Chromatography Forum

I am doing my project on derivatizing the acids compounds by BSTFA/pyridine. Including injector, column and also the MS detector are renewed just before I start my project, the standard calibration curves look very ideal and reasonable. However, when I start doing the real samples, there occurs a big solvent peak even after 10mins solvent delay. And I cannot find any target compounds. I tried the two standard calibration levels later and I cannot find any peaks at the lowest concentration level. The absolute abundance is not good for the higher concentration level when compared with the previous result.

I am worried whether there are anything wrong with the sample collected on filter paper(e.g. yeast and bacteria grow on the filter paper) and whether those contaminants could quickly deterioate my column.

Any crude biological sample has large quantities of things that you do not want in your GC column, and some kind of sample cleanup is nearly always necessary.

Have you done intlet maintenance since the problem appeared ? - it is more likely that the inlet is contaminated than that the column is, but if you carry on injecting dirty sampels the column will suffer also.

Peter

Would you mind suggesting some ways for me to solve the current situation? I have asked the university technician and he doesn't agree with trimming the new column.

I am not sure about whether there is any significant amount of biological samples present on the filter paper as I only use methanol for extraction. I am not sure the extracting efficiency of methanol is high enough to extract those things.

Have you encountered any similar situation? How can I decide the broad solvent peak may due to the presence of biological samples or its metabolites?

eunicewg,

The university technician is wrong. Trimming the column is part of routine maintenance. Sometimes daily, sometimes not so often depending on what you are running. Also, you certainly need to have a look at the liner to see what kind of shape it is in. BSTFA/pyridine is a nasty combination for inlets in my opinion.

Ten minute solvent delay and you still see a solvent peak suggests something else is wrong. Are you sure it is methanol? I would be surprised you are scanning that low since you would also be seeing nitrogen.

Best regards.

AICMM,

After my extraction by methanol from filter paper, I dried my sample under ultrapure nitrogen before both BSTFA and pyridine is added. After the addition, the mixture is sealed and heated at 70 degree for derivatization. I apply this technique in both calibration standards and real samples, however, the broad solvent peak only appears in real sample but not in the standards. I am not sure what it really is.

Would you mind suggesting some ways for me to improve the current situation, for example, maybe injecting something that may help the column?

Regards,
eunicewg

Eunicewg,

Clean your inlet. Replace your liner and gold seal. Make sure your column is in the right position in the liner. Try the low level calibration. Doesn't work, trim a meter off the front of your column. Yes, a meter. Try your low level calibration. Doesn't work, reverse your column. Try your low level calibration. Doesn't work, buy a new column and hold on to your old one. Doesn't work, you are now the proud owner of one new and one suspect column. If you immediately toast your new column, take a serious hard look at your sample prep and what may be causing the column to toast. If your old column is toasted, do the whole world a favor (okay maybe not the whole world but there are lots of people who will understand this comment) and throw the old column AWAY.

In a university situation where a technician is telling you not to trim, I have to wonder what the history of this column and inlet are. Some people suggest rinsing but that is, in my opinion, only useful when you have a good handle on the column history.

Problems like this are never easy, sorry. Divide the system in half and keep plugging away.

Best regards.