Isocratic reversed-phase

Nyambaka, H. and Ryley, J., An isocratic reversed-phase HPLC separation of the stereoisomers of the provitamin A carotenoids (a- and (3-carotene) in dark green vegetables, Food Chem., 55, 63, 1996. 

The isocratic reversed phase solvent system consists of water (polarity, p = 10.2), the most polar solvent in RPLC, as a primary solvent to which water-miscible organic solvents such as methanol (p = 5.1), acetonitrile (p = 5.8), or tetrahydrofuran (p = 4.0) are added. In order to optimize the speed of separation for an analyte pair, the proportions of water to nonpolar solvent are chosen such that the capacity factor of the last-eluting analyte of interest has a value of about 2.13... 

Hafkenscheid, T.L., Tomlinson, E. (1983) Correlations between alkane/water and octan-l-ol/water distribution coefficients and isocratic reversed-phase liquid chromatographic capacity factors of acids, bases and neutrals. Int l. J. Pharmaceu. 16, 225-239. 

Isocratic system This consists of a solvent delivery for isocratic reversed-phase and gel filtration chromatography. 

Figure 4.12 Analysis of biosynthetic human growth hormone by isocratic reversed-phase chromatography. To generate all possible degradation products, a production lot of HGH was exposed to 40°C. The profile of the unstressed HGH is shown in the upper trace. (Reproduced from R.M. Riggin, G.K. Dorulla, and DJ. Miner, Anal. Biochem., 167 199 [1987]. With permission from Elsevier Science.)...

Wei JQ, Wei JL, Zhou XT. 1990. Optimization of an isocratic reversed phase liquid chromatographic system for the separation of fourteen steroids using factorial... 

FIGURE 21 (a) An isocratic reversed-phase HPLC method developed for assay, with which 15%... 

Isocratic reversed-phase HPLC determinations of twelve natural corticosteroids in serum with online UV and fluorescence detection. (163)... 

A variety of gradient and isocratic reverse phase HPLC systems-have been used to ehromatograph dorzolamide, and the salient characteristics of these are summarized in Table 6. 

Dinkci, N., Akalin, A., Gone, S., and Unal, G. (2007). Isocratic reverse-phase HPLC for determination of organic acids in Kargi Tulum cheese. Chromatographia 66, 45—49. 

Air drawn through a cartridge containing silica gel coated with acidified DNPH derivative eluted with acetonitrile and determined using isocratic reverse phase HPLC with UV detection at 360 nm (U.S. EPA 1988, Method TOll) recommended flow rate 1 L/min sample volume 100 L. 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

A list of the 64 analytes and their method performance is shown in Table 6.6. The increased number of analytes is possible because of improvements to the collision region of the MS/MS system that provide increased sensitivity and reduced memory effects. In addition, robotic systems for sample handling and on-line solid-phase extraction (SPE) of plasma samples were integrated with the LC/MS/MS system (Figure 6.22). An isocratic reversed-phase HPLC method provided a cycle time of 4.5 min per sample. The on-line sample preparation and short analysis resulted in an increased sample throughput that required less time from the scientist. The... 

R. E. B. Ketchum and D. M. Gibson, Rapid isocratic reversed phase HPLC of taxanes on new columns developed specifically for taxol analysis, J. Liquid Chromatogr., 16 12 (1993). 

This consists of a solvent delivery for isocratic reversed phase and gel filtration chromatography. The isocratic system (Fig. 1.1) provides an economic first step into high performance liquid chromatography techniques. The system is built around a high performance, dual-piston, pulse-free pump providing precision flow from 0.01 to 5mL min-1. 

A. R. Ivanov, I. V. Nazimov, and L. Baratova, Determination of biologically-active low-molecular-mass thiols in human blood. I. Fast qualitative and quantitative, gradient and isocratic reversed-phase high-performance liquid-chromatography with photometric and fluorescence detection, J. Chromatogr. A. 895, 157-166 (2000). 

Fucoxanthin and its metabolites Egg yolk Normal phase, Silica gel column Acetone hexane gradient Normal phase, Silica gel column Hexane, Dichlormethane, 2-Propanol and Ethyl-diisopropylamine (90.9 7 3 0.1 v/v/v/v) isocratic Reverse phase, C18 column Methanol and Water (67 33 v/v) isocratic Bonded nitrile column Hexane, Isopropyl acetate, Acetone, Methanol (76 17 7 0.1 v/v/v/v) isocratic Strand et al., 1998... 

If only a few specific components need be quantified, the analysis time can be considerably shortened. Dong and DICesare (50) developed an high speed isocratic reverse phase separation for 2,5-dimethyl-4-hydroxy-3(2H)-furanone. This compound is commonly known as pineapple ketone and can be quantified in food flavoring products using one of the new 3x3, C-18 columns in as little as 2 minutes. Other workers (51J have used rapid gradients (6 min.) to separate caffeine, trigonelle and theobromine in coffee and cocoa products. 

Figure 9.37 Isocratic reversed-phase HPLC chromatogram showing separation and detection of dihydroquinozolinium (DHQ) and o-aminobenzaldehyde (OAB). The column was LiChrosorb Qg, and 4.6 mm X 250 mm, and the solvent system was methanol-water (1 2), pumped at 1.5 mL/mm. Detection was at 254 nm with an Altex 310 chromatograph. (From O Donnell et al., 1978.)...

Pilorz, K. and Choma, I. Isocratic reversed-phase high-performance liquid chromatographic separation of tetracyclines and flmnequine controlled by a chaotropic effect. J. Chromatogr. A. 2004, 1031, 303-305. 

The pH dependence of the isocratic reversed-phase chromatographic retention of ionogenic solutes has been reviewed by Schoenmakers and Tijssen [ 110[ mostly for the purpose of optimisation of the chromatographic separations. 

The ch5unobypsin-catalyzed condensation of 37 (10 mM) with 38 (20 mM) was performed in 0.2 M sodium veronal buffer (pH 9) containing 40% DMSO (0.5 mL). The ch5miobypsin concentration was 1.6 X 10 M. The reaction course was monitored by isocratic reverse-phase HPLC using a C-18 column... 

The performance of the SEC method was compared to that of an isocratic reversed phase (RP)-HPLC method (UV detection). 

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