Introduction

Trimethylsilyl (TMS) ethers are a convenient way to derivatize a variety of functional groups prior to GC analysis. The reagent we use is bis(trimethylsilyl)trifluoroacetamide (BSTFA). It will react quickly and quantitatively with alcohols, acids, and amines, and the by-products are volatile and can be injected directly into the GC without extraction. The primary drawback to this method is that the TMS derivatives are unstable, and will commonly last only a few days before significant hydrolysis becomes a problem.

Materials

• BSTFA (should be stored in freezer)
• anhydrous pyridine
• methanol, DCM (for rinsing)
• syringe with Teflon-tipped plunger (note A)
• heating block (65C)

Warnings

• BSTFA is toxic, corrosive, and smells horrible. Always wear gloves and keep it in the hood.
• Capped vials have occasionally burst while being heated. Keep the heating block in the hood with the sash lowered while samples are being heated.

Procedure

1. Transfer sample to a GC vial in an appropriate aprotic solvent (DCM, ether, hexane, etc.). Remember that aprotic solvents like methanol cannot be present, as they will react with the reagent. (note B). Add any internal standard.
2. Add 25 uL BSTFA and 10 uL pyridine (note C) to the sample. This amount is sufficient for a sample containing <100ug of total derivatizable material and dissolved in ~100uL of solvent. If your sample is bigger, you will need to scale up the amount of reagents being added, or take a smaller aliquot of your sample for the reaction.
3. Cap the vial tightly and heat at 65C for ~20 minutes. The heating step is simply to ensure that the reaction goes to completion, so the the exact time and temperature are not critical. You should not need to heat samples for any longer than 30 minutes.
4. Let the sample cool to room temperature then inject on the GC/MS. Store derivatized samples in the freezer to help lengthen their lifespan.
5. Important! BSTFA is very corrosive to metal syringe needles and plungers. Be sure to clean all syringes that come in contact with BSTFA thoroughly, first using methanol then DCM. This includes the autosampler syringe on the GC/MS.

Notes

1. BSTFA will quickly corrode syringe plungers, leading to a stuck syring. Using a teflon-tipped plunger will help to minimize this, as will careful and immediate cleaning of the syringe after every use. The glass capillary autopippetor is an even better option. It may be helpful to designate a single syringe which is only to be used with BSTFA.
2. The BSTFA itself can be used as a solvent with good success. In cases where minimal dilution is desired, the sample can be transferred to a GC vial using DCM, the solvent completely removed by evaporation, then the sample redissolved (with sonicating) in BSTFA + pyridine.
3. BSTFA will react very slowly with tertiary or otherwise hindered hydroxyl groups, such as those often found on sterols. The addition of anhydrous pyridine as a catalyst will greatly speed the reaction of these groups. Addition of pyridine for other compounds is not necessary, but for most samples the addition of pyridine can only help matters, thus its addition is included as part of the standard protocol.