Chromatography Forum

I have noticed that our BSTFA derivatized blanks, standards, and samples all have a significant stearic acid TMS peak when run on the GCMS. This is present in both SPE extracted and non-extracted samples. The neat standards/blanks we prepare only involve drying down a standard solution, reconstituting in BSTFA and ethyl acetate, and then injecting after derivatization. The peak is still there. Does anyone know where the stearic acid would be coming from? Gloves are always worn and we use disposable test tubes. It seems like it could be preventing the actual analytes of interest from being fully derivatized and therefore reducing sensitivity.

It might be carryover - is it still there if you run only solvent ?

Equally likely is that with your gloves on you touch bare skin, or something that has touched bare skin, so the outside of the gloves is contaminated.

Peter

Thanks for the reply. Our blanks are derivatized too, so I actually don't know what just solvent would look like. We'll give that a try today. I guess I figured the contamination was happening prior to or during the addition of BSTFA since the peak is stearic acid TMS, not just stearic acid.

We've observed this for years here, for palmitic and for stearic acid, thought it was mainly us with our type of business/products.

The retention times are also consistent with their TMS retention times. Since we inject solutions from bar soaps and consumer products, we feel we've contaminated the liner and/or beginning of the column with fatty acids and soaps, so when an injection that contains derivatizing agent hits that, then the TMS esters are formed.

To minimize this, along with trimming beginning of column and good scrubbing of the inlet, weldment, etc., sometimes we inject several times a mix of DMF containing 0.5% HCl and about 25% derivatizing agent, to acidify soaps/derivatize to clean out of the system with accompanying oven heating.

That is interesting - thanks. We are a toxicology lab and do not inject soaps. It is just extracted and derivatized blood/urine. We do trim the column often and recently did a thorough cleaning of one of our inlets and the peaks are still there.

Blood and urine: there you have your source of fatty acids and neutralized fatty acids.

It makes sense now - thanks. I'll try out the cleaning injections you recommended.

I see that too with all my derivatizing reagents (MSTFA, ethyl chloroformate) though both palmitic and stearic. I suspect perhaps some are coming from dishwashing detergent. I don't think it is in the liner as with ethyl chloroformate the reagent is neutralized through reaction with NaHCO3 before injection (it forms diethyl carbonate).

I checked the ingredients of our cleaning solution for glassware and didn't see anything that would indicate that being the source. Wherever it is coming from, it is already stuck in the system somewhere as blanks also show the peaks which haven't touched any glassware or sample matrix. When blank ethyl acetate is injected, the peak is essentially gone. As soon as BSTFA is introduced it is back. I figure the boiling point is so high it just sits there until it derivatizes and becomes more volatile.

The derivatives have higher MWs and lower vapour pressures than the native acids, but they are less adsorptively active. Which brings me to my next questions - what kind of inlet liner do you use, and when did you last do inlet maintenance ?

Peter

We change our liners/septum before the start of every new run. We did a thorough cleaning of the inlet using Agilent's procedure at the end of December. We use Agilent 4mm deactivated single taper liners with glass wool. The column on this same instrument was just changed on 1/14.

Wherever it is coming from, it is already stuck in the system somewhere as blanks also show the peaks which haven't touched any glassware or sample matrix. When blank ethyl acetate is injected, the peak is essentially gone. As soon as BSTFA is introduced it is back. I figure the boiling point is so high it just sits there until it derivatizes and becomes more volatile.

That is our thinking/experience as well.

The inlet maintenance sounds good.

Can you exclude the possibility that the BSTFA is contaminated ? Maybe try a fresh bottle ?

Peter

We use a fresh ampule for every run that we do, and have been seeing this since we started working on the Agilent instruments (~ 1 year ago). I think we have always used the same source of BSTFA. I suppose trying another brand couldn't hurt.

We use a fresh ampule for every run that we do, OK, not likely from the reagents then


and have been seeing this since we started working on the Agilent instruments (~ 1 year ago). aha ! From numerous posts on the forum it looks as if Agilents have a problem with persistent deposits in inlet plumbing, and flashback of vapours from the liner. Agilent users recommend cleaning or replacing the split vent lines and filter.

I think we have always used the same source of BSTFA. I suppose trying another brand couldn't hurt. I would try the inlet cleaning first

Peter