Procedure for Microscale Flash Column Chromatography

In microscale flash chromatography, the column does not need either a pinchclamp or a stopcock at the bottom of the column to control the flow, nor does it need air-pressure connections at the top of the column. Instead, the solvent flows very slowly through the column by gravity until you apply air pressure at the top of the column with an ordinary Pasteur pipet bulb.

(1) Prepare the column.

The column is packed using a simple dry-pack method.
Plug a Pasteur pipet with a small amount of cotton; use a wood applicator stick to tamp it down lightly. Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out. Add dry silica gel adsorbent, 230-400 mesh -- usually the jar is labeled "for flash chromatography." One way to fill the column is to invert it into the jar of silica gel and scoop it out . . .
. . . then tamp it down before scooping more out. Another way to fill the column is to pour the gel into the column using a 10 mL beaker.
When properly packed, the silica gel fills the column to just below the indent on the pipet. This leaves a space of 4–5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand using a small 3-pronged clamp.
Whichever method you use to fill the column, you must tamp it down on the bench top to pack the silica gel. You can also use a pipet bulb to force air into the column and pack the silica gel.

(2) Pre-elute the column.

The procedure for the experiment that you are doing will probably specify which solvent to use to pre-elute the column. A non-polar solvent such as hexanes is a common choice.

Add hexanes (or other specified solvent) to the top of the silica gel. The solvent flows slowly down the column; on the column above, it has flowed down to the point marked by the arrow. Monitor the solvent level, both as it flows through the silica gel and the level at the top. If you are not in a hurry (or busy doing something else), you can let the top level drop by gravity, but make sure it does not go below the top of the silica. Again, the arrow marks how far the solvent has flowed down the column.
Speed up the process by using a pipet bulb to force the solvent through the silica gel - this puts the flash in microscale flash chromatography. Place the pipet bulb on top of the column, squeeze the bulb, and then remove the bulb while it is still squeezed. You must be careful not to allow the pipet bulb to expand before you remove it from the column, or you will draw solvent and silica gel into the bulb. When the bottom solvent level is at the bottom of the column, the pre-elution process is completed and the column is ready to load.
If you are not ready to load your sample onto the column, it is okay to leave the column at this point. Just make sure that it does not go dry -- keep the top solvent level above the top of the silica (as shown in the picture to the left) by adding solvent as necessary.

(3) Load the sample onto the silica gel column.

Two different methods are used to load the column: the wet method and the dry method: wet and dry. Below are illustrations of both methods of loading a crude sample of ferrocene onto a column.

In the wet method, the sample to be purified (or separated into components) is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent. This solution is loaded onto the column.

Wet loading method

The column at the left is being loaded by the wet method. Follow the thumbnails below to see close-up details of the sample as it is allowed to sink into the column. Once it's in the column, fresh eluting solvent is added to the top and you are ready to begin the elution process (see step 4).

Sometimes the solvent of choice to load the sample onto the column is more polar than the eluting solvents. In this case, if you use the wet method of column loading, it is critical that you only use a few drops of solvent to load the sample. If you use too much solvent, the loading solvent will interfere with the elution and hence the purification or separation of the mixture. In such cases, the dry method of column loading is recommended.

Dry loading method

First dissolve the sample to be analyzed in the minimum amount of solvent and add about 100 mg of silica gel. Swirl the mixture until the solvent evaporates and only a dry powder remains. Place the dry powder on a folded piece of weighing paper and transfer it to the top of the prepared column. Add fresh eluting solvent to the top -- now you are ready to begin the elution process (see step 4). Follow the thumbnails below to see close-up details of this process.

(4) Elute the column.

Force the solvent through the column by pressing on the top of the Pasteur pipet with a pipet bulb. Only force the solvent to the very top of the silica: do not let the silica go dry. Add fresh solvent as necessary.

The photo at the left shows the solvent being forced through the column with a pipet bulb.

The series of 5 photos below show the colored compound as it moves through the column after successive applications of the pipet bulb process.

The last two photos illustrate collection of the colored sample. Note that the collection beaker is changed as soon as the colored compound begins to elute.

The process is complicated if the compound is not colored. In such experiments, equal sized fractions are collected sequentially and carefully labeled for later analysis.

(5) Elute the column with the second elution solvent.

If you are separating a mixture of one or more compounds, at this point you would change the eluting solvent to a more polar system, as previously determined by TLC. Elution would proceed as in step (4).

(6) Analyze the fractions.

If the fractions are colored, you can simply combine like-colored fractions, although TLC before combination is usually advisable. If the fractions are not colored, they are analyzed by TLC (usually). Once the composition of each fraction is known, the fractions containing the desired compound(s) are combined.

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