CHROMATOGRAPHY

Chromatography Chromatography is from two Greek words chroma meaning "color" and graphein meaning "to write”, that is Chromatography means "color writing". Chromatography may be defined as the separation technique based on the differential distribution of a mixture of substances between two phases one of which is percolated through the other.

Chromatography One of these phases is termed the stationary phase and the other the mobile phase. The stationary phase may be a finely divided solid or a liquid that has been bound to some inert supporting material. The mobile phase may be a liquid or a gas (pure or homogeneous mixture).

Terms used in Chromatography Chromatography: It is a physical method of separation (i.e. the sepation technique). Chromatograph: A Chromatograph is an equipment or instrument that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation. Chromatogram: It is the visual output of the chromatograph. In the case of an optimal separation, different peaks on the chromatogram correspond to different components of the separated mixture.

Mobile phase : The mobile phase may be a liquid or a gas Mobile phase : The mobile phase may be a liquid or a gas. The mobile phase moves through the chromatography column which is packed with the stationary phase. Stationary phase: The stationary phase is the substance which is fixed in a column or on a flat plate or surface. The mobile phase is passed through the stationary phase where the sample interacts and is separated. Examples are silica gel, aluminum oxide or cellulose. Elution : Purification or separation by washing. Eluent : A liquid used for elution.(Mobile Phase) Eluate : A liquid obtained by eluting. It is the mobile phase leaving the column.

Retention time : The retention time is the characteristic time it takes for a particular substance to pass through the system (from the column inlet to the detector) under set conditions.

Classification of chromatographic methods Chromatographic methods can be classified in a number of ways: Chromatographic methods can be differentiated into two broad categories based on the physical means of bringing the stationary and mobile phases into contact Planar Chromatography Column Chromatography

Planar Chromatography: the stationary phase is supported on a flat plate or in the fibres of a paper. Here the mobile phase moves through the stationary phase by capillary action or by gravity. Examples include: Paper chromatography Thin layer chromatography (TLC).

Column Chromatography can be divided further into two broad categories based on the physical state of the mobile phase Gas Chromatography if the mobile phase is gas. Liquid Chromatography if the mobile phase is liquid.

Gas Chromatography is further differentiated into two groups based on the type of stationary phase Gas solid Chromatography (GSC) where the stationary phase is solid. Gas liquid Chromatography (GLC) where the stationary phase is liquid. Liquid Chromatography is differentiated further on the basis of mechanism of separation into the following: Adsorption Chromatography Partition Chromatography Ion-Exchange Chromatography Size Exclusion Chromatography Affinity Chromatography

Based on the purpose of separation the chromatography may be Preparative or Analytical Preparative chromatography : The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). It is done normally with larger amounts of material. Analytical chromatography : The purpose of analytical chromatography is to measure the relative proportions of analytes in a mixture. It is done normally with smaller amounts of material.

Column chromatography Column chromatography is generally used to separate chemical compounds from a mixture. It is often used for preparative purpose. The classical preparative chromatography column, is a glass tube with a diameter from 1cm to 10cm and a height of 20 cm to 50cm with a tap at the bottom. Column chromatography is differentiated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, it is called flash chromatography.

Column Chromatography Procedure A. Packing a column with a stationary phase Place a peace of cotton at the bottom of a long glass column using a long glass rod. Press the cotton evenly to make sure that the tube at the bottom of the column is securely closed. Clamp the column to a ring stand and add enough sand to fill the curved portion of the column. Tap the side of the column gently with the stir rod to ensure that the sand has settled evenly 3. Add enough solvent (or initial eluent) to the column (using a funnel) to reach a height of about 10cm.

Prepare a slurry of adsorbent in the initial eluent in a beaker and carefully pour the slurry into the column. Open the tap and drain the excess solvent into a clean flask. Tap on the side of the column with a stir rod to help the silica settle uniformly. 6. Once the silica has settled, carefully add sand to the top of the column. 7. Be sure that a layer of solvent always just above the top layer of sand, otherwise cracks will develop in the column.

B. Loading a sample onto the column Drain eluent from the column until no solvent remains above the surface of the sand. Using a long Pasteur pipet, carefully add the sample solution to the column. Drain eluent from the column until no sample remains above the surface of the sand. Use ~ 1 mL of eluent to rinse the container and add the solution to the sand. Drain eluent from the column until no liquid remains above the surface of the sand. 12. Repeat step 11 two or three times to completely transfer your sample onto the adsorbent (stationary phase).

C. Eluting the sample Once the sample is added onto the silica, carefully add eluent to the top of the column. 14. Add more eluent as necessary. The composition of the eluent can be changed as the column progresses. Do not let the level of the solvent fall below the top layer of sand at any point. D. Collecting the eluent from the column 15. Collect the eluent as it is released from the column in small volumes of 5, 10 or 15ml in test tubes.

Isolation of compounds by Column Chromatography E. Analyzing the fractions 17. Analyze the fractions by thin-layer chromatography. 18. Similar fractions are combined. If the fraction contains a single compound the solvent is evaporated to obtain the pure compound. If it contains more than one component they are separated using other suitable technique Isolation of compounds by Column Chromatography

Loading the sample 2 , 3. Eluting the sample 4, 5. Collecting the eluents

The Adsorbent The most commonly used adsorbents for column chromatography are Silica gel (SiO2) and alumina (Al2O3). These adsorbents are sold in different mesh sizes, as indicated by a number on the bottle label such as silica gel 60 or silica gel 230-400. This number refers to the mesh of the sieve used to size the silica. Actually it is the number of holes in the mesh or sieve through which the crude silica particle is passed in the manufacturing process. The larger the mesh size, the smaller the adsorbent particles. Silica gel 70–230 is used in gravity column chromatography and Silica gel 230–400 is used in flash chromatography.

The Solvent Choice of an eluting solvent is very important for the successful separation of a mixture of chemical compounds. Polar solvents elute polar molecules more rapidly and the components may not be separated at all. On the other hand if a very non polar solvent is used, compounds will not be elute from the column. Therefore if a silica column is used, the initial solvent is usually the petroleum ether. The polarity of this solvent is gradually increased by adding increasing amounts of polar solvents.

Advantages of Column Chromatography Column chromatography is advantageous over most other chromatographic techniques because it can be used to separate and purify substantial quantities of components from a mixture. Column chromatography can also be used for analytical purposes.  

Disadvantages of Column Chromatography Packing of the column requires some technical skill. The technique is time-consuming and tedious, especially for larger samples. It requires constant attention while the experiment is being performed: collection vessels must be frequently switched and solvent should be added continuously to the top of the column at a rate sufficient to cover the adsorbent.