Chromatography

solvent

Vapor Phase Chromatography. This is accomplished by constructing or buying complicated and expensive equipment. Although this method is very effective, it is superseded by the simple, inexpensive and effective column chromatography.

Thin Layer Chromatography. Thin layer chromatography is primarily a tool for small qualitive analysis (deciding which solvents elute which substances, etc.). A microscopic amount of sample is applied at one end of a small plate covered on one side with a thin absorbent coating. The plate is then dipped into a shallow pool of solvent which rises on the coated layer, permitting the compounds of the sample to move with the solvent to differing heights. The individual components can then be detected as separate spots along the plate. Unfortunately this process can only be scaled up to do several grams at a time, again making column chromatography the champion of chromatography.

If, however, you wish to use thin layer, consult your local library on methods. I chose not to go into depth on thin layer because it is so inferior to column style.

Column Chromatography. The main idea here is to dissolve your mixture and put it on the adsorbent, at the top of the column. Then you wash the mixture down the column using at least one eluent (solvent), perhaps more. The compounds of your mixture are carried along by the solvents and washed out of the column at different rates and collected into separate flasks. Why do you want to do this? Let us say you have a substance that needs to be purified, but it cannot be distilled because it decomposes at a low temperature, or you wish to extract one of many mixable liquid substances that have been mixed together, etc. A column chromatography can separate, purify and extract.

Preparing The Column. Alumina or silica.gel is supported by glass (see Figure 7) with a valve to control the flow of eluent. Right above this valve place a fritted glass disc or a wad of glass wool or cotton to keep everything from falling out. Do not use too much, and do not pack it too tightly, or too loosely.

Fill the column half full with the least polar eluent that you will use. If your particular formula does not give the eluents to use (this is rare) then you will have to look up the directions on

solvent

- sand alumina, etc.

fritted glass disc product alumina, etc.

fritted glass disc product thin layer chromatography or find an effective eluent (remember this is just another name for solvent) through trial and error with this small scale method.

Slowly put sand over the cotton until you have at least one centimeter. Next, very slowly add the adsorbent (alumina, silica). Adsorbents liberate heat, possibly causing the eluent to boil, ruining the column. Add the adsorbent slowly. Use about 25 g of adsorbent for every 1 g of mixture you want to separate. When the alumina settles, add another 1 cm of sand to the top. During the entire procedure the level of the eluent must be higher than any solid material placed in the column,

Now you may open the valve until there is a little over 1 cm of solvent above the top layer of sand. If there are any cracks or air bubbles in the adsorbent, dump everything and start over.

Dissolve the mixture (your substance) in the same solvent you are going to put through the column, keeping the amount as small as possible (this is called the analyate). You should be using the least polar solvent that will dissolve your substance. Now you may add the analyate very carefully; do not disturb the sand. Open the valve until the level of the column is the same as it was before you added the analyate (1 cm above the sand). At no time let the solvent level drop below the sand! Add the required eluent (solvent) to the column, not disturbing the sand. Open the valve to slowly let the eluent run through the column until the first compound comes out. Collect the different compounds in different flasks. At no time let the solvent drop below the top of the sand! If necessary, stop the flow, add more eluent, and start the flow again.

Should the compounds be colored, you can watch them travel down the column and separate, changing collection flasks as the colors change. If your compound is clear then you will have to use one of the following steps:

1. Occasionally let one or two drops of eluent fall onto a microscope slide. Evaporate the solvent and see if there are any properties of the compound that should be coming through, such as crystal shapes, tastes, smells, viscosities if oil, etc.

2. Occasionally use several drops to spot, develop, and visualize a thin layer chromatography plate. Although thin layer is very similar to column, you should read up on it as I do not have time to go into the complete operation.

If you find the eluents are taking an excessive amount of time to wash down the compounds, then switch to the next most polar solvent. If you had two compounds and one of them is already collected, then go ahead and get some really polar solvent and get that last compound pronto.

List of solvents arranged in order of increasing polarity. Note: This is a very small list of many solvents.

(Least polar)

(Most polar)

petroleum ether cyclohexane toluene chloroform acetone ethanol methanol

Chromatography 17

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