Micron Spherical Silica Gel Powder for Cosmetics Additive and Column Chromatography
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Application Examples of Column Chromatography Silica Gel

Extraction, isolation and characterization of small biological molecules from natural products is one of the main ways to obtain lead compounds in the development of new drugs. Chromatography is one of the most commonly used methods for the isolation of natural products. Chromatography was first used to separate some plant pigments based on the color of the compounds. Today, chromatography has evolved into a highly sensitive and effective method for separating and identifying compounds. Column chromatography generally includes atmospheric pressure column chromatography, medium pressure column chromatography and high pressure column chromatography. According to column packing, it can be divided into column chromatography silica gel, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, etc. Among them, column chromatography silica gel (normal/reverse phase) is the most widely used. Compared with other chromatography methods, column chromatography has the advantages of simple operation, economy and large sample carrying capacity. It is one of the preferred methods for the separation and purification of natural products.

1 Mix sample

Sample mixing is an important step in the silica gel dry sample loading for column chromatography. The amount of silica gel G used for mixing

the sample should be appropriate. Generally, the sample mass:silica gel G mass = 1:3 is appropriate. If there is too much silica gel G, the

column chromatography sample layer will be too thick, and each component will easily overlap. If there is too little silica gel G, the sample

will not be completely adsorbed in the silica gel G, resulting in serious sample loss. In addition, uneven sample mixing is also an important

factor affecting the experimental results. The specific methods are as follows:

1) Weigh 1g of crude extract, and weigh 3g of silica gel G into an evaporating dish according to the mass of crude extract: mass of silica gel

G = 1:3.

2) Completely dissolve the sample with organic reagents, add a small amount of the sample dropwise into the silica gel G in the fume hood,

stir the sample evenly with a medical spoon, wait until the sample evaporates to dryness, and continue adding the sample dropwise until the

sample is completely mixed into the silica gel G. middle. During this period, constantly stir the silica gel G with a medical spoon to prevent

uneven mixing.

3) Place the evaporated silica gel G in a fume hood overnight to allow the organic reagent to completely evaporate.

2 Packed columns

Column packing is the most important part of column chromatography silica gel. The quality of the column directly determines the separation

effect and yield of the sample. First, make sure the column is vertical, otherwise the sample band will be tilted during elution. In severe cases,

the elution product will be impure and require re-purification. Secondly, the silica gel G must be compacted without leaving any air bubbles,

otherwise the sample strips will be dispersed and affect the separation effect. Specific steps are as follows:

1) Mount the glass column vertically on the iron stand.

2) Weigh 40g of silica gel G according to the mass ratio of sample:silica gel G=1:40. Pour silica gel G into the silica gel column, tap the

column body to compact the silica gel powder, and continue to rotate the column during this period to make the silica gel G evenly filled.

3) Pour the mixed silica gel G into the silica gel column, tap the column body and rotate the column continuously to make the sample layer


4) Spread a layer of silica gel G about 0.5 cm thick on the sample layer to prevent the sample layer from splashing when adding

chromatography solution.


3 Sample reception

Prepare 3 times the column volume of chromatography solution and sonicate for 10 minutes. Open the glass column valve and slowly pour the

chromatography solution into the silica gel column until it is full. Choose a test tube of appropriate size to receive the effluent. Generally,

10~20 mL/tube is appropriate, and the flow rate is 1 mL/min. If the flow rate is too fast, the sample will be seriously tailed, and if it is too

slow, the sample band will be dispersed. If the polarity of the target product and the impurity are not much different, a gradient chromatography

system can be used. Gradient chromatography, also known as gradient elution, refers to using eluents of different polarities to elute samples

separately to achieve separation effects. Normal phase column chromatography silica gel If gradient chromatography is required, pour the

chromatographic liquid into the silica gel column in order from small to large polarity. Note that the difference in polarity of the chromatographic

solutions should not be too large, and before changing the polarity of the chromatographic solutions, drain as much of the chromatographic

solution above the silica gel as possible.

4 Detection and merging

Collect the effluent at 20 mL/tube, and collect 10 tubes in total. Perform TLC detection on these 10 tubes of effluent respectively. The developing

solvent is dichloromethane:ethyl acetate=9:1 (Figure 3). Combine tubes 1 to 4, tubes 5 to 7, and tubes 8 to 10, and rotary evaporate until dry.

Calculate the sample mass, which is 7 mg, 10 mg, and 4 mg respectively. Transfer to EP tubes respectively, number samples 1, 2, and 3, and store

at -20°C. If the sample volume is sufficient during merging, in order to ensure purity, the receiving solution with low purity can be discarded; if the

sample volume is small and insufficient for subsequent product identification experiments, all receiving solutions containing the target substance

should be combined and further purified.