Micron Spherical Silica Gel Powder for Cosmetics Additive and Column Chromatography
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Operating technology of silica gel column chromatography

Operation methods and precautions of silica gel column chromatography


Key points for operation: a layer of absorbent cotton shall be padded at the bottom of the column before loading to prevent the leakage of adsorbent. There are two methods of dry loading and wet loading:

(1) Dry column loading: silica gel is loaded into the column through a funnel without interruption, forming a trickle and slowly adding into the tube. The rubber mallet can also be used to gently tap the silica gel column to make the silica gel filling continuous, uniform and tight. After the column is installed, open the lower piston, and then pour in eluent for elution to exhaust the air in the column and maintain a certain liquid level.

(2) Wet loading: load the eluent to be used initially into the column, open the lower piston, and make the eluent flow out slowly. Then pour silica gel into the column slowly and continuously (or mix silica gel and an appropriate amount of eluent into a suspension and slowly add it into the column). Silica gel will settle freely in the column driven by gravity and eluent. During this time, the eluting agent flowing out will be added back to the column to maintain a certain liquid level until silica gel is added and the sedimentation in the column is no longer changed. Then add a small piece of filter paper or a little absorbent cotton on the silica gel. The eluent level shall be controlled to a certain height according to the sample dosage.

(3) Homogenization method: stir into homogenate. Add a solvent twice the volume of dry silica gel and stir it fully with a glass rod. If the eluent is petroleum ether/ethyl acetate/acetone system, mix it with petroleum ether; If the eluent is chloroform/alcohol system, chloroform shall be mixed. If it cannot be stirred into homogenate, it means that the water content in the solvent is too large, especially ethyl acetate/acetone. If it is not compatible with water to distribute the chromatography, it must be dried with anhydrous sodium sulfate for a long time in advance. Chloroform is dried with anhydrous calcium chloride to remove 1% alcohol. If the sample is sensitive to acid, do not use chloroform system to pass the column.

Loading sample

Dissolve the sample to be separated in a small amount of eluent for column loading to prepare a small volume and high concentration sample solution, and add it to the silica gel surface of the chromatographic column. If the sample is not soluble in the eluent used for loading the column, dissolve the sample in a volatile solvent, add an appropriate amount of silica gel (not more than 1/10 of the total amount of silica gel in the column) and mix with it, remove the solvent, evenly add the silica gel mixed with the sample to the top of the column (always maintain a certain level of eluent), and then cover it with a layer of silica gel.

During sample loading, slowly add along the inner wall of the column, and always keep the surface of the upper end of the silica gel flat; The loading amount is 1/60~1/30 of silica gel.


The selection of eluent can be selected through thin layer chromatography. Generally, the solvent system with Rf value of 0.2~0.3 during TLC development is the best elution system, which is eluted by gradient elution method.

First open the piston at the lower end of the column and keep the flow rate of eluent at 1-2 drops per second. The eluent is continuously added at the upper end (the addition speed can be controlled by the separating funnel to be close to the outflow speed at the lower end). If the elution effect of a single solvent is not good, mixed solvent can be used (generally no more than three solvents), and gradient elution is usually used. The elution ability of the eluent increases gradually from weak to strong.

Collection and processing

Collect eluent in equal parts, and the amount of each part is about the same as the amount of silica gel used. Each eluent shall be subject to thin layer qualitative inspection, and the eluents with the same composition shall be combined. A certain monomer can often be obtained through concentration and recrystallization. If it is still a mixture of several monomer components, it is not easy to precipitate the crystallization of monomer components. Further chromatography or other methods are required for separation.