Isocratic mode, HPLC

Figure 2. HPLC chromatograms (isocratic mode, 60% methanol, 40% water) of sediment extracts from 15 study sites in west Florida coastal waters. Migration profile are compared among sediment extracts and crude extract of Nannochloris sp. cell-free culture [See Moon and co-workers (.26) for specific sites].

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

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%... 

Many industrial laboratories conducting significant amounts of additive analyses have developed a universal HPLC method which may be used to separate most of the additives of interest. Thomas [417] has reported a method that can separate over 20 common primary and secondary stabilisers. Verdurmen et al. [197] employ a gradient ranging from 60 % acetonitrile/40 % water to 100% acetonitrile subsequently, all components are eluted off the column in isocratic mode. Irganox 1063 is used as a suitable internal standard since this compound is not frequently encountered in commercial polymers, elutes without overlap to other additives and shows good UV absorbency. In order... 

Ion chromatography instruments have the same components as those found in HPLC (see Fig. 4.1). They can exist as individual components or as an integrated instrument. The components of the system are made out of inert materials because the mobile phase is composed of acids or alkaline entities that can be highly corrosive. Instruments that operate in the isocratic mode are used more often than those allowing gradient elution. 

Figure 8. Separations of rhGH from its methionyl analog by reversed-phase HPLC. The chromatography was done at the indicated pHs using phosphate-containing mobile phases with propanol organic modifier on a Vydac C4 column. Elutions were run in an isocratic mode.

Radhofer-Welte and Dittrich [35] described a rapid and sensitive HPLC method for the determination of lornoxicam in plasma samples of humans and laboratory animals. After addition of the internal standard, tenoxicam, the plasma was acidified and extracted by dichloro-methane via Extrelut columns or by solid-phase extraction using Cis columns. After evaporation of fhe solvenf, fhe separation is performed on a Ci8 column in isocratic mode wifh a mobile phase consisting of 0.1 M phosphafe buffer (pH 6)-methanol, and defection at 372 nm. The limit of deferminafion was set to 10 ng/ml using 0.5 ml of sample but can be extended down to 2 ng/ml plasma. Using solid-phase extraction with Cis columns both lornoxicam and its main metabolite 5 -hydroxylornoxicam can be determined while extraction via Extrelut was used in studies where only lornoxicam was to be determined. The method was used in several thousand samples of pharmacokinetics and bioavailability studies in animals and humans. 

Application of RP-HPLC in isocratic mode. In HPLC, the most frequently used chromatographic retention parameter for characterising hydrophobicity is the logarithmic value of the retention factor (logA = log((fR - Ud/tn). where /r is the retention time of the analyte and to is the retention time of the unretained compound). The retention factor can be related directly to the chromatographic partition coefficients (A chr) according to Eiq. (12.6) ... 

The HPLC pump is operated in either an isocratic or gradient mode (Figure 6-16). In the isocratic mode, the mobile phase composition remains constant throughout the chromatographic run. This mode is usually used for simpler separations and separations of those compounds with similar structures and/or retention times. An isocratic mobile phase is a single solvent (e.g., methanol) or a prepared mixture of several solvents (e.g., methanol, acetonitrile, and water) delivered from a single solvent reservoir. Alternatively a multisolvent mobile phase can be metered and proportioned from two or more reservoirs. Most HPLC separations are performed under isocratic conditions. 

The HPLC system can be operated in either the isocratic mode, i.e. with the same mobile phase composition throughout the chromatographic run,... 

In the isocratic mode of RP-HPLC, the use of retention factor extrapolated to pure water (log kw) is very common in practice instead of retention factor itself, to obtain a more comparative hydrophobic index and avoiding the effect of changing the organic modifier. The extrapolation is based on the following Soczewinski-Snyder relationship 4 4 ... 

The HPLC run time dictates the gradient program. If the run time is required to be very short, a mobile phase in the isocratic mode should be considered, with 30-50% organic solvents. 

Pro-Phe-NH2, in human and rabbit plasma. In this procedure, after the addition of deuterated IS, the sample was desalted, proteins were precipitated, and the drug was extracted with solid-phase extraction (SPE). The HPLC separation was performed in the isocratic mode with a mobile phase that consists of 1% methanol and 20% acetonitrile in 10 mM ammonium formate (pH 5.2). A flow splitter was used to allow only 5% of the flow to enter the ESI source. 

Most RP-HPLC separations are done in the isocratic mode (i.e., where the composition of the mobile phase is held constant during the analysis). This approach is suitable when the sample consists of analytes having similar properties or where their hydrophobicities encompass a small or moderate range. Under these conditions, all solutes in the sample will be eluted over a reasonable time span (i.e., not too short to prevent resolution of individual analytes and not too long to result in an inconvenient analysis period). Therefore, proper selection of the mobile-phase composition is essential in the development of any RP separation method. Fortunately, due to the decades of... 

Fig. 5. Possibilities for improving resolution in the RP-HPLC (isocratic mode).

It should perhaps be noted here that these pneumatic pumps can however only operate for a limited time before re-filling of the solvent reservoir is necessary. Further, whilst they are excellent devices for operating HPLC columns in the isocratic mode they are not suitable for gradient mode elution. In spite of these drawbacks, the simplicity and cheapness of this type of eluant-pressurizing system make them very attractive to a number of laboratories and especially those on limited budgets. 

Mdrquez, N., R. E. Anton, A. Usubillaga, J. L. Salager, HPLC analysis of APE in microemulsion systems isocratic mode with mixed solvents. Sep. Sci. TechnoL, 1993,28,1769-1782. 

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