Hydrolysis stability of reverse fillers
Q: Some manufacturers say that the pH of their columns can be 9 or 10, while others suggest that it should not be greater than 8. Recently, I have a reversed-phase column to use PH9, because only under this condition can my sample be completely separated. This is beyond the scope of application stated by the manufacturer, but the service life of the column is acceptable. Now I want to know how we should treat the pH application range recommended by the manufacturer.
Experiments have come up with the best answer:
If the column life is acceptable then it can be used outside the recommended pH range. But I wonder if the results are the same with different brands of columns. If it is, then there is no problem with using it this way.
The pH stability of fillers is a complex problem, which is difficult to be explained by a simple rule. Let me explain it in some detail just to make sense of it.
In alkaline pH, OH- attacks and breaks down silica gel. The decomposition rate is related to the concentration of OH- in the mobile phase, the channel of OH- to the packing surface and the solubility of dissolved silica gel in the mobile phase. As you can see, the pH concentration in the mobile phase is only one factor. And all of this is temperature-dependent. It may work well at room temperature, but column life may be significantly reduced at 60 ° C.
OH- to packing channel plays an important role in the stability of packing, packing surface covered with dense C18 or C8 can improve the stability. End sealing is also important. The surface of the packing is covered with hydrophobic groups to protect the packing from OH- attack, and its density is a ruler to measure the protection ability. Therefore, we can say that the packing with high surface coverage is more stable than the packing with low surface coverage. In addition, the quality of end sealing is also very important.
At acidic pH, silica gel breaks down on its own. Thus, the properties of the bond play only a secondary role. At the same bonding level, there is no difference in the stability of single-function silane and triple-function silane. However, the OH- to filler channel is the most important, so the stability of the large isopropyl side chain of the single-function bond is weaker than that of the standard bond phase because of its low maximum coverage.
If the column has been using the same mobile phase without washing with organic solvent, the desorption and decomposition of the bonded phase is very slow, so the retention time does not change. But the silica gel is slowly decomposing. As a result, the column may collapse suddenly without warning.
Of course, the packing density is also very important in this case. Silica gel with large pore volume is not as stable as silica gel with small pore volume because its skeleton is more fragile. The porosity of silica gel is generally 40% – 70%, but its strength changes 10 times. Therefore, the difference of bonding phase can be inferred according to the filler density. In addition, as the pore diameter of the filler becomes larger, the surface area will decrease. Therefore, if other conditions are consistent, the filler with large pore diameter is more stable than that with small pore diameter.
The characteristics of mobile phase components are also very important to the stability of the filler. At the same pH, organic buffers such as aminobutanol buffer [Tris: (hoch2) 3cnh2], citric acid buffer and hydroxyethyl piperazine ethylthiosulfonic acid (HEPES) buffer are less aggressive than the commonly used phosphoric acid buffer. In addition, glycine and boric acid are very mild even at pH 1 0.
It should be pointed out that the known theoretical studies on the stability of fillers are carried out under equal degree conditions. When you change to organic solvent to clean the pollutants in the column, the groups adsorbed on the filler without bonding may also be washed off. Therefore, the cleaning process will also affect the stability of the column.
The above studies are for C18 and C8 columns. Many polar columns, such as CN based columns, have much less stability even in normal operation. At pH 7, the hydrolysis rate of CN based fillers is 1000 times that of C18 and C8 fillers.
In this way, as long as the operation is reasonable, the service life of the column is OK even if it exceeds the recommended pH range. The most stable column is the matrix of high-density silica gel, bonded with high-density C18 or C8, and sealed at the end. The properties of mobile phase components have a great impact on the service life of the column, which should be carefully selected. However, if the analysis is needed and the life of the column is acceptable, then dare to challenge the limit of the column!
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