Hydrophobic collapse and application of AQ chromatography column

Hydrophobic collapse and application of AQ chromatography column

As we all know, pure water does not wash your hair in life, because pure water will make the hair stick to each other, and you must use shampoo to separate the hair one by one in order to wash it thoroughly. This phenomenon also exists in reversed-phase high-performance liquid chromatography systems. When pure water systems (including pure water or pure salt solutions) are used as the mobile phase, the long carbon chains of C18 and C8 columns will choose to avoid water and stir to each other. At the same time, the retention capacity of the chromatographic column is reduced or even no retention. This phenomenon is called hydrophobic collapse. Although this situation is reversible (it can be washed with methanol or acetonitrile and other organic solvents), it will also cause certain damage to the column, so we will try to avoid using pure water as the mobile phase or using pure water to wash the column.

However, when HPLC analysis is performed on some substances with better hydrophilicity or greater polarity, in order to keep it better, the mobile phase of high water system has to be used. The mobile phase specified in the pharmacopoeia of samples such as kanamycin sulfate and gallic acid is the organic phase below 5%. Also, when doing some gradient experiments, the initial flow is too high compared to the water content. In these experiments, such high water content often leads to hydrophobic collapse.

Here is an example of fludarabine sample:

The phosphate and methanol used have a gradient, and the methanol content in the initial mobile phase is low. Therefore, the hydrophobic collapse phenomenon will be caused during the preview of the baseline of the equilibration column.

In order to solve these problems, it can be done by adjusting the mobile phase, but modern HPLC analysis is more inclined to solve the problem by improving the chromatographic column. These technologies generally exist as company secrets, and here is just an overview. According to the author's personal summary, there are roughly three types of this technology:

1. The surface modification technology of the silica gel substrate, that is, making an article on the silica gel substrate, and performing carbon hybrid modification on the surface of the silica gel substrate to make the silica gel surface more hydrophilic.

2. Amide group embedding technology, that is, making an article on the bonded phase, embedding the amide group on the end of the traditional C18 or C8 column at the carbon chain connected to the silica gel matrix, the amide group can form a hydrogen bond with water to form a Water film. This type of column not only prevents hydrophobic collapse, but also has better retention and better peak shape for samples containing nitrogen and hydroxyl groups, and even has different selectivity from traditional C18 and C8 columns.

3. The tail sealing technology of hydrophilic groups, that is, the article on the residual silanol of silica gel, using hydrophilic groups and residual silanol on the basis of traditional C18 or C8 columns to make the chromatography column better for polar hydrophilic samples This kind of column is a widely used C18-AQ (C8-AQ) column.

Our company's Comatex AQ column is a typical application of the third technology. It is suitable for hydrophilic samples with high water mobile phase, such as kanamycin sulfate, caffeine metabolites, and water-soluble vitamins.