Derivatives reaction vials

Derivatives may be formed prior to analysis in a tube or reaction vial, or some derivatives may be formed directly on the gas chromatographic column at the start of the analysis. Chapter 12 of this volume deals more extensively with this topic and the reader is referred to the appropriate section. 

A similar approach has been used for the synthesis of 7900 products derived from reactions of piperidone 6 (Scheme 2), of 7500 compounds derived from a piperazine template 9 (Scheme 3), and of 6000 products derived from 4-amino-benzylamine [8]. Thus, over 20000 compounds have been synthesized by using solution-phase chemistry and liquid-liquid extraction work-up procedures. For acylations, the authors used a work-up procedure that typically involved robotic addition of an aqueous solution of NaHCOj to the reaction vials, agitation, and robotic removal of the organic layer. Reductive aminations required a work-up that con-... 

After fixation of [ N]N2 for the specified times, the su ensions were extracted with 80% methanol and the extract subjected to sequential vacuum and steam distillation to recover free and amide-derived NHs. Cpm of in distillate was determined by scintillation spectroscopy, corrected (as was the A Ci added) to the time of the start of fixation and normalized to equal microcuries of [13N]N2 in the 1.0-mL reaction vial. 

Dimethyl Disulfide Derivatization. Dimethyl disulfide (DMDS) derivatization was performed on the unsaturated anacardic acids in order to locate the position of the double bond (2.). A solution of the anacardic acid (0.05-0.1 mg) was dissolved in hexane (1.75mL). DMDS (2.5mL) and iodine solution (0.25mL of a 60mg la/mL ether) were added to the reaction vial, and the reaction was allowed to run overnight at 40°C. After the reaction was complete, 5mL of a 5% (w/v) solution of sodium thiosulfate was added to the reaction mixture to remove the excess iodine. The organic layer was removed, and evaporated to leave the crude DMDS derivative, which was purified by hpic, before being submitted for mass spectrometric analysis. 

Figure 1. The effect of polytetrafluoroethylene on perfluoroacyl derivatization. The profile in curve A was obtained from derivatization of / -phenylethylamine (PE) with pentafluorobenzoyl chloride (PFBzO-Q) in an zill-glass system, that in curve B from the same reaction in a reaction vial sealed with a polytetrafluoroethylene-lined cap. Peak 1 PE derivative peak 2 derivative of the internal standard, tolylethylaminc (Ref. 13). Note the major peak of pentafluorobenzoic acid, the other by-products, and the reduced yield of derivatives in profile B.

Preparation of the TMS derivatives of nucleic acid bases, nucleosides and nucleotides [251] After thoroughly drying the sample overnight in a vacuum desiccator in the presence of P2O5, transfer a 50 pg portion to a suitable reaction vial, and add BSTFA containing 1% TMCS (40/il) and pyridine (10/il). Heat the tightly capped reaction vial at 100 °C for ih. After cooling to room temperature the reaction mixture can be... 

Figure 7 shows the spectrum obtained from the methyl ester of leucine-enkephalin. The ester was prepared by dissolving approximately 300 yg of dry peptide directly in 150 y of a fresh solution of HCl-saturated methanol (pH a 1). The reaction vial was sealed and heated at 42 °C for 3h, and the mass spectrum was obtained from I /A of the reaction mixture (a 2yg of the derivative) dissolved in glycerol. 

Trimethylsilyl derivatives Add 0.05 g sample and 0.5 g N,0- (trimethylsilyl) acetamide to a glass micro reaction vial of about 3 mL capacity. Maintain at 40 C for 15 min. For some compounds, higher temperatures and longer reaction times may be required. 60 C and 30 min is recommended for amine derivatives. 

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... 

Preparation of the TMS derivative Add 0.5 ml of TRI-SIL Z reagent (trimethylsilylimidazole in pyridine) to 1-5 mg of the sample. (This derivatizing preparation does not react with amino groups and tolerates the presence of water.) Heat in a sealed vial at 60° until the sample is dissolved. An alternate method is to let the reaction mixture stand at room temperature for at least 30 minutes (or overnight). This procedure is not appropriate for amino sugars. 

Microwave-assisted reactions allow rapid product generation in high yield under uniform conditions. Therefore, they should be ideally suited for parallel synthesis applications. The first example of parallel reactions carried out under microwave irradiation conditions involved the nucleophilic substitution of an alkyl iodide with 60 diverse piperidine or piperazine derivatives (Scheme 4.22) [76]. Reactions were carried out in a multimode microwave reactor in individual sealed polypropylene vials using acetonitrile as solvent. Screening of the resulting 2-aminothiazole library in a herpes simplex virus-1 (HSV-1) assay led to three confirmed hits, demonstrating the potential of this method for rapid lead optimization. 

GA and Cr, add 100 pi of PFBBr solution (7% v/v in acetonitrile) and 10 pi of tri-ethylamine, after which the reaction takes place at room temperature (RT) in 15 min. After the addition of 200 pi 0.5 M HC1, the derivatives formed are extracted with 1 ml hexane. The hexane layer is pipetted into a GC vial, blown to dryness at RT with nitrogen, and the dry residue is re-dissolved in 100 pi of hexane. 

HFB derivatives were also applied to the determination of phenols in water [12] at the 10 ng/ml level. A 25 ml sample of water was acidified with concentrated hydrochloric acid to pH 1 and 25 ml of benzene were added. The mixture was agitated for 15 min and allowed to stand until the layers separated. Portions of 2 ml of the benzene extract were dried by passage through a 5 cm X 5 mm glass column packed with anhydrous sodium sulphate. A 1-ml volume of the eluent was taken into a 4-ml glass vial and 5 pi of HFB— imidazole reagent were added. The vial was closed and the reaction solution was heated... 

The silylation of amino acids with BSTFA was studied in detail by Gehrke and coworkers [254—256]. BSTFA—acetonitrile (1 1) was applied first and fourteen amino acids were silylated at 135°C for 15 min. Glu, Arg, Lys, Trp, His and Cys, however, require up to 4 h, in order for measurable peaks to be obtained in the chromatogram. Despite such a long reaction, Gly and Glu gave two peaks and also it was difficult to separate the tris-TMS derivative of Gly from the derivatives of lie and Pro. The influence of polar and non-polar solvents was demonstrated later and was decisive mainly with respect to uniformity of the products. Only the bis-TMS derivative was produced in hexane, methylene chloride, chloroform and 1,2-dichloroethane bis- and tris-derivatives were produced in six more polar solvents. On the other hand, Arg did not provide any peak in the less polar solvents that were used and only one peak in the six more polar solvents. The best and most reproducible results were obtained when silylating seventeen amino acids with BSTFA—acetonitrile (1 1) at 150°C for 15 min 2.5 h at 150°C were necessary for the reproducible derivatization of Gly, Arg, and Glu. These reaction conditions were recommended for the analysis of all twenty amino acids. The TMS derivatives of amino acids were found to be stable on storing them in a sealed vial at room temperature for 8 days, with no decomposition. 

Trimethylsilyl derivatives are prepared by treatment with BSA alone [310,311] or with the addition of TMCS [312,314] in a suitable solvent (acetonitrile, pyridine, tetra-hydrofuran) or even without a solvent. For completion of the reaction, 10—20 min at 50°C are necessary [312], but as little as 30 min at 150°C has been reported for a stoppered vial with the use of a solvent [311], BSA alone can be used to advantage if pico-mole amounts are to be derivatized. The reaction products are said to decompose in dilute solutions even though pure BSA is used for dilution. At concentrations around 1 ng//d, up to 40% decomposition of the products is observed if diluted with BSA— acetonitrile (1 4), 100% decomposition occurs in 20 min. Of other silylating agents, e.g., HMDS and TMCS have been tested, but conversion into derivatives was not complete [311]. Silicone stationary phases of the SE-30, OV-1 and similar types have been used in the analysis. In most instances, temperature programming is required. Using the FID (in almost all instances), the detection limit is about 20 ng for T4 and 5—20 ng for T3, whereas with the aid of an ECD amounts about two orders of magnitude smaller can be detected [310,314]. Fig. 5.21 demonstrates a typical separation of five iodoamino acids and Tyr on 0.5% of SE-30. 

Gehrke and Patel [460] looked for optimal reaction and chromatographic conditions in the analysis of silyl derivatives of nucleosides prepared by reaction with BSTFA. They recommended performing the derivatization with a 225-fold molar excess of BSTFA at 150°C for 15 min in a closed vial and analysis on a 1 m X 4 mm ED. column packed with 4% of OV-11 on Supelcoport (100—120 mesh) with temperature programming at 5°C/ min from 140°C. 

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