Column Chromatography ➢ What is column chromatography?

Column chromatography may be defined as the selective adsorption and separation of a mixture of chemical substances on a column through which a suitable solvent has passed. No chemical reaction takes place during the process and once separated from the mixture any starting material is recovered chemically unchanged. ➢ Classification of chromatography: a. By mechanism: 1. Adsorption Chromatography • Column chromatography • TLC 1. Partition Chromatography • Paper Chromatography • Gas Chromatography 1. Ion-exchange Chromatography 2. Size exclusion Chromatography • Gel permeation • Gel filtration 1. Affinity Chromatography a. By the nature of the stationary and mobile phase: 1. LSC (Liquid Solid) 2. GSC (Gas Solid) 3. LLC (Liquid Liquid) 4. GLC (Gas Liquid ) 5. VLC (Vapor Liquid)

➢ Properties of adsorbent: 1. They should be insoluble in solvent. 2. They should be chemically inert. 3. They should be active but not so active that no movement of components occurs. 4. They should be colorless to facilitate the observation of zones (bands). 5. They should be allowing suitable flow of mobile phase. 6. They should have reproducible properties. 7. There particle size should be between 75-150 µ. ➢ Classification of adsorbents: a. Weak affinity adsorbents 1. Sucrose 2. Starch 3. Insulin 4. Talc 5. Na CO a. Medium affinity adsorbents 1. CaCO 2. MgCO 3. MgO 4. Ca(OH) a. Strong affinity adsorbents 1. Activated alumina 2. Activated silica 3. Activated charcoal 4. Activated MgSiO 1

➢ Advantages of Column chromatography 1. It can be used in both analytical and preparative applications. 2. It is used to determine the number of components of a mixture. 3. It is also used to separate and purify substantial quantities of those components for subsequent analysis. ➢ Disadvantages of Column Chromatography 1. Properly setting up the column requires some technical skill and manual dexterity. 2. It is very time consuming and tedious, especially for large samples. 3. Collecting vessels must be frequently switched and solvent levels need to be topped up. ➢ Application of Column Chromatography For analytical purpose, column chromatography has an unlimited value, although it is used to determine amino acid contents of protein hydrolyte. Some other applications are---1. Separation of---a. 17-ketosteroid glucuronides b. Urinary 17-ketosteroids c. Plasma cortisols d. Mixtures containing stereo isomers or related compounds e. Geometrical isomers 1. Purification of technical products 2. Purification of homogeneity of colored compounds. ➢ Why silica is used as adsorbent? Silica is used as adsorbent because-----1. Fine 2. Inert 3. Porous ➢ Solvents used in the Column Chromatography Adsorption is most powerful from non-polar such as petroleum, ether or benzenes and a single solvent may also often be effective in developing the chromatogram. The rate of movement of the compounds down the column can be increased by the addition of a second solvent is usually to the mobile phase. The second solvent is usually more polar then first. List of solvents used in column chromatography is given below:-

➢ (Q) Suppose you want to separate a mixture of two colored compounds-one yellow, one blue. The mixture looks green. The blue is more polar than the yellow one. Explaining what is happening? What initiative should be taken if you want to collect the blue compound? 2

Answer: Suppose we wanted to separate a mixture of two colored compounds - one yellow, one blue. The mixture looks green. Then we have to make a concentrated solution of the mixture preferably in the solvent used in the column. First I have to open the tap to allow the solvent already in the column to drain so that it is level with the top of the packing material, and then add the solution carefully to the top of the column. Then I have to open the tap again so that the colored mixture is all absorbed into the top of the packing material, so that it might look like this:

Then I have to add fresh solvent to the top of the column, trying to disturb the packing material as little as possible. Then open the tap so that the solvent can flow down through the column, collect it in a beaker or flask at the bottom. As the solvent runs through, I have to keep adding fresh solvent to the top so that the column never dries out. The next set of diagrams shows what might happen over time.

Explaining what is happening The blue compound is obviously more polar than the yellow one - it perhaps even has the ability to hydrogen bond. I can tell this because the blue compound doesn't travel through the column very quickly. That means that it must adsorb more strongly to the silica gel or alumina than the yellow one. The less polar yellow one spends more of its time in the solvent and therefore washes through the column much faster. The process of washing a compound through a column using a solvent is known as elution. The solvent is sometimes known as the eluent. What if you want to collect the blue compound as well? 3

It is going to take ages to wash the blue compound through at the rate it is travelling at the moment! However, there is no reason why I can't change the solvent during elution. If I replace the solvent and I have been using by a more polar solvent once the yellow has all been collected. That will have two effects, both of which will speed the blue compound through the column. • The polar solvent will compete for space on the silica gel or alumina with the blue compound. Any space temporarily occupied by solvent molecules on the surface of the stationary phase isn't available for blue molecules to stick to and this will tend to keep them moving along in the solvent. There will be a greater attraction between the polar solvent molecules and the polar blue molecules. This will tend to attract any blue molecules sticking to the stationary phase back into solution.

The net effect is that with a more polar solvent, the blue compound spends more time in solution, and so moves faster. ➢ What if everything in your mixture is colorless? If we are going to use column chromatography to purify the product of an organic preparation, it is quite likely that the product that we want will be colorless even if one or more of the impurities is colored. Let's assume the worst case that everything is colorless. There is no quick and easy way of doing this! What we do, is collect what comes out of the bottom of the column in a whole series of labeled tubes. How big each sample is will obviously depend on how big the column is - you might collect 1 cm3 samples or 5 cm3 samples or whatever is appropriate. We can then take a drop from each solution and make a thin layer chromatogram from it. You would place the drop on the base line alongside a drop from a pure sample of the compound that you are making. By doing this repeatedly, we can identify which of our samples collected at the bottom of the column contain the desired product, and only the desired product. ➢ Definitions:

1. Elution: the process of washing a compound through a column using a solvent is known as the
elution. The solvent is sometimes known as the eluent.

2. Stationary Phase: The Phase which is a fixed bed at large surface area may be porous or finely
divided solid or a liquid that has been located in a thin layer of an inert supporting material. a pure liquid or a mixture of solutions or may be gas.

3. Mobile Phase: The Phase which moves through or over the surface of the stationary phase may be
➢ Why we always want to top up the mobile phase then the stationary phase in the column? Answer: - There may be two causes1. If the stationary phase is topper then the mobile phase, the air may be affect the stationary phase. And causes the damage the packing of the stationary phase. 2. If the stationary phase is in top and then we adding sample in the column, it causes the disturbance the packing of the stationary phase.

Thin Layer Chromatography (TLC)

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