixture to be separated. All components are thus displaced down of the column. In displacement development, the solvent interact most strongly with chromatographic material (adsorbent) then the compound to be separated down the column, thus displacing the bound molecule.

C. Elution development: The components of applied sample are separated by continuous flow of mobile phase through the column and this is called elution development. In this method the solvent interact with the column material more weakly than the solute molecules and drags the bound molecule gradually eluting them from the column.

5. Collection and analysis of fraction

The fraction from the column is collected into the series of test tubes either manually or with fraction collector. Each fraction is then analyzed for the presence of compound. For colored compounds, detection can be achieved simply by visual observation. But for colorless compound detection can be done by Spectrophotometric method or by Colorimetry method or by UV adsorption method. E.g. Protein can be detected and quantified at 190-220 nm and at 260-280 nm. For amino acids, quantization is done at 570 nm by reacting with ninhydrin.

Application of Column Chromatography

i.    Adsorption chromatography has been used primarily for the separation of organic compounds and separation based on adsorption chromatography depends upon the equilibrium that governs the distribution of various solute species between the solvent and the surface of solid. Large difference exists in the tendencies of compounds to be adsorbed. The tendencies to be adsorbed decreases in following manner;

Acid > Alcohol > Carbonyl compounds > Ester > Hydrocarbon

ii.   It is useful for separating non polar or fairly polar organic molecules.

iii. Commonly used adsorbents: Silica, Alumina, Carbon etc are commonly used adsorbents. Silica adsorbents are acidic and good for the separation of basic materials whereas the alumina is basic and better suited for the separation of acidic materials.

iv. Adsorption chromatography is most commonly used to separate non-ionic, water insoluble compounds such as triglycerides, vitamins and many drugs.

v.   It has widely been used for the chromatography of polycyclic compounds, phenols and amines.

vi. It is used to determine amino acid content of protein hydrolysate.

vii. It is used in the separation of urinary ketosteroids, separation of plasma cortisol and separation of mixtures containing stereoisomers or related compounds.

viii. It is used in testing the homogeneity of colored compounds.

ix. It is used in removing small impurities whose structures differ widely from that of major components. The impurities get adsorbed.

x.   In establishing the identity and non identity of two substances: E.g. two substances whose identity is to be determined are mixed and the solution is allowed to pass through a vertical adsorbent column. On elution, bonds are developed corresponding to substances. If only one bond produced it shows that the two substances are different.

NOTE: It should be noted that success of column chromatography depends upon the following factors.

·         Nature of adsorbents

·         Nature of solvents

·         Rate of flow of solvent through column

·         Dimension of column

·         Particle size of adsorbents

Temperature of column during separation

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THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY

The technique of TLC was first introduced by Izmailov and Shrabier in 1938. TLC as procedure for analytical absorption chromatography was first introduced by Stahl in 1958. He was mainly responsible for bringing out standard equipment for the preparation of thin layer apparatus.

TLC is defined as a method for chromatographic analysis on thin layer of absorbent held on a glass plate. TLC is also known as open column chromatography or drop strip or spread layer or surface chromatography.

Principle:

A thin layer of stationary phase is formed on a suitable flat surface such as glass or plastic plate. Since the layer is so thin, the movement of mobile phase across the layer occurs generally by simple capillary action and is rapid. As the mobile phase move across the layer from one edge to opposite edge, it transfers and analyzes the compounds placed on the thin layer. The analyte movement stops either when the mobile phase reaches the end of the layer and capillary action closed up when the plate is removed from the mobile phase i.e. solvent phase. The movement of analyte is expressed by retardation factor.

Types of TLC:

TLC is mainly of 4 types which are as follows:

a) Partition TLC: In this technique, stationary and mobile phase are in liquid. The stationary phase is liquid which is held on suitable solid such as cellulose.

b) Adsorption TLC: Stationary phase is solid and mobile phase is liquid. Stationary phase e.g. silica gel, alumina etc. is used. Mobile phase is organic solvent.

c) Reversed Phase partition TLC: Plate is coated by silica which is hydrophilic in nature and it is again coated by wax or paraffin making it hydrophobic.

d) Ion exchange TLC: A reversible exchange of ions is possible in between ions in a liquid phase and a solid insoluble substance containing ionic state (ion exchange resins).

Technique of TLC:

1. Adsorbents (coating material used in TLC):

Silica gel, alumina or aluminium powders, cellulose powder, kieselgurh (natural adsorbent) are used as adsorbent in TLC. In general silica gel and alumina are used for adsorption TLC and silica gel, cellulose and kieselguhr are used for partition TLC.

Silica gel:  It is the most commonly used adsorption and slightly acidic in nature. A binding agent usually gypsum salt (CaSO₄~13 %) is often added with silica gel to hold the adsorbent firmly on the plate. Silica gel is active absorbent is applicable to separation of all classes of compounds.

Alumina: They are slightly basic in nature used in separation of bases and steroids.

Kieselguhr (Diatomaceous earth): It is a natural adsorbent and is weakly absorbing than silica gel and alumina. It is an inactive type of adsorbent. It can be added to silica gel to give a less active mixed adsorbent. It has been used for separation of sugars.

Cellulose: It is used only as support for the stationary liquid phase in partition TLC in the source of as a paper of cellulose act as support in paper. Modified cellulose paper such as diethyl aminoethyl, triethyl aminoethyl and carboxy methyl cellulose are useful for separation of phospholipids, nucleolipids and pigments.

2. Preparation of plates / chromatoplates:

In TLC, the adsorbent supports are usually glass plates of square or rectangular plates and the size of the plates generally 20 x 20 cm.  Generally glass plates are used.

The glass plates should be cleaned i.e. prewash to remove dirt and dust particles. Glass plates should be cleaned with detergent and ethanol.

3. Preparation of slurry:

Slurry is normally made in water. Required amount of adsorbent powder is weighed out and dissolve in water e.g. silica gel powder are mixed with water in a slurry of silica gel with water in a ratio of 1:2. But 0.02 M Na acetate is used instead of water in case of silica to avoid cracking of adsorbent. Proper stirring is required during slurry preparation for homogenous preparation of the slurry (without grains). Magnetic stirrer can be used for mixing the slurry. The slurry should not be too thick or too thin. If it is too thin it will run too quickly and produce extensively thin layers and if it is too thick spreading produces lines along plate.

Two types:           Thin layer:       0.25 mm

Thick layer:     1.20 mm

A layer of thickness of about 0.25 mm is used for analytical purpose. By mixing 25 gm of silica gel with 50 ml distilled water, a layer of 0.25 mm can be prepared. Thick layer of silica gel with water in ratio 25:40 is used instead of 25:50 ratio.

4. Preparation of Thin layer on plate:

Application of thin layer of adsorbent can be made by different ways/methods like by spreading method, Spraying method and Dipping method.

Dipping method: In this method, slurry of adsorbent is prepared then glass plates are dipped in the slurry (Adsorbent). After about 20 sec the plates are carefully withdrawn and allowed to dry. This method is not of much used.

Spraying method: In this method, slurry of adsorbent is prepared and then sprayed on to the glass plates with the help of an ordinary spray.

Spreading method: Most commonly used method to obtain thin and uniform layer. The slurry is placed in an applicator. This is either moved over the stationary plate or it is held stationary and plate is pushed or pulled through. In one method the adsorbent reservoir containing the slurry is held stationary and the plate to be coated is moved underneath. In another method the reservoir is moved over the plate.

5. Activation of Adsorbent:

After making thin layers on plate, the next job is to remove liquid associated with the layer as completely as possible. This is done by drying the thin layer plate for 30 min in air and then in an oven at 110°C for another 30 minutes and this drying makes the adsorbent layer active. Since water or other polar solvents greatly affect the development by adsorption chromatography. They should be removed from the chromatoplates and this is known as activation.

6. Sample Activation:

Methods of application of sample on chromatoplates are almost similar to these used in paper chromatography. Using micropipette, microsyringe or Pasteur pipette, the sample is applied in small spot above the edge (i.e. 2 cm) and 1.5 cm apart. Different amount of sample from 1-50 ml can be applied. The amount of sample that can be applied depends on thickness of plate and principle of chromatography employed. For example in adsorption TLC, thickness of 1.2 mm is used in which 10-50 ml of sample can be loaded. For 0.25 mm thickness TLC, ideal volume is 0.5 -5.0 ml.

7. Choice of Solvent

Mixtures of two or more solvents of different polarity are used. The choice of solvents depends on the two factors

       I.            Nature of substance to be separated

    II.            Material on which separation is to be carried out

For e.g. if the mixture to be separated consists of hydrocarbon, then active adsorbent is required together with non polar solvent.

Mixture of two or more solvents of different polarity often gives better separation than chemically homogenous solvent, i.e. one solvent is polar and another is non polar. Generally polar substances like alcohol, carboxylic acid, and amine solution should be made in polar solvent and cellulose or silica gel layers are selected. Polar solvent produce greater migration and thus give better separation. It has been observed that a combination of two solvents give better separation than obtained with single solvent.

Examples of Solvents system used in TLC

Solvent mixture                                 Ratio

·         Cyclohexane: ethyl acetate                 95:5

·         Benzene: ethyl acetate                        95:5

·         Benzene: methanol                              95:5

·         Cyclohexane: ethyl acetate                 85:15

·         Benzene: ethyl acetate                        90:10

·         Chloroform:  ethanol                           85:15  

·         Ethyl acetate:  methanol                     99:1

·         Benzene: acetone                                1:1

Solvent system for specific compounds

Compounds               Solvent system (Ratio)

·         Fruit juices                  Ethyl acetate: isopropanol: water: pyridine (26:14:7:2)

·         Amino acids                Butanol: acetic acid: water (4:1:5)

·         Lipids                          Petroleum ether: diethyl ether: acetic acid (80:20:1)

·         Free cholesterol           Chloroform: methanol (2:1)

·         Phosphotidyl choline Chloroform: methanol (4:1)

(Phospholipid)             Chloroform: methanol: acetic acid: water (70:90:25:1)

·         Neutral Lipids             Hexane: diethyl ether: acetic acid (90:15:1)

Figure1: Separation of sugars in the Thin Layer Chromatography

8. Development of Chromatogram

It is usually carried out by ascending method in which solvent is allowed to rise to the height of about 10-16 cm above the origin. Then, the plate is removed from solvent tank and solvent front is carefully marked.

9. Visualization of spot

Location of compounds in TLC is easy because layers can be treated with corrosive substance without any adverse effect on thin layers. E.g. colorless compounds can be located with locating reagent. A solution of locating reagent of desired concentration is used where the plate is dipped or fine sprayed with such reagent.

       I.            Iodine is non destructive locating reagent and is very useful in identifying a large number of organic compounds. Either solution form or direct crystal forms of iodine can be used for locating. 

a.       Iodine is first dissolved in an organic solvent and then solution is sprayed on to the chromatoplate for detection of compound. Iodine is dissolved in 10 % methanol and this solution used for spray.

b.      Cystals of iodine are placed in the tank during the development. Components on chromatogram may directly be exposed to iodine vapors contained in a tank for 10 min.

    II.            Sulfuric acid is also a good locating reagent but is corrosive in nature. It produces colored compound which are visible in daylight as well as in UV light.

 III.            Ninhydrin: Ninhydrin can be used as locating agents for amino acids.

Typical Spray Reagent:

Types of compounds                                     Spray reagents

·         Amino acids                                        Ninhydrin

·         Lipids                                                  Iodine vapor

·         Reducing sugars                                  Aniline diphenylamine reagent

·         Carboxylic acids                                 Bromocresol green

·         Aldehydes and ketones                      2,4 dinitrophenol hydrazine

·         Phenols                                                Ferric chloride

·         Steroids, glycosides, Vitamins A        Antimony trichloride

10. Qualitative Analysis of Spot

After spot are visualized, R_f values are calculated immediately because of the possibility of fading. Evaluation of R_f values can be used for qualitative purpose.

11. Quantitative Analysis

Quantitative evaluation of chromatogram can be carried out by 2 methods

a.       Direct

b.      Indirect

a. Direct method:

Qualitative determination is undertaken directly on the layer. It includes visual comparison of spot. Photodensitometry method and Flourimetry method are used in direct method.

b. Indirect method

Spots are scrapped off from chromatoplates and then eluted with solvent e.g. ethanol is used.

Advantages of TLC:

TLC not only combines the advantage of paper and column chromatography but it is considered to be superior to either of these techniques.

a.       It utilizes simple equipments. The technique is simple and cost is very low.

b.      It is a versatile technique and can be applied to a wide variety of compound both organic as well as inorganic compound.

c.       In TLC, development time is one hour or less. Excellence separation can be obtained within 15-20 mins. Identification and separation by TLC can be carried out quickly.

d.      Wide choice of stationary phase can be used. The method may be employed for adsorption, partition or ionic exchange chromatography.

e.       Sensitivity: The separation achieved by TLC is superior to those obtained by paper chromatography (10-100 times more sensitive). Due to its high sensitivity, compounds which are encountered in trace amount can be detected by TLC.

f.       TLC gives sharper distinct spot. Diffusion of spot in TLC is low.

g.      Easily recovery of component: It is possible to remove powdery coating of plate by scrapping with the knife.

h.      Easy visualization of separated components.

i.        Sometime, fluorescent indicators such as sodium salt of fluorescence, eosin or zinc silicates are added to the silica gel. Thin layer thus produce fluorescence on exposure to UV light. The main advantage is that non fluorescence but UV absorbent, substance can be obtained on a thin layer containing fluorescent indicator.

j.        Corrosive reagent and acid can be spread on thin layer chromatography without adverse effect.

k.      Capacity of thin layers of an adsorbent is higher than that of paper chromatography.

Limitation: The only limitation of TLC is that it can only be used for small scale production.

Application of TLC:

• ·         TLC is applicable for qualitative and quantitative analysis.
• ·         TLC has been used as analytic tool especially in organic chemistry. The main reason for popularity as an analytical tool in organic chemistry:
• o   It can be used for most of the chemical compound
• o   It has high speed separation i.e. development time is rapid in comparison to paper chromatography.
• ·         TLC can be used as a purification process. Micrograms of sample are sufficient for checking their purity by TLC. Because TLC has 10-100 times more sensitivity in comparison to paper chromatography. Due to its high sensitivity it often enables impurities to be observed in pure sample.
• ·         TLC can be used to assess that a reaction is complete or not.
• ·         TLC can be used for identifying organic compound for e.g. acids: citric acid and tartaric acid have been separated on layers of silica gel G by using a butyl acetate, acetic acid and water mixture.
• ·         Amino acids, proteins and peptides: A mixture of 34 amino acid, proteins and peptides has been successfully isolated from urine by using silica gel plate.
• ·         Vitamins, antibiotics and food products have been separated by TLC on Thin layer of silica gel G by using 2 solvent systems acetone and methanol or Benzene and formamide.
• ·         Antibiotics: Erythromycin has been separated on silica gel G using Methyl chloride, methanol, Benzene, formamide. Tetracycline by using 10 % citric acid, Butanol, methanol as a solvent system.
• ·         Vitamins A, D and E can be separated on silica gel G using 80 % cyclohexane and 20 % diethyl ether.
• ·         TLC can be used to separate inorganic ions, cations, anions and organometallo compounds.
• ·         TLC has been used in the analysis of urine and blood in pathological lab.