HPLC analysis conditions, reaction

The subsequent Claisen-Schmidt reaction was originally performed on a 10-pmol scale using 20-fold excess of both acetophenone and LiOH to achieve complete formation of the chalcone 8. This result could be verified on a small scale however, employing the same conditions on a 35-mmol scale resulted in no conversion even after 22 h, as revealed by IR spectroscopy. By cleaving a resin sample with 20% TFA in dichloromethane, only -formylbenzamide 11 was detected by HPLC. This result may be explained by the low solubility of LiOH in DME under dry/aprotic conditions. Therefore, a small amount of EtOH was added, which initiated a fast reaction (Chiu et al. 1999) and the formation of the desired chalcone 8 together with 20% of the Michael adduct 10 (Fig. 2). This was confirmed by sample cleavage from the resin and LC-MS analysis. Short reaction screening resulted in considerable im-... 

Batch conditions are used with a ratio enzyme/substrate of 2 (weight), during 1 to 24 hours at 25 °C. Progress of the reaction is measured by proton NMR with an internal standard, and enantiomeric excess is obtained through chiral HPLC analysis of the product. 

Using similar reaction conditions with rapeseed oil, fatty acids were treated with various supercritical alcohols. From the HPLC analysis, it was shown that selective reactions could be obtained. Figure 5 presents the yields of alkyl esters of five fatty acids treated in various supercritical alcohols at 300°C. In the case of methanol, the reaction time for the complete conversion... 

The chiral anisole derivative 37 has been used in the synthesis of several asymmetric functionalized cyclohexenes (Table 9) [22]. In a reaction sequence similar to that employed with racemic anisole complexes, 37 adds an electrophile and a nucleophile across C4 and C3, respectively, to form the cyclohexadiene complex 38. The vinyl ether group of 38 can then be reduced by the tandem addition of a proton and hydride to C2 and Cl, respectively, affording the alkene complex 39. Direct oxidation of 39 liberates cydohexenes 40 and 41, in which the initial asymmetric auxiliary is still intact. Alternatively, the auxiliary may be cleaved under acidic conditions to afford /y3 -allyl complexes, which can be regioselectively attacked by another nucleophile at Cl. Oxidative decomplexation liberates the cyclohexenes 42-44. HPLC analysis revealed high ee values for the organic products isolated both with and without the initial asymmetric group. 

The oxidation reactions were performed in a glass batch reactor, equipped with magnetic stirrer (mechanic for L-sorbose oxidation), reflux condenser and thermometer. The reaction conditions are summarized in Table I. Before reaction the catalyst was pre-reduced in situ in a nitrogen atmosphere ( 20 min) with the alcohol reactant in 30-40 ml alkaline water (and dodecylbenzenesulfonic acid sodium salt detergent for water-insoluble reactants). The reactor worked in a mass transfer limited regime, controlled by the air flow rate (7.5-20 cm3min 1) and the mixing rate (1500-1800 min 1). The reactions were followed by GO or HPLC analysis. 

It is important to determine early on whether the reaction conditions previously developed for the assay of a given activity can be adapted for use with HPLC assay. For example, is the reaction mixture of sufficient volume to permit the withdrawal of multiple samples For assays carried out in volumes of a few microliters, it is virtually impossible to withdraw samples of sufficient volume for analysis on the HPLC system. Thus, unless dilutions can be made after sampling, HPLC analysis must be ruled out in such cases. 

Figure 9.146 HPLC analysis of PAP in an aryl sulfotransferase IV reaction mixture. The reaction mixture and incubation conditions were 50 fiM 1-naphthalenemethanol and 2.9 fig of AST IV. The mobile phase for HPLC analysis contained 12% methanol in 75 mM potassium phosphate (pH 5.45), 100 mM ammonium chloride, and 1.0 mM 1-octylamine. The flow rate was 2.0 mL/min, and detection was at 254 nm, with a full scale sensitivity of 0.02 AU. Sample injection is indicated by an arrow. (From Duffel et al., 1989.)...

In addition, sometimes a normal-phase HPLC method at subambient temperature must be applied for analytes that are extremely prone to hydrolysis. In the synthesis of leukotriene D4 antagonist, accurate quantitation of mesylate intermediate is essential for process optimization. Owing to its inherent instability, analysis of mesylate intermediate must be carried out under normal-phase conditions with nonprotic solvents however, significant cycliza-tion of mesylation was stiU observed in such condition at room temperature. The authors concluded that the on-column reaction of the mesylate was silica-catalyzed cyclization. By conducting the normal-phase HPLC analysis at -30 C, it was demonstrated that on-column cyclization was adequately inhibited [30]. 

Prior to the incorporation of protected Cys residue into polymers, the stability of the Npys group was studied in the presence of pentafluorophenol. This was caused by the formation of this compound as byproduct during the reaction between Boc-Cys(Npys)-OPfp and the a-amino groups of the N-terminal amino acids of branched polypeptides. In a model experiment, Boc-Cys(Npys)-OH was kept in DMF-water (9 1, v/v) mixture in the absence or presence of pentafluorophenol. Based on HPLC analysis we found that the Npys group is stable under these conditions. 

The mathematical treatment of FMC data can be accomplished by standard procedures via the solution of mass balance equations, on condition that the data were converted to reaction rate data with Eq. (21). As mentioned above, this requires the determination of the transformation parameter a. Two approaches based on calibration were developed and tested. In the first approach, thermometric signals are combined with the absolute activity of IMB, which had been determined by a separate measurement using an independent analytical technique. Figure 5 shows a calibration for the cephalosporin C transformation catalyzed by D-amino acid oxidase. The activity of the IMB was determined by the reaction rate measurement in a stirred-tank batch reactor. The reaction rate was determined as the initial rate of consumption of cephalosporin C monitored by HPLC analysis. The thermometric response was measured for each IMB packed in the FMC column, and plotted against the corresponding reaction rate. From the calibration results shown in Fig. 5 it can be concluded, independently of the type of immobilized biocatalyst, that the data fall to the same line and that there is a linear correlation between the heat response and the activity of the catalyst packed in the column. The transformation parameter a was determined from... 

Similar results as just described can be obtained by changing the ratio of Cu /Cu2 or even by using only Cu or only Cu, as outlined in Table 20.1, although some change in the distribution of the products has been observed. If the polyynes solution in heptane is left in contact with the aqueous solution of copper chlorides under acidic conditions for one week, a plethora of new products can be detected by HPLC analysis. Probably, coupling reactions and intramolecular cyclization as well as addition reactions have taken place. 

Figure 6. Membrane assisted condensation of amino acids. HPLC analysis (at 289 nm) of the products of the oligomerization of NCA-Trp assisted by POPC liposomes. There are several products (for reaction conditions and other details see ref. 21). It is important to notice the difference between the liposome-assisted oligomerization (A) and the control experiment, in the absence of liposomes (B). In this second case, the highest product has a oligomerization degree n = 7 in the case of liposomes we reach n = 29, although in minimal amounts.

It is clear now that irradiation of phenyl azide at room temperature gives dehydroazepine. At high concentration of azide, the dehydroazepine polymerizes rapidly in competition with its slow isomerization to triplet phenyl nitrene. The major product formed from photolysis of phenyl azide under conditions where its quantum yield for disappearance is claimed to be greater than unity is poly-1,2-azepine [48], not azobenzene. Of course, the polymer does not elute from an HPLC, and analysis of reaction mixtures by chromatography will show only two components. 

The partition value p can be determined by differnt methods l107 109l. In the presence of a large excess of nucleophile ([N] [A]0) the decrease in the nucleophile concentration during the reaction course can be ignored. Under these conditions vh/ vA = [fy/Iffi]- The determination of p can be established out from the product ratio obtained by HPLC analysis according to Eq. (7). 

We have observed that T.B.T.U. gives a little dehydration of the side chain of asparagine. This side reaction has been confirmed by synthesis of an authentic cyanoalanyl peptide and HPLC analysis. Optimisation conditions have been found to decrease the level of this side reaction low temperature, minimisation of the quantity of diisopropylethylamine used during the coupling step and use of hydroxysuccinimide ester of asparagine as the activated aminoacid. The cyano alanyl peptide can be eliminated by preparative HPLC at the end of the synthesis. 

Figure 21.6 shows the kinetic profile under the actual process conditions. The reaction profile (combination of the anion and free acid of 3 versus time) obtained by high-performance liquid chromatography (HPLC) analysis also matched the onfine IR data. Again, the use of online IR coupled with principal component analysis provided the means to profile HA and A during the reaction. Formation of the free acid form 3 (HA) was immediately observed upon addition of a catalytic amount of TFA. This clearly shows that constant liberation of the free acid form HA... 

A series of rapid chemical quench experiments under single enzyme turnover conditions using radiolabeled S3P or PEP revealed that the tetrahedral ketal phosphate enzyme intermediate was formed as a new peak upon HPLC analysis with detection of the radiolabel. The time course revealed that the formation of the tetrahedral intermediate species paralleled the disappearance of PEP substrate and formation of the EPSP product thus establishing that it was a kinetically competent species. Isolation of the tetrahedral ketal phosphate intermediate using C-2 PEP and S3P as substrates coupled with rapid chemical quench was carried out in conjunction with H-, C-, and P- NMR to provide a definitive structure proof Thus with these studies we have satisfied the criteria for a true reaction intermediate in terms of a chemically plausible mechanism, structure proof, and kinetic competence. Additional studies support the mechanism for EPSP synthase described (Scheme 4, pathway a) including observation of the intermediate bound to the enzyme at internal equilibrium using solution NMR and C-2 PEP as well as using rapid freeze-quench/solid-state NMR studies. ... 

Titration curves for rosmarinic acid in (he presence of different concentrations of iron ions indicate a stoichiometry of 1 to 1 for the complex. These data were confirmed by elemental analysis and inductively coupled plasma emission spectrometry (70. This 1 to 1 complex seems to be the only absorbent species in the reaction media. Support of this assertion come from graphical analysis of the consecutive spectra according to Coleman et al. (Jd) and from HPLC analysis of the reaction media (Figure 6). From this figure, it can also be deduced that, under the assay conditions, almost all rosmarinic acid is forming part of the complex, since absorbance at 333 nm for the expected retention time of rosmarinic acid is very low. 

Treatment of (C59N)2 with diphenylmethane under photolytic conditions at room temperature was expected to produce 37. However, after five hours of irradiation, HPLC analysis of an aliquot of the reaction mixture showed no formation of 37, but instead revealed the formation of material with proposed M-oxide structure 38 (25%), next to recovered (C59N)2 (75%). It was assumed that trace amounts of 62 initiate this conversion (Fig. 20). Because fullerenes are excellent sensitizers for the conversion of triplet to singlet oxygen, any oxygen in the reaction flask is converted to 62 under these reaction circumstances [66]. 

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