Column Chromatography

Column Chromatography ➢

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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 mech mechan anis ism m: 1. Adsor Adsorpti ption on Chro Chroma matog tograp raphy hy Column chromatography TLC 1. Parti Partitio tion n Chro Chroma matog tograp raphy hy Paper Chromatography Gas Chromatography 1. Ion-e Ion-exch xchan ange ge Chroma Chromatog tograp raphy hy 2. Size Size exclus exclusion ion Chrom Chromato atogra graph phy y Gel permeation Gel filtration 1. Affin Affinity ity Chrom Chromato atogra graph phy y a. By the natu nature re of the the station stationary ary and and mobile mobile phase phase:: 1. LSC LSC (Li (Liqu quid id Soli Solid) d) 2. GSC GSC (Ga (Gas s Sol Solid id)) 3. LLC LLC (Li (Liqu quid id Liqu Liquid id)) 4. GLC GLC (Ga (Gas s Liq Liqui uid d) 5. VLC VLC (Va (Vapo porr Liq Liqui uid) d) • •

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Properties of adsorbent: 1. They They should should be insoluble insoluble in solve solvent. nt. 2. They They shou should ld be che chemi mical cally ly inert inert.. 3. They should should be active active but not not so active that no movemen movementt of components components occurs. occurs. 4. They should should be colorless colorless to facilitate the observation observation of zones zones (bands). (bands). 5. They They should should be allowi allowing ng suitabl suitable e flow of of mobile mobile phase. phase. 6. They They should should have have repro reproduci ducible ble prope properties rties.. 7. There There particl particle e size should should be between between 75-15 75-150 0 µ. Classification of adsorbents: a. Weak Weak affin affinity ity adsor adsorbe bents nts 1. Sucrose 2. Starch 3. Insulin 4. Talc 5. Na CO a. Mediu Medium m affin affinity ity adsor adsorbe bents nts 1. CaCO 2. MgCO 3. MgO 4. Ca(OH) a. Stron Strong g affin affinity ity adsor adsorbe bents nts 1. Acti Activa vate ted d alum alumin ina a 2. Activ ctivat ated ed sil silic ica a 3. Acti Activa vate ted d cha charc rcoa oall 4. Activ ctivat ated ed MgS MgSiO iO 1

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Advantages of Column chromatography 1. It can be used used in both analytical analytical and preparative preparative applications. applications. 2. It is used used to determin determine e the number number of compo componen nents ts of a mixture. mixture. 3. It is also used used to separate separate and and purify substantial substantial quantities quantities of those compone components nts for subsequen subsequentt analysis. Disadvantages of Column Chromatography 1. Properly setting up the column requires some technical skill and manual dexterity. 2. It is very time time consuming consuming and tedious, tedious, especially for large large samples. samples. 3. Collecting vessels must must be frequently frequently switched switched and solvent levels levels need to be topped 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. Sepa Separa rati tion on of-of----a. 17-ke 17-ketos toster teroid oid gluc glucuro uronid nides es b. Urina Urinary ry 17-ke 17-ketos toster teroid oids s c. Plas Plasma ma co cortis rtisol ols s d. Mixtures containing stereo isomers or related compounds e. Geom Geomet etri rica call isome isomers rs 1. Purif Purifica icatio tion n of techni technical cal prod product ucts s 2. Purifica Purification tion of homo homogen geneity eity of colore colored d compoun compounds. ds. 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. 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. •

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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 cm 3 samples or 5 cm 3 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: Elution: the process of washing a compound through a column using a solvent is known as the elution. elution. The solvent is sometimes known as the eluent. eluent .

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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.

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Mobile Phase: The Phase which moves through or over the surface of the stationary phase may be a pure liquid or a mixture of solutions or may be gas.

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 stationary phase phase is topper topper then the mobile mobile phase, the air may may be affect affect the stationary stationary phase. And causes the damage the packing of the stationary phase. 2. If the stationary stationary phase phase is in top top and then then we adding adding sample sample in the column, it causes the the disturbance the packing of the stationary phase.

Thin Layer Chromatography (TLC)

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