By : James W Zubrick
Email: j.zubrick@hvcc.edu
This is, as you may have guessed, chromatography carried out on a column of adsorbent, rather than a layer. Not only is it cheap, easy, and carried out at room temperature but you can separate large amounts, gram quantities, of mixtures.
In column chromatography, the adsorbent is usually alumina but can be silica gel. Except that alumina tends to be basic and silica gel, acidic, I don’t know why the former is used more often. Remember, if you try out an eluent (solvent) on silica gel plates, the results on an alumina column may be different.
Now you have a glass tube as the support holding the adsorbent alumina in place. You 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 carried along by the solvent are washed entirely out of the column, into separate flasks. Then you isolate the separate fractions.
PREPARING THE COLUMN
1. The alumina is supported by a glass tube with either a stopcock or a piece of tubing and a screw clamp to control the flow of eluent (Fig. 98). You can use an ordinary buret. What you will use will depend on your own lab program. Right above this control you put a wad of cotton or glass wool to keep everything from falling out. Do not use too much cotton or glass wool, and do not pack it too tightly. If you ram the wool into the tube, the flow of eluent will be very slow, and you’ll be in lab till next Christmas waiting for the eluent. If you pack it too loosely, all the stuff in the column will fall out.
2. At this point, fill the column half-full with the least polar eluent you will use. If this is not given, you can surmise it from a quick check of separation of the mixture on a TLC plate. This would be the advantage of an alumina TLC plate.
3. Slowly put sand into the column through a funnel until there is a 1-cm layer of sand over the cotton. Adsorbent alumina is SO FINE, it is likely to go through cotton or glass wool but NOT through a layer of fine sand.
Fig. 98 Wet-column chromatography setup.
4. During this entire procedure, keep the level of the solvent above that of any solid material in the column!
5. Now slowly add the alumina. Alumina is an adsorbent and it sucks up the solvent. When it does, heat is liberated. The solvent may boil and ruin the column. Add the alumina slowly! Use about 25 g of alumina for every 1 g of mixture you want to separate. While adding the alumina, tap or gently swirl the column to dislodge any alumina or sand on the sides. You know, a plastic wash bottle with eluent in it can wash the stuff down the sides of the column very easily.
6. When the alumina settles, you normally have to add sand (about 1 cm) to the top to keep the alumina from moving around.
7. Open the stopcock or clamp and let solvent out until the level of the solvent is just above the upper level of sand.
8. Check the column! If there are air bubbles or cracks in the column of alumina, dismantle the whole business and start over!
COMPOUNDS ON THE COLUMN
If you’ve gotten this far, congratulations! Now you have to get your mixture, the analyate, on the column. Dissolve your mixture in the same solvent you are going to put through the column. Try to keep the volume of the solution of mixture as small as possible. If your mixture does not dissolve entirely, and it is important that it do so, check with your instructor! You might be able to use different solvents for the analyate and for the column, but this isn’t as good. You might use the least polar solvent that will dissolve your compound.
If you must use the column eluent as the solvent, and not all the solvent will dissolve, you can filter the mixture through filter paper. Try to keep the volume of solution down to 10 ml or so. After this, the sample becomes unmanageable.
1. Use a pipet and rubber bulb to slowly and carefully add it to the top of the column (Fig. 99). Do not disturb the sand!
2. Open the stopcock or clamp and let solvent flow out until the level of the solution of compound is slightly above the sand. At no time let the solvent level get below the top layer of sand! The compound is now “on the column.”
3. Now add eluent (solvent) to the column above the sand. Do not disturb the sand! Open the stopcock or clamp. Slowly let eluent run through the column until the first compound comes out. Collect the different products in Erlenmeyer flasks. You may need lots and lots of Erlenmeyer flasks. At no time let the level of the solvent get below the top of the sand! If necessary, stop the flow, add more eluent, and start the flow again.
Fig. 99 Putting compounds on the column by pipet.
VISUALIZATION AND COLLECTION
If the compounds are colored, you can watch them travel down the column and separate. If one or all are colorless, you have problems. So:
1. Occasionally let 1 or 2 drops of eluent fall on a clean glass microscope slide. Evaporate the solvent and see if there is any sign of your crystalline compound! This is an excellent spot test, but don’t be confused by nasty plasticizers from the tubing trying to put one over on you, pretending to be your product.
2. Put the narrow end of a “TLC spotter” to a drop coming off of the column. The drop will rise up into the tube. Using this loaded spotter, spot, develop, and visualize a TLC plate with it. Not only is this more sensitive, but you can see whether the stuff coming out of the column is pure (see Chapter 19, “Thin-Layer Chromatography”). You’ll probably have to collect more than one drop on a TLC plate. If it is very dilute, the plate will show nothing, even if there actually is compound there. It is best to sample 4 or 5 consecutive drops.
Once the first compound or compounds have come out of the column, those that are left may move down the column much too slowly for practical purposes. Normally you start with a nonpolar solvent. But by the time all the compounds have come off, it may be time to pick up your degree. The solvent may be too nonpolar to kick out later fractions. So you have to decide to change to a more polar solvent. This will kick the compound right out of the column.
To change solvent in the middle of a run:
1. Let old solvent level run down to just above the top of the sand.
2. Slowly add new, more polar solvent and do not disturb the sand.
You, and you alone, have to decide if and when to change to a more polar solvent. (Happily, sometimes you’ll be told.)
Fig. 100 A growth of crystals occurs as the eluent evaporates.
If you have only two components, start with a nonpolar solvent, and when you are sure the first component is completely off the column, change to a really polar one. With only two components, it doesn’t matter what polarity solvent you use to get the second compound off the column.
Sometimes the solvent evaporates quickly and leaves behind a “fuzz” of crystals around the tip (Fig. 100). Just use some fresh solvent to wash them down into the collection flask.
Now all the components are off the column and in different flasks. Evaporate the solvents (No flames!), and lo! The crystalline material is left.
Dismantle the column. Clean up. Go home.
