Chromatographic reaction spectra

The electrospray spectrum from the corresponding chromatographic response in the LC-MS analysis of the tryptic digest of the protein after reaction with the inhibitor is shown in Figure 5.24. In addition to the three species found in the digest of the parent protein, two additional polypeptides, with molecular weights of 2439.36 zb 0.07 and 2457.43 zb 0.02 Da, i.e. 70 and 88 Da above... 

The quantitation of products that form in low yields requires special care with HPLC analyses. In cases where the product yield is <1%, it is generally not feasible to obtain sufficient material for a detailed physical characterization of the product. Therefore, the product identification is restricted to a comparison of the UV-vis spectrum and HPLC retention time with those for an authentic standard. However, if a minor reaction product forms with a UV spectrum and HPLC chromatographic properties similar to those for the putative substitution or elimination reaction, this may lead to errors in structural assignments. Our practice is to treat rate constant ratios determined from very low product yields as limits, until additional evidence can be obtained that our experimental value for this ratio provides a chemically reasonable description of the partitioning of the carbocation intermediate. For example, verification of the structure of an alkene that is proposed to form in low yields by deprotonation of the carbocation by solvent can be obtained from a detailed analysis of the increase in the yield of this product due to general base catalysis of carbocation deprotonation.14,16... 

An increase in absorbance at 351 nm and a concomitant decrease in absorbance at 380 nm in the ultraviolet visible spectrum of methylcobalamin during the abiotic transfer of the methyl group to Hg2+ are characteristic for the loss of the methyl group and formation of aquocobalamin. In experiments monitored by both analytical techniques, gas chromatographic measurements of methylmercury formation were in good agreement with the spectropho-tometric measurement of aquocobalamin formation from methylcobalamin at 351 nm. Aerobic versus anaerobic reaction conditions had no measurable effect on either the methyl transfer rates, the stability of the reactants, or on the reaction products. 

A spectrophotometric method for determination of primary and secondary amines requires development for each particular compound, determining the kinetics of reaction of the amine with sodium l,2-naphthoquinone-4-sulfonate (143) and the UVV absorption spectrum of the product, under a set of fixed conditions. The procedure was applied to determination of ephedrine (30) and amphetamine (28) in pharmaceutical samples339. Reagent 143 in a FLA. system was used for the fast determination of lysine (144) in commercial feed samples by multivariate calibration techniques, without need of chromatographic separation340. 

Controlled release epoxy formulations in which tin is chemically anchored as tributyltin carboxylate to the polymer chain are discussed. NMR evidence is presented to establish that rapid exchange exists in tributyltin carboxylates. Consequently, even the interfacial reaction between tributyltin carboxylates and chloride is very fast equilibrium constants are reported for the reaction between tributyltin acrylate in hexane and sodium chloride in water. IR spectra, gas chromatographic retention time, chloride assay, and the complex intensity pattern of the molecular ion peaks in the mass spectrum show that the product of the reaction is tributyltin chloride, suggesting that it is the chemical species responsible for antifouling activity in marine environment. 

The various fractions of the forerun were analyzed employing a gas chromatography column packed with silicone gum, No. XE-60, suspended on Chromosorb P and heated to 248°. The components found (with the retention times indicated) were benzyl bromide (9.0 minutes), 2-methylcyclohexanone (5.3 minutes), and, in some cases, bibenzyl (22.6 minutes). The bibenzyl, formed by reaction of the benzyl bromide with the excess methyllithium, was identified from the infrared spectrum of a sample collected from the gas chromatograph. 

The uniquely distinguished axial alcohol at C5 was activated by triflation and the resultant triflate was displaced by the action of tetra n-butylammonium azide in benzene at room temperature. Compound 25 was thus available in 86% yield from alcohol 24. Hydrogenolysis ((H24 d(OH)2C) followed by acetylation afforded compound 26 in 96% yield. TTie richly detailed NMR spectrum (490 MHz) of 26 was identical in every respect with that of the L-antipode derived from reaction of NeuSAc, first with acidic methanol and then with benzoyl chloride under the influence of DMAP. The infrared and mass spectra, as well as the chromatographic characteristics of the two materials, were identical. 

It was not certain that the aquation product would be identical in spectrum to that obtained by thermal reaction, and analysis was therefore carried out chromatographically, rather than spectrophotometrically. The procedure was that described by Woldbye, highly standardized to ensure maximum reproducibility. Parallel separations were carried out on unirradiated solutions, and the chromatographic yields of the cis and trans were in some cases determined by using Cr5i... 

The epoxidation reaction was monitored by high performance liquid chromatograph (HPCL) on a reverse phase column. The reaction product was identified by IR, proton NMR and carbon-13 NMR spectroscopy. In the IR spectrum, absorption bands at 904 cm-1 and 842 cm-1 were attributed to the epoxy groups (12) peaks at 2.60, 2.78, and 3.15 ppm on the 1H-NMR spectrum were assigned to the three protons of the epoxide group (13) and in the 13C-NMR spectrum, the three glycidyl carbons were identified at 44.5, 50.6, and 70.6 ppm (14). 

The amino add analysis of all peptide chains on the resins indicated a ratio of Pro Val - 6.6 6.0 (calcd. 6 6). The peptides were then cleaved from the resin with 30% HBr in acetic acid and chromatographed on sephadex LH-20 in 0.001 M HC1. 335 mg dodecapeptide was isolated. Hydrolysis followed by quantitative amino acid analysis gave a ratio of Pro Val = 6.0 5.6 (calcd. 6 6). Cyclization in DMF with Woodward s reagent K (see scheme below) yielded after purification 138 mg of needles of the desired cyc-lododecapeptide with one equiv of acetic add. The compound yielded a yellow adduct with potassium picrate, and here an analytically more acceptable ratio Pro Val of 1.03 1.00 (calcd. 1 1) was found. The mass spectrum contained a molecular ion peak. No other spectral measurements (lack of ORD, NMR) have been reported. For a thirty-six step synthesis in which each step may cause side-reactions the characterization of the final product should, of course, be more elaborate. 

A gas-chromatographic analytical procedure is available (Ref 13). Mass spectra have been published for all the isomers (Ref 10), In all cases, initial reaction in obtaining the spectrum involves interaction between the methyl group and an adjacent nitro group, except in the case of the 3,4,5-isomer, which lacks this requirement. The isomers can be separated from each other by adsorption chromatography over silica gel (Ref 16), or by treatment of the mixt with aq urea (Ref... 

Method. To the residue in a test-tube is added 1 ml of 5% sodium hydroxide followed by 30 pi of anisoyl chloride. The tube is shaken for 1 min and left to stand at room temperature for 20 min in order to complete the reaction. At this time, 9 ml of water are added and the contents of the tube are shaken for 2 min. The mixture is then shaken with three 10-ml volumes of hexane. After centrifugation, the hexane solution is transferred quantitatively to a clean test-tube and evaporated to dryness. 1.0 ml of n-butyl chloride is added in order to dissolve the residue. An aliquot portion of the resulting solution is injected into the liquid chromatograph. The chromatographic apparatus consists of a stainless-steel column (2 ft. X 2.3 mm I.D.) which is packed with silica (particle diameter, 36-40 pm). The mobile phase is hexane-n-butyl chloride (11 9) at a flow-rate of 0.7 ml/ min. UV detection is carried out at 254 nm. The absorption spectrum of the HCP dianisate... 

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