Hi Guys! Excuse my ignorance but I have little experience with RP-phases. I've managed to initially purify my peptide product using normal phase flash-chromatography. Unfortunately, Ihad to use a significant % of MeOH to elute my peptide. I achieved 95% purity however I'm certain that my product contains silica gel dissolved by MeOH and collected with the target fraction (the consistency of the solid material confirms that). Now I plan to purify it completely using a RP-HPLC (C18 column) instrument (H2O/AcCN eluents). Question is this: is the presence of normal phase silica gel a danger for the C18 column ?
Thanks for advice in advance.....
Michael.
Misinformation and poor terminology. *SILICA or silica gel will not "dissolve" in methanol. Unless it is treated with base, it will be suspended in the solution. If you have filtered the solution, then you should not have any particulate left (filtered out, as it will remove any fines left over) so will be fine to use the sample, dissolved in the mobile phase, for HPLC analysis.
- Michal asked: "Question is this: is the presence of normal phase silica gel a danger for the C18 column ?"
Answer: YES, any particulate matter left in your solution is a problem and must be filtered out before using a technique such a s liquid chromatography. HPLC analyis is not for solids or particulate matter and introducing such material may foul the column, plug the tubing or injector.
As far as I know, but I had never faced this problem, it should not be a problem. However, considering your sample, a guard column is recommended, as well monitoring sudden changes in pressure.
Install a guard column to avoid hypothetical damage to the C18 column.
To William Letter,
In my case, passing a sample through 0.45 um ptfe filter did not solve the problem with silica completely. Probably, some particles were smaller than pores of the filter.
Try a smaller filter, 0.22 u. If that does not work, than the problem is related to something else such as precipitation .... Unless high pH conditions are used (which may dissolve the silica and are generally not appropriate for HPLC use), silica should not be soluble in the mobile phase and easily removed from the sample. You may be precipating sample (or something else) out using a too high, organic solvent concentration. As you said, "I have little experience with RP-phases" and you need some formal, local, help with this.
If the silica is dissoved in methanol as a solution, no filter will help as it will go thru with the liquid. Silica is a backbone of the column, so it would not harm the column. The filter will get rid of any particulates, you can use the disposible column filter installed in front of the column if you don’t want to filter as the filter element may absorb your analytes. It will last many injection before it is clogged. Upchurch Scientific or Thermo is on of the supplier You may use centrifugation instead.
Thank you all very much. This is exactly it - I don't worry 'bout solid particles, it's been filtered, but yes - I was worried about the dissolved silica gel flowing through C18 phase. The injection will be also prepared in MeOH so everything will dissolve along with the gel. But If you Narong claim that nothing gonan happen than Im relieved - in the end - the eluent itself dissolves silica gel also so it will.....eluate.... hope so. Thanks all.
Thank you for answers. One adjunct today just suggested that a significant amount of silica gel can be filtered through a 0,22 um syringe filter even if dissolved in neat MeOH ??? is that true ?
Misinformation and poor terminology. *SILICA or silica gel will not "dissolve" in methanol. Unless it is treated with base, it will be suspended in the solution. If you have filtered the solution, then you should not have any particulate left (filtered out, as it will remove any fines left over) so will be fine to use the sample, dissolved in the mobile phase, for HPLC analysis.
- Michal asked: "Question is this: is the presence of normal phase silica gel a danger for the C18 column ?"
Answer: YES, any particulate matter left in your solution is a problem and must be filtered out before using a technique such a s liquid chromatography. HPLC analyis is not for solids or particulate matter and introducing such material may foul the column, plug the tubing or injector.
OK, William, now I understand - a suspension can be thoroughly filtered if only a fine-enough filter will be used. 0.22um filter suggest that we can even call it colloid. I think this misinformation issue is a common thing in academic education. But this clinic helped.
The way we stop misinformation in our field is to continue to ask questions (just as you did. Thank you.). Don't just accept information, question it to find the correct answer, then help spread the correct info to stop others from spreading misinformation, as appeared here.
Will do!!! My students will be corrected on Monday immediately! Anyone who will say "dissolved" after that will face firing squad.
I had a little trouble understanding how silica would dissolve in methanol. It is quite frequently used as an eluent in reverse phase chromatography. Silica fines on the other should be rinsed out before hooking the column (normal or reversed phase) up to a detector or introducing the sample by directing the outlet of the column to a waste cup for a half an hour or so.
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