HPLC chromatographic

Distribution of benzodiazepines in I-octanol - water system was investigated by a direct shake flask method at the presence of the compounds used in HPLC mobile phases the phosphate buffer with pH 6,87 (substances (I) - (II)), acetic and phosphate buffer, perchloric acid at pH 3 (substances (III) - (VI)). Concentrations of substances in an aqueous phase after distribution controlled by HPLC (chromatograph Hewlett Packard, column Nucleosil 100-5 C, mobile phase acetonitrile - phosphate buffer solution with pH 2,5, 30 70 (v/v)). 

HPLC is often reported to be the technique of best choice for the quantification of food colorants. According to European Directive 94/36/EC, the quantities of synthetic colorants to be added to foods are restricted and thus reliable methods for their quantification must be established. Approved colorants, defined by E-coded numbers (Table 6.6.2), are permitted for non-alcoholic beverages, confectionery products, and even for caviar (dying fish roe). For example, a specific HPLC chromatographic method for the quantization of 14 synthetic food colorants belonging to azo dye, triphenyhnethane, or quinophthalone classes (E 102,104, 110, 122,123, 124, 127, 128, 129, 131, 132, 133, 142, 151) was reported to check their contents in caviar. ... 

Chromatographic retention times of 23 doubly substituted chalcones, as determined by 8 HPLC chromatographic methods using heptane as the mobile phase. The methods differ by addition of 0.5 percent of a chemical modifier. 

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... 

The purpose of the HPLC analysis of cleaning samples is to prove with data that the equipment and cleaning procedures work, and that the surfaces of the equipment are indeed clean. The HPLC chromatographic finish is extremely reproducible and is the easiest part of the analytical... 

Gas (GC) and Liquid (HPLC) Chromatographs These are similar to spectrophotometers in that they are calibrated via a detector response to some property of analyte. The analyte may either be in solution or, in the case of GC (Figure 5.9), in pure form. Again, a calibration (or "standard") curve of detector response vs. either concentration or amount of pure chemical used is plotted and unknowns determined by correlation with the known stan-... 

The extract was then diluted with 0.5 mL with 0.2 M sodium acetate buffer, pH 4.7 and analyzed by HPLC. Chromatographic conditions were the same as for the determination of benzidine in hair dye formulations. For the particular lot of diarylide yellow studied 46 Ug/kg of DCB was found. In an attempt to confirm the identity of the chromatographic peak, its response as well as the response for the authentic DCB standard was determined at several different electrode potentials. These data, shown in Figure 7, illustrate the ability of HPLC/EC to yield qualitative as well as quantitative information for unknown components. 

An HPLC chromatograph was used together with a mass detector (when required, a stream splitter (app. 10 1) was inserted between the column and the detector). A column (250 X 4.6-mm ID) of NUCLEOSIL 5SA was flushed with 1% aqueous ammonium nitrate solution at a flow rate of 0.5 ml/min for 1 h, then with distilled water at 1 ml/min for 1 h. Silver nitrate (0.2 g) in water (1 ml) was injected onto the column in 50-/zl aliquots at 1-min intervals silver began to elute from the column after about 10 min. Twenty minutes after the last injection, the column was washed with methanol for 1 h, then with 1,2-dichloroethane-dichloromethane (1 1) for another hour. The three solvent reservoirs contained the following (A) 1,2-dichloroethane-dichloromethane (1 1) (B) acetone and (C) acetone-acetonitrile (9 1). For linoleic acid-rich seed oils, gradients of A were employed to 50% A-50% B over 15 min, then to 50% B-50% C over a further 25 min and held there for 5 min. For linolenic acid-rich seed oils, C was changed to acetone-acetonitrile (4 1), and the flow rate was increased to 1 ml/min gradients of A were utilized to 50% A-50% B over 10 min, then to 70% B-30% C over 20 more min, and finally to 100% C over another 30 min. 

They studied the effect of the mass detectors drift tube temperature on the low-molecular-mass TGs. Solutions of 10 mg/ml of tributyrin, tricaproin, tricaprylin, tricaprin, and trilaurin were injected twice at each of the following drift tube temperatures 20,25, 30,45, and 60°C. Five replications of the HPLC analysis were performed for one sample of ewe s milk fat to determine the reproducibility of the HPLC method. The TG composition was estimated in accordance with the method based on the calculation of the equivalent carbon numbers (ECNs) of the HPLC chromatographic peaks and in the molar composition in fatty acids, analyzed by GLC, collected at the HPLC chromatograph outlet. The HPLC fractions were collected every 40 s at the outlet of the column after 14 min there were no peaks before that time. 

The HPLC chromatograph (Waters Associate Model A.I.C. 202) was used with solvent system of 60% hexane and k0% chloroform. A UV detector at 25 nm was used at an atenuation of x8 for camphor and x 6h for parachlorophenol and phenol. 

TABLE 10.4 HPLC Chromatographic Conditions for the Fixed Combination Tablet Purity Method... 

Chromatographic System Set up the system with reference to High-Performance Liquid Chromatography under Chromatography, Appendix IIA. The HPLC chromatograph has a 254-nm detector and a 4.6-mm x 15-cm column that contains 5- to 10-mm porous microparticles of silica bonded to octylsilane (Zorbax 8, or equivalent). The flow rate is about 2 mL/min. Chromatograph three replicate injections of the Standard Preparation, and record the peak responses as directed under Procedure. The relative standard deviation is not more than 2.0%, and the resolution factor between nitrilo-triacetic acid and Disodium EDTA is not less than 4.0. 

Katz E, Eksteen R, Schoenmakers P, Miller N, eds. Handbook of HPLC. Chromatographic Science Series Vol. 78. New York Marcel Dekker, 1998. 

Because this method makes use of standard HPLC chromatographic equipment (pump, sample cell with filters, on-line detectors), there is a tendency to call it chromatographic or frontal chromatographic . We suggest that this tendency should be resisted it is misleading since there is no chromatographic effect or phenomenon. 

Figure 9.89 shows HPLC chromatographic analysis of an enzymatic reaction. 

Figure 9.99 Separation of the components of the reaction studied (catalysis by nucleoside phosphorylase of a purine riboside-base conversion) by HPLC. Chromatographic conditions isocratic elution flow rate, 2 mL/min 0.02 F KH2P04 (pH 4.2) and 3% methanol. Peaks 1, uric acid 2, hypoxanthine 3, xanthine 4, inosine. (From Halfpenny and Brown, 1980.)...

Experiments have been carried out to demonstrate the method described above by means of a multienzyme complex. This complex was assayed first for the AMP kinase activity. A reaction mixture was prepared containing unlabeled ATP (1 mM) and radioactive [3H]AMP only. The reaction was started by the addition of the complex, and samples were removed and analyzed by HPLC. Chromatographic profiles, each representing the analysis of a sample removed from an incubation mixture at increasing times after the start of the incubation, are shown in Figure 10.4. Both optical density and radioactivity were determined. 

Figure 19-6. RP-HPLC chromatographic profile of an in-process sample from E. coli recombinant DNA derived IFN-a-2b. Peak 1 is IFN-a-2b. Isoform peak 2 and 3 are putative scrambled disulfides. Isoform peak 4 is a putative open disulfide. The HPLC was run under a linear gradient of 49-65% B (10 90 H2O CH3CN/0.1% TEA) over 24 minutes with the UV set at 214 nm. The mobile phase A was water with 0.1% TEA and the flow rate was set at 0.2 mL/min. The column used was Vydac C8 column at 30°C (2.1mm X 50mm, 5 pm, 300 A).

A fine example of the use of pulse radiolysis technique in combination with steady-state product distribution is the recent study by Schuler and co-workers to probe charge distribution in aromatics. Their initial studies on biphenyf which were extended recently to phenof have been concerned with the determination of relative yields at o-, m- and -positions to examine the substituent effects on charge distribution. For example, Fig. 2 shows the contour plot of HPLC chromatographic data obtained in the OH radical reaction following the irradiation of phenol in the presence of the oxidant ferricyanide. Figure 3 shows the transient absorption spectra recorded in the reactions of OH and Ng radicals with phenol. The absorption... 

The first exposure to spectroscopy for most scientists is ultraviolet/ visible absorbance. As virtually every HPLC chromatograph employed in the pharmaceutical industry uses UV absorbance as the detection method, it is no wonder that the most popular hyphenated technique is HPLC-DAD. DAD spectrographs have been coupled to all liquid-based chromatographic systems including HPLC (preparative, analytical, and microbore), capillary electrophoresis (CE) and supercritical fluid chromatography (SFC). There have been several successes with TLC plates,18 but it is more common for developed plates to be scraped and the sample analyzed offline. 

The method has been used for the determination of competitive isotherms in cases in which the deviation from the Langmuir model is moderate [115]. An HPLC chromatograph configured so as to permit injecting into the column a wide rectangular pulse, e.g., by pumping into it either the pure mobile phase or, for a known time, a solution of the compound of interest, was used to make the measurements described [115]. Excellent agreement was observed with other experimental data and with the experimental band profiles recorded in overloaded elution for binary samples of various compositions [48]. 

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