Column chromatography is found important in the labs due to its factors of easy packing procedure, low pressure of operation and low costs as well. Depend on the way that the solvent flows down the column, we can find two different kinds of column chromatography: one is gravity column chromatography when the solvent flows down the column by gravity, the other is flash column chromatography when the solvent is pushed down the column by positive air pressure.
In traditional column chromatography, we place a sample that is going to be purified or separated on the top of the column which contains silica gel or other solid support. After the column is filled with the solvent which runs through the solid support by gravity, the various components of the solvent will go through the column at a different speed. And we can collect them separately as we find them at the bottom of the column.
Compared with gravity column chromatography, flash column chromatography is basically an air pressure driven hybrid of medium pressure which can speed up the flow of solvent and save the time of sample purifying in flash chromatography. Usually, it takes less than 10-15 minutes to make the column and run the separation.
By using the injection formula, i.e. J = Z x 0.208% x n (1) the injection quantity is represented by J, Z is the silica gel filling quantity; 2) the difference of ORF is proposed to be accurate to 0.01, then the difference of 0.01 is set to 1 unit, which is represented by N; 3) the injection coefficient is 0.208%, which is based on the silica gel with 40-60UM particle size and 60A pore diameter, under the specific pressure condition and ideal flow rate, the ideal injection quantity is calculated Sample coefficient. Considering the factors such as uneven sample loading and large operation error in actual use, 0.2% is used instead, which is closer to reality.)
For example, how many samples can be separated by the 80g column in the following conditions? If the thin layer data: RF1 = 0.19, RF2 = 0.33, then n = (0.33-0.19) + 0.01 = 14, according to the formula, j = 80 x 0.2% x 14 = 2.24g, then according to the thin layer data, the maximum injection quantity of 80g column is 2.24g.
For another example, the thin layer data: how many samples can a column with RF1 = 0.25, RF2 = 0.21120g be divided into? N = (0.25-0.21) – 0.01 = 4, injection volume J = 120 x 0.2% x 4 = 0.96g.
It is not difficult to see that the smaller the ORF difference, the less the injection volume. In the process of experiment, we can choose which kind of column to use according to the separation degree (ORF difference) of target and impurity. Therefore, the injection formula can be optimized as J = zx0.2xorf, that is: injection amount = silica gel amount (actual grams) x0.2xrf difference.
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