Chromatography Forum

Hi

I analyse soil lipid extracts by GC and typically methylate samples first with diazomethane (to methylate any acids) before drying and treating with BSTFA (+1% TMCS) 100 ul, a couple of drops of pyridine and some DCM (if needed to solublise the sample) and reacting at 60oC for 1.5 h to silylate any alcohols.

The problems i have been experiencing are twofold;

1) Sometimes, upon drying the sample under N2 to remove excess BSTFA, white crystals form of what i can only assume to be polysiloxanes. These do not dissolve well in DCM. Has anyone else experienced this problem and know why it happens and how it can be avoided?

2) when re-silylating a sample for a repeat analysis where, in the first analysis all my fatty acids were present in the GC chromatogram as single peaks corresponding to their methyl esters, i get the appearance of additional silyl esters analogues. I am sure that this is not down to incomplete methylation of the sample, however, trans esterification of FAMEs seems unlikely without a considerable kick from some acid. Does anyone have any advice to offer on this problem?


Thanks,

Matt

We use BSTFA for the cholesterol analysis. We do not remove the BSTFA (which is an acid).

procedure:

Use internal standard.
Evaporate solvent from sample extract.
Add 200 µL pyridine.
Add 100 µL BSTFA.
React for 60 min. in a waterbath at 80°C.
Let cool and inject directly into GC.

I've used BSTFA for over 30 years. Not once have I ever tried to remove it.

Thanks

I did try analysing my samples with the BSTFA still present at first, but this caused some problems with fouling of the FID detector and the front ends of the GC column which needed trimming more often. Furthermore, samples which have been stored a while show evidence in the GC chromatograms of polysiloxanes forming over time.

Hello.
1) How do you make the extraction from the soil? Details of extraction is not described , can be a mystery. Maybe it's the sand does not dissolve.

Something may be fouling the head of your column, but I don't think it's the BSTFA. I run an awful lot of BSTFA injections without problems. It's likely something in the sample itself. The FID can get fouled with silica, but it's usually not too bad if you keep the detector temperature high enough. The HF produced when burning BSTFA in the FID is supposed to help with cleaning; not sure if that's just marketing.

Something may be fouling the head of your column, but I don't think it's the BSTFA. I run an awful lot of BSTFA injections without problems. It's likely something in the sample itself. The FID can get fouled with silica, but it's usually not too bad if you keep the detector temperature high enough. The HF produced when burning BSTFA in the FID is supposed to help with cleaning; not sure if that's just marketing.

Agree with Dave H