We have detected a low self-life of the chromatography column for cis/trans fatty acid methyl esters separation, and producers have said us it is because of the low pH of the injected solution. They also indicated that we should neutralize the solution before injection, but they have not said how.
Thank you.
Personally, I haven't experienced such a problem with the analysis of FAMEs with GC, mostly due to the need for extraction of analytes from liquid matrices into organic solvents in the presence of K2CO3 (strong alkaline solution) prior to GC analysis. However, columns for separation of FAMEs and especially separation between cis and trans, have polar stationary phases like DB-WAX. This columns are more sensitive and unstable compared to columns with non-polar stationary phases. I don’t have good insight into analysis of FAMEs with HPLC or LC, but I think you can simply use buffer solutions for neutralization.
use bicarbonate and separate the salts using a mild acidic resin
Thank you all for your answers.
Isaiah, thank you. I think I'll follow your suggestion.
André, thank you as well. We analyze the FAMEs by GC-FID-MS, and we have problems only with columns for cis/trans FAMEs separation capillary columns, but not with common capillary columns usually used for 22 main FAMEs separation. The methylation/transesterification protocol we use is like yours. But it seems that the pH of the final FAMEs solution in hexane is too much low for those especial columns used for cis/trans FAMEs separation, so we need to neutralize the acidity.
Many protocols for FAME analysis on GC-MS use the saturated NaCl : Hexane separation after the transesterification, as André explained. I understand the pH may be an issue for cis/trans FAMEs analysis, but did you perform the NaCl:hexane isolation step in specific? The dried hexane phase should be of low polarity, whereas the polar material is retained in the saturated NaCl aqueous phase (the phase we get rid off). Shouldn´t the H+ ions be retained/enriched in that aqueous phase? Maybe this is not a clear cut. I was just trying to understand this question, because I have isolated FAMEs this way in several experiments and did not encounter degradation issues.
José, thank you for the question.
We've analyzed several hundred meat samples separating trans-18:1 isomers using a 100m CPSil 88 column and hydrogen as a carrier gas. We methylate lipid extracts with 1.5M methanolic HCl under anhydrous conditions (1 h at 80C), then add water and extract FAME into hexane. We've never had a problem with column degradation due to acidity. As others have indicated, the HCl stays in the aqueous phase.
MetOH-HCl can be removed with a vacuum evaporation (HCL is gas), reconstitute ambient pressure with Ar or N2, repeat twice with MetOH, then extract FAME with hexane (for GC).
Usually it is not recommended to inject samples with acid in non polar silicate columns which are popular for analysis of FAME. The colums will bleed and have a short shelf-life as you observed. In addition, the traces of acid may encourage trans/cis isomerization leading to erroneous/non natural/artificial ratios. You can use weak acid resins in the esterification process to avoid all the problems you have listed
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