Introduction to HPLC Columns
1. Type of column
Commonly used column packing agents are silica gel and chemically bonded silica gel, ion exchange resin, gel or glass microspheres. Among the chemically bonded silica gels, cetylsilane-bonded silica gel is most commonly used, followed by octylsilane-bonded silica gel, and cyano or amino-bonded silica gel is also used. In recent years, due to the rapid development of separation and purification technologies for biological macromolecules such as proteins, the application of ion exchange chromatography columns and gel chromatography columns has become more and more widespread.
2 Factors affecting column life
2.1 Mobile phase
When an aqueous solution is used as the mobile phase, microorganisms such as bacteria in the aqueous solution are easily grown. When the column is protected with an aqueous solution or an organic acid buffer, some mold may grow in the column and block the gap between the particles of the stationary phase. Due to the advent of wet filling technology, the commonly used column packings are generally less than 10um in diameter, and the particulate impurities in the mobile phase are easily deposited on the column head and then slowly block the column. The pH of the mobile phase also affects the column, especially for chemically bonded silica gel. The pH of the aqueous solution is between 2 and 7.5. When the pH is 8, the silica gel will release floccules and block the column. Recovery, the efficiency of the column is quickly reduced, or even completely failed. When an organic solvent such as methanol or acetonitrile is added to the buffer, the solubility of the salt is lowered, salt precipitation is precipitated, and the column is blocked. At the same time, the organic solvent and salt in the mobile phase corrode the sieve plate of the column head, resulting in a stigma depression. The polarity of the mobile phase also has an effect on the column. For silica gel columns, more polar substances such as methanol, water, and glacial acetic acid will destroy the filler. Conversely, for chemically bonded silica, the less polar substances such as n-butanol and dichloro Methane and the like also play the same role. The alkaline solution destroys the cation exchange resin column, and the acidic solution easily damages the anion exchange resin column.
2.2 Sample and stationary phase
If a small or small amount of sample is not able to dissolve in the mobile phase, the column will be blocked by the stationary phase. Particulate impurities in the sample can also block the column. When sample components such as sugars, proteins, etc. pass through the column, irreversible adsorption occurs, so that the active sites of the stationary phase are covered. The sample components also react with the stationary phase, especially for the amino column, because the amino group easily forms a Schiff condensation with the anhydride and the ketone to reduce the activity point and shorten the retention time. Column bonded phase group shedding, stationary phase decomposition, and reactive group denaturation as well as significant changes in column efficiency and retention time. Most of the bonded phases are bonded to the silicon substrate via a silicon-silicon bond (Si-O-Si) to form a stationary phase with organic groups, Si-O-Si-R (R is CnH2n+1), silicon. The siloxane bond may be hydrolyzed in an aqueous solution to break, causing the bonding group to fall off.