BSTFA

For low molecular weight aliphatic acids, try TMSDEA reagent. Otherwise, use MSTFA, BSTFA, or TRI-SIL BSA (Formula P). For analysis of the keto acids, methoxime derivatives should be prepared first, followed by the preparation of the trimethylsilane (TMS) derivatives using BSTFA reagent. This results in the meth-oxime-TMS derivatives. 

MTBSTFA is the recommended reagent for silylating the amine functionality because it forms a more stable derivative than MSTFA. BSTFA, or BSA. Because amines can be difficult to silylate, the solvent used is important. 

Derivatives Add 0.25 ml of methoxime hydrochloride in pyridine and let stand at room temperature for 2 hr. Evaporate to dryness with clean, dry nitrogen. Add 0.25 ml of BSTFA, MSTFA, or BSA reagent and let stand for 2 hr at room temperature. 

Extract with three 1-ml volumes of diethyl ether (top layer) followed by three 1-ml volumes of ethyl acetate. Combine the extractions and evaporate to dryness with clean, dry nitrogen. Add 10 /jl of pyridine and 20 /d of BSTFA reagent. Cap the vial and heat at 60° for 7 min. 

Add 0.25 ml of DMF (./V,iV-dimethylformamide) and 0.25 ml of TRI-SIL TBT reagent to the sample in a screw-cap septum vial. If TRI-SIL TBT reagent is not readily available, add 0.25 ml of acetonitrile and 0.25 ml of BSTFA reagent instead. Heat at 60° for at least 1 hour for ribonucleosides or for a minimum of 3 hours for deoxyribo-nucleosides. After cooling to room temperature, inject 1-2 /a 1 of the reaction mixture directly into the GC. The resulting derivatives have been reported to be stable for weeks if capped tightly and refrigerated.1... 

Alternative silylating reagents such as N,0-bis(trimethylsilyl)acetamide 22a (BSA) [39-43], N,0-bis(trimethylsilyl)trifluoracetamide 22b (BSTFA) [44], or N,N-bis(trimethylsilyl)formamide 22c (BSF) [41, 46], in which the N- and O-trimethyl-silyl groups are in equilibrium [45] (Scheme 2.4), are much more powerful silylating reagents [40, 45] but are more expensive than FIMDS 2, because they are usually prepared by heating formamides or acetamides with TCS 14/triethylamine... 

This method requires about 40 g of tobacco which are extracted with ethyl acetate in the presence of ascorbic acid. A trace amount of C-NDELA is added as an internal standard for quantitative analytical work. The filtered extract is concentrated and NDELA is enriched by column chromatography of the concentrate on silica gel. The residues of fractions with p-activity are pooled and redissolved in acetonitrile. Initially, we attempted to separate NDELA on a 3% OV-225 Chromosorb W HP column at 210 C using a GC-TEA system with direct interface similar to the technique developed by Edwards a. for the analysis of NDELA in urine (18). We found this method satisfactory for reference compounds however, it was not useful for an optimal separation of NDELA from the crude concentrate of the tobacco extract (Figure 4). Therefore, we silylated the crude concentrate with BSTFA and an aliquot was analyzed by GC-TEA with direct interface. The chromatographic conditions were 6 ft glass column filled with 3% OV-... 

Acetone, Resi-Analyzed, J.T. Baker Acetonitrile, Resi-Analyzed, J.T. Baker BSTFA, Pierce... 

BSTFA should be used within 10 min after opening the ampule to ensure complete derivatization. BSTFA readily absorbs moisture, which will interfere with derivatization. 

If final sample solutions will be stored for several days, the derivatization of the HMS metabolite may reverse. If the derivatization has reversed, the HMS method recovery would be low and an additional broad peak (underivatized HMS) would be visible after the derivatized HMS peak. In this case, add 10 qL of fresh BSTFA to the final sample solution in the GC vial, vortex the sample for several seconds and re-inject the sample solution. 

BSTFA or BSA Reagents of choice for the formation of N-TMS derivatives. May promote the formation of enol-TMS ethers unless ketone groups are protected. 

BSTFA Produces volatile byproducts which do not interfere with the GC analysis of low molecular weight compounds. Reactivity is similar to BSA and is generally preferred to BSA for most applications. 

There seem to be few applications for which the use of weaX "silyl donors" is either necessary or desirable. Other imwrtant considerations for the selection of the correct reagents for a particular application are summarized in. Table 8.15. The strongest silylating reagent of all is a mixture of TMSIM-BSTFA-TNCS (1 1 1). 

The most general method for the simultaneous analysis of oxyanions by gas chromatography is the formation of trimethylsilyl derivatives. Trimethylsilyl derivatives of silicate, carbonate, oxalate, borate, phosphite, phosphate, orthophosphate, arsenite, arsenate, sulfate and vanadate, usually as their ammonium salts, are readily prepared by reaction with BSTFA-TMCS (99 1). Fluoride can be derivatized in aqueous solution with triethylchlorosilane and the triethylfluorosilane formed extracted into an immiscible organic solvent for analysis by gas chromatography [685). 

Repeating the reaction in HMDS without BSTFA gave 78% assay yield. The reaction could also be performed with BSTFA in other solvents. Of the solvents screened, dioxane gave the highest yields and cleanest reaction. The oxidation was also successful with amide 10 and ketone 31, as shown in Scheme 3.10. Reactions... 

Development of the dehydrogenation reaction There remained obstacles in making the dehydrogenation reaction practical for manufacturing. The reaction worked best in dioxane, not a suitable solvent for production. BSTFA and DDQ were both expensive and difficult to obtain in the purity required. Considerable effort was invested at this stage in search of a reagent/solvent system that was practical for scale-up. 

BSTFA provided the highest yield of product in comparison to other silylating agents. BSA [bis(trimethylsilyl)acetamide] reacted with DDQ. TMSOTf with luti-dine or collidine in toluene offered the best alternative but the yield was lower than the yield achieved with BSTFA. 

The reaction with valerolactam 24 was also investigated, with surprising results. The reaction with BSTFA gave the silyl lactam 78 rather than the silyl imidate 25, as shown in Scheme 3.33. Subsequent reaction with DDQ gave a C-N adduct 79... 

Physical chemical studies provided insight into the mechanism of the reactions and aided in optimization of key parameters. The order of the reaction in BSTFA,... 

The dehydrogenation reaction was generally monitored by taking samples for reversed phase H PLC analysis. Diode array detectors for H PLC were relatively new at that time and proved valuable for quickly getting structural information on products of the reaction produced under different conditions. Key reaction parameters for adduct formation, overall concentration, BSTFA, TfOH, and DDQ charges, were optimized using a thermostated HPLC autosampler to sample reactions directly for analysis. Comparison of reaction profiles provided rate and reaction time information that was used to select a more limited number of reaction conditions that were scaled up to compare yields. 

In the early 1990s, FTIR was being evaluated at Merck for the in situ monitoring of reactions. This new technology was expected to provide a powerful means to study a reaction as well as a method for analytical control in production [28]. Both silyl imidate formation and the reaction with DDQ could be conveniently monitored by FTIR, as shown in Figure 3.13. Silyl imidate formation was indicated by the appearance of an absorbance at 1667.5 cm4 with concomitant disappearance of the absorbance corresponding to BSTFA at 1324.0cm-1. A new absorbance... 

---