Trifluoroacetates chromatography

Nonvolatile analytes must be chemically converted to a volatile derivative before analysis. For example, amino acids are not sufficiently volatile to analyze directly by gas chromatography. Reacting an amino acid with 1-butanol and acetyl chloride produces an esterfied amino acid. Subsequent treatment with trifluoroacetic acid gives the amino acid s volatile N-trifluoroacetyl- -butyl ester derivative. 

Conversion to acetates, trifluoroacetates (178), butyl boronates (179) trimethylsilyl derivatives, or cycHc acetals offers a means both for identifying individual compounds and for separating mixtures of polyols, chiefly by gas—Hquid chromatography (glc). Thus, sorbitol in bakery products is converted to the hexaacetate, separated, and determined by glc using a flame ionisation detector (180) aqueous solutions of sorbitol and mannitol are similarly separated and determined (181). Sorbitol may be identified by formation of its monobensylidene derivative (182) and mannitol by conversion to its hexaacetate (183). 

Ebumamonine was assembled utilizing a Pictet-Spengler cyclization of hydroxy-lactam 52 in the presence of trifluoroacetic acid at low temperature to give a mixture of diastereomers 53 in 95% yield. These compounds were readily separated by chromatography and the a-epimer was further elaborated to give the natural product. 

Figure 5.5 Trifluoroacetate determination in calcitonin acetate (a) without and (b) with heait-cut column switching. Reprinted from Journal of Chromatography, 602, S. R. Villasenor, Matrix elimination in ion cliromatography by heart-cut column switching techniques ,pp 155-161, copyright 1992, with permission from Elsevier Science.

An alternative technique to NMR spectroscopy is chromatography. The partially functionalized sample is completely fimctionahzed with a group different from the one present, the product carefully de-polymerized, its structure examined with a chromatographic technique. For example, partially substituted CA was further derivatized with methyl vinyl ether, the product hydrolyzed, the monomers produced examined with gas chromatography [241]. HPLC has been advantageously applied for the determination of substitution pattern for CAs with DS 0.8 to 3.0, by employing the same approach, i.e., further derivatization of the partially derivatized polymer with methyl trifluoroacetate, followed by de-polymerization. The results obtained by this technique compared favorably with those obtained by NMR [242]. 

It is noteworthy that trifluoroacetic acid was introduced as the most appropriate acidifler for column chromatography and solid phase extraction techniques, i.e., low boiling point due to its high acidity, requiring low amounts to reach the respective pH. Also, its high volatility allows easy evaporation thus minimizing the thermal load and acidification during concentration. 

Niki and Kuwatsuka reported a method involving trifluoroacetylation of the amino derivatives of chlornitrofen, nitrofen and chlomethoxyfen. A 1-mL volume of 10 M NaOH solution was added to 50 g of soil and the mixture was extracted with 100 mL of benzene. After separation and drying over anhydrous Na2S04, the benzene solution was trifluoroacetylated by adding successively 1 mL of 0.1% trifluoroacetic anhydride in benzene and 1 mL of 0.1% triethylamine in benzene. The mixture was shaken for 30 s and diluted to 10 mL with benzene. To remove the excess of trifluoroacetic anhydride, about 2 mL of water were added to the mixture and shaken for 30 s. The benzene layer was dried over anhydrous Na2S04 and injected for gas chromatography/flame ionization detection (GC/FID). 

Chloroform, sodium chloride, anhydrous sodium sulfate, sulfuric acid (97%), hydrochloric acid (36%), sodium bicarbonate, trifluoroacetic acid, tris(hydro-xymethyl)aminomethane (Tris), special grade Water, high-performance liquid chromatography grade 0.1 M Phosphate buffer solution (pH 7.0)... 

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

Peroxide ring closures were effected by stirring the 2,3-dibromocycloalkyl hydroperoxides with silver trifluoroacetate, and the bromo-substituted bicyclic peroxides were isolated by silica chromatography at —25 °C. Yields (based on 2-cycloalkenyl hydroperoxide) of 56 and 38% were achieved respectively for the [3.2.1]- and [4.2.11-compounds, but only 16% of the [2.2.1]- and 13% of the [5.2.1]-peroxide was obtained. The main reason for the low yield of the [2.2.1]-peroxide was that substitution by trifluoroacetate, which competes with the desired dioxabicyclization, is particularly prevalent with the 5-membered ring. 

Apffel, A., Fischer, S., Goldberg, G., Goodley, P.C., Kuhlmann, F.E. (1995). Enhanced sensitivity for peptide mapping with electrospray liquid chromatography—mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases. J. Chromatogr. A 712, 177-190. 

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

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