Chromatography Forum

Hi,

I had derivatised my sample using BSTFA in pyridine. Realising I need to dilute my solution, I added in a solution of BSTFA/pyridine (3:2) into the vial to achieve the dilution factor I need.

I ran my samples over a few days (long sequence) and realised the signal intensity and area for the analyte is very much lower, but the rest of peaks in the chromatogram has very much higher intensity.

Would the excess BSTFA be the reason for this?

Regards,
Wan

The answer is: could be.

The excess BSTFA will pass through the column - and can give some degredation products. So, if you are looking at that portion of the chromatogram, yes.

If you have derivatized a biological sample, injected it, diluted it and injected it again, you may see the result of slower derivitization of some specides present, For example ketones can form enols. While equilibrium far favors the ketone form, the enol is derivatized as it forms, so over the space of hours and perhaps even days, the peak for the enol grows into the chromatogram. If you are looking at these compunds - possibly not.

There is a nice paper by J. Little describing artifacts formed in derivatization reactions. I ran across it esterday, but do not have the full reference with me - it is at work... (I am at home.)

Hi Don,

Thank you for your reply.

I ran a search and believe the article ref is [J.L. Little, J. Chromatogr. A, 844 (1999) 1-22]?

I will try dilution with only pyridine and check back again.

Thanks again!

Regards,
Wan

That is the reference.

Hi Wan, I strongly recommend using a retention gap before your GC column when injecting derivatitation agents directly. It helps protect your analytical column since those agents can literally wash out the stationary phase. Also, if it is feasible for your compounds try MSTFA which is supposed to be more “column-friendlyâ€

I've used excess BSTFA when injecting samples for over 30 years in GC work, including 20 years with Agilent GCMS, and not noticed any issues. I'd say half our GCMS work involves BSTFA derivatization. We typically use DB-1 and DB-5 type columns when we derivatize with BSTFA.

Hi,

We realised it might not be the problem with BSTFA/pyridine. We'll try cleaning the ion source today and see if that helps.

carras: If the stationary phase is washed out by excess BSTFA, won't the retention time be more affected than the signal intensity? If so, I did not notice significant changes in the RT, but more of the abundance.

Consumer Products Guy: We're using DB5 and an Agilent GCMS, so I guess I can safely say, given your experience, it might not be the problem of excess BSTFA.

Thank you so much for all the comments/help! I'll get back after we clean the ion source.

Regards,
Wan

If you are running biological extracts, I would suggest using an inlet liner with a large plug of deactivated glass wool. And change the liner frequently. The problem with a column is accumulation of stuff on the column head. And with the glass wool plug to collect nonvolatile material, the column lasts longer. Select your derivatizing agent on the ability to do the job. BSTFA is considered to be a stronger derivatizing agent under some circumstances.

You might want to solvent wash your injection port shell every once in a while. Agilent has an article about injection port maintence on their web site that details the process.
I've done it and it works. Note to protect the EPC remove the split vent line to start with.

Hi,

We've changed the ion source and that seemed to help thus far.

Don: Could you elabortate on "BSTFA is considered to be a stronger derivatizing agent under some circumstances."? Our samples are food extracts. We had cleaned up the samples using liquid-liquid extraction and SPE.

Bigbear: Thanks for telling. I'll go look up on that.

Regards,
Wan

On strength of derivatizing agents, I'll have to direct you the literature. The various vendors have application notes and there are discussions in published papers. The last place I recall BSTFA being described as a stronger derivatizing agent is in the paper by Little, mentioned above.

You have to try these out for your application, however. Each application offers unique challanges.

Hi Don,

Thank you for your reply. I'll go read up on those. Thank you again!

Regards,
Wan