Isocratic HPLC

Kilpatrick, L Jones, M. and Phillipson, O. A semiautomated analysis method for catecholamines, indoleamines, and some prominent metabolites in microdissected regions of the nervous system An isocratic HPLC technique employing eoulometric detection and minimal sample preparation. J Neurochem 46(6) 1865-1876, 1986. 

Normal-phase chromatography is still widely used for the determination of nonpolar additives in a variety of commercial products and pharmaceutical formulations, e.g. the separation of nonpolar components in the nonionic surfactant Triton X-100. Most of the NPLC analyses of polymer additives have been performed in isocratic mode [576]. However, isocratic HPLC methods are incapable of separating a substantial number of industrially used additives [605,608,612-616], Normal-phase chromatography of Irgafos 168, Irganox 1010/1076/3114 was shown [240]. NPLC-UV has been used for quantitative analysis of additives in PP/(Irganox 1010/1076, Irgafos 168) after Soxhlet extraction (88%... 

Isocratic HPLC Yes/Yes Simple Low Receiving site have extensive familiarity with both product and methodology Knowledge based only... 

Isocratic method. Whenever possible, use isocratic HPLC condition, as this is affected less by the variation in flow rate, temperature, and dwell volume. If gradient HPLC conditions have to be used, a simple linear gradient is preferred over multistep gradients. Complicated gradient conditions are more susceptible to differences between HPLC instruments (e.g., flow rate, dwell volume). 

Measurement of tHcy in Plasma Using Isocratic HPLC-Fluorescence Detection... 

The reaction of AdoMet and AdoHcy with monochloroacetaldehyde produces the etheno derivatives, which are strongly fluorescent (Fig. 2.2.6). These derivatives are separated by isocratic HPLC and detected and quantified by fluorescence. Our methods have been modified from those of Wagner et al. [28]. 

Isocratic HPLC System for Blue-Fluorescing Pterins... 

Isocratic HPLC system with electrochemical detection (5010 Electrode, ESA, Chelmsford, USA). Upstream Electrode, 50 mV, downstream electrode, 450 mV. 

Isocratic HPLC system with 5011 ESA electrode, downstream electrode 450 mV. 

Modular isocratic HPLC with ED detection HPLC pump 515 (Waters), Injector 7725i (Rheodyne), Pulsation Reduced (Portman Instruments), Coulochem Detector Model 5100A (ESA), Analytical Cell 5011 (ESA), and PC with chromatography software (SCPA). 

Isocratic HPLC system with fluorescence detection (excitation 404 nm, emission 618 nm). 

Figure 25-12 Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 mL/min on a 0.46 x 25 cm Hypersil ODS column (C,8 on 5-jxm silica) at ambient temperature ( 22 C) (1) benzyl alcohol (2) phenol (3) 3, 4 -dimethoxyacetophenone (4) benzoin (5) ethyl benzoate (6) toluene (7) 2,6-dimethoxytoluene (8) o-methoxybiphenyl. Eluent consisted ot aqueous buffer (designated A) and acetonitrile (designated B). The notation 90% B in the first chromatogram means 10 vol% A and 90 vol% B. The buffer contained 25 mM KH2P04 plus 0.1 g/L sodium azide adjusted to pH 3.5 with HCI.

Figure F2.3.1 Isocratic HPLC separation of carotenoid standards using Basic Protocol 1. Conditions 5-pm x 250-mm x 4.6-mm Vydac 201TP column, 90 10 methanol/acetonitrile mobile phase, 1.0 ml/min flow rate, visible detection at 450 nm, column temperature 25°C.

Bernhard et al., on the other hand, selected 5-fim spherical Nucleosil-NH2 as the stationary phase (43). In order to improve the resolution of the early-eluting components and to reduce the overall time needed per analysis, they set up an isocratic HPLC system, including two separate columns. By means of a switch valve, the second 175-mm column could be excluded after the elution of PC, SPH, LPC, /V-methyl PE, PG, and PE. This enabled the rapid elution of the acidic phospholipids PI and PS from the 50-mm column and hence significantly decreased the total run time (Fig. 3). The mobile phase contained 1460 ml A, 500 ml M, 30 ml W, and 600 jj, 1... 

Rustan et al. (123) used an isocratic HPLC method for the determination of alpha-, gamma-, and delta-tocopherol, BHT, BHA, PG, OG, DG, NDGA, TBHQ, ascorbyl palmitate, and beta-carotene in foods. An RP18 column was used in experiments, and seven mobile phases based on various combinations of acetonitrile, methanol, water, and tetrahydrofuran were tested. Trials with carrot juice, dried milk formula for infants, and aperitif cakes showed that all 12 antioxidants could be determined by a single isocratic HPLC analysis. The optimum mobile phase... 

The OPP malathion is widely used because of its low persistence in the environment and its high insecticide activity. Pure malathion has moderate toxicity, but crude malathion and its formulations contain impurities, which are far more toxic to mammals. These impurities not only are formed during commercial production but can also develop in the grains during storage. The most toxic of these products is the oxidation product malaoxon. An isocratic HPLC method indicated the degradation of malathion in stored wheat (55) and the presence of malathion and malaoxon in maize and bean samples (56). Isomalation and malation monocarboxylic acid metabolites were also detected. 

Most cases of isocratic HPLC reported were carried out on reversed-phase chromatography on silica-based ODS (C,8 bonded phase) columns (1) for citrus juices. For mobile phases utilizing C,8 columns, isocratic solvents of 80-82% aqueous acetic acid with acetonitrile were commonly used. A ternary mobile-phase system of water-acetonitrile-glacial acetic acid... 

Table 5 Isocratic HPLC Analysis for Flavanone Glycosides HPLC condition...

MFM Borges, FMF Roleira, MMM Pinto. Simultaneous isocratic HPLC separation of the dias-tereoisomers of caffeic, ferulic, and isoferulic acids and related coumarins. J Liquid Chromatogr 16 149-160, 1993. 

Figure 6 Isocratic HPLC chromatogram obtained on a sample of drag substance stressed in pH 7 phosphate buffer/ACN.

Chollet et al. [39] developed an isocratic HPLC method for simultaneous determination of vigabatrin and gabapentin in human serum after precolumn derivatization with o-phthaldialdehyde and fluorimetric detection at 435 nm with excitation at 235 nm. A column (25 cm x 3.0 mm) of Nucleosil Ci8 (5 /im) with a mixture of 0.022 M phosphoric acid (pH 2)-acetonitrile (45 55) as a mobile phase (flow rate 0.6 ml/min) was used. The calibration graph was rectilinear from 2.0 to... 

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