First, the sample aspect
1. Remove particles and impurities
2. Understand the solubility of the sample in the mobile phase
If the solvent of the dissolved sample is not the mobile phase, the mobile phase must be tested for solubility to prevent precipitation in the mobile phase.
3. Understand whether the sample interacts with the matrix/filler of the column.
Second, the mobile phase
Remove particles
Purity requirements: ultrapure water, the content of organic impurities in the water is low during the gradient test; the pH value of the buffer is within the allowable range of the filler; the concentration of the buffer (salt); the organic solvent: chromatographically pure, the impurity content is as low as possible, And match the filler
2. Flow relative to the solubility of the sample
Organic solvent or water ratio
Third, the column cleaning
Have a good understanding of the sample you are doing, and clean it with the mobile phase that has the strongest elution ability.
The general method of the silica gel column is to first wash away the polar impurities with methanol, and then sequentially activate with dry dichloromethane and n-heptane 100~200ml.
The general method of bonding a phase column (alkyl group) is to sequentially rinse with 20 volumes of methanol-chloroform-methanol-water.
(Remember: There are specific methods for each column, so be sure to read the instructions first!)
Fourth, the storage of the column
Pre-storage treatment to remove impurities, buffer salts
Suitable storage solvent
Avoid drying up the column bed
Avoid mechanical vibration
Prevent bacterial growth
Pay attention to the stored temperature
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