Isocratic mode

For preparative or semipreparative-scale enantiomer separations, the enantiose-lectivity and column saturation capacity are the critical factors determining the throughput of pure enantiomer that can be achieved. The above-described MICSPs are stable, they can be reproducibly synthesized, and they exhibit high selectivities - all of which are attractive features for such applications. However, most MICSPs have only moderate saturation capacities, and isocratic elution leads to excessive peak tailing which precludes many preparative applications. Nevertheless, with the L-PA MICSP described above, mobile phases can be chosen leading to acceptable resolution, saturation capacities and relatively short elution times also in the isocratic mode (Fig. 6-6). 

As in isocratic mode, the estimate of log P is indirect and based on the construction of a linear retention model between a retention property characteristic of the solute (logkw) and a training set with known logP ci values. To assess the most performing procedures, the three hydrophobicity indexes (( )o, CHI and logkw) were compared on the basis of the solvation equation [41]. These parameters were significantly inter-related with each other, but not identical. Each parameter was related to log P with values between 0.76 and 0.88 for the 55 tested compounds fitting quality associated with the compound nature. 

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... 

As in isocratic mode, the variation in the percentage of organic modifier in the mobile phase can be described by the linear Soczevfinski-Snyder model... 

Nevertheless, even if the accuracy of the gradient methods is slightly lower than with isocratic conditions [105], it offers the ability to extend lipophUicity range determination without too high a loss in resolution. Therefore, it was recommended to preferentially use isocratic mode for expected log P between 0 to 4 and to use gradient mode for more lipophilic compounds [105]. 

When the mobile phase has a fixed composition (isocratic mode) a single pump is sufficient. However, if the composition of the mobile phase has to vary with time, as in a gradient elution, one of the methods described below can be chosen. The instrument must compensate for differences in solvent compressibility in order to attain the desired composition at a given pressure. 

The refractive index detector, considered to be almost universal, is often used in series with a UV detector in the isocratic mode to provide a supplementary chromatogram. This detector, which is not highly sensitive, has to be temperature controlled, as does the column (0.1 °C). The baseline of the chromatogram has to be set to an intermediate position because it can lead to either positive or negative signals (Fig. 3.18). The detector can only be used in the isocratic mode because in gradient elution the composition of the mobile phase changes with time, as does the refractive index. Compensation, which is easily obtained in the case of a mobile phase of constant composition, is difficult to carry out when the composition at the end of the column differs from that at the inlet. 

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. 

In summary, a chemical suppressor containing an anionic resin (e.g. ArCH2(NR)3OH) is associated to a cationic separation column (ArS03H) in order to neutralise the mobile phase. The limitation of this type of suppressor lies in its very large dead volume that diminishes separation efficiency by remixing ions before their detection. It must be periodically regenerated and can only be used in the isocratic mode. 

Isocratic mode, 47 Isotope ratio, 318 Isotopic abundance, 317 Isotopic dilution, 330 Isotopic source, 241... 

If the separation at critical conditions is impossible because of very high retention volumes of the functional macromolecules, it is necessary to take an adsorbent with wider pores or proceed to the exclusion region (eab > e ) and perform the chromatography in the isocratic mode, or to use gradient elution. If this is not successful, another adsorbent has to be chosen which is less selective with respect to functional groups. 

Crude extract was also separated and collected on another Waters system, which consisted of a 600 pump, a 2996 Photodiode Array Detector, and a 2767 fraction collector. The detection wavelength was set in the ultraviolet (UV) between 190 and 400 nm. The column used was a 150 x 21 mm long ACE AQ with 10-mm particles (Advanced Chromatography Technologies, Aberdeen, UK). The system was operated at room temperature. The injection volume was 1500 pL. The mobile phase consisted of 1 3 acetonitrile water with 0.01% trifluoroacetic acid, which was flowing at a rate of 10 mL/min. The system was operated in the isocratic mode. Fractions of 1.25 mL were collected every 7.5 s. 

Analytical methods for the determination of one antidepressant and/or its metabolite(s) were usually performed in isocratic mode, with total run times from seconds to a few minutes. However, as previously mentioned, multianalyte procedures are preferable, particularly if the method is intended for clinical or forensic analysis. Gradient separation was usually applied when the most common antidepressants were included in the methodology however, total chromatographic run times varied widely, from 5 to 40 min [57, 76], depending on column length, extraction technique (offline vs. online techniques), biological matrix or the specific application of the method. 

Fig. 3 Chiral separation of atropine (racemic mixture of S- and //-hyoscyamine). Analysis of buffered serum dilution was performed according to John et al. [49] using two consecutively coupled AGP columns (150 mmx2.0 mm I.D., 5 pm) in isocratic mode with solvent A (0.01 M NH4FA, pH 8.0) and solvent B (0.01 M NH4FA in 25 % v/v ACN, pH 8.0) in 85 15 ratio at 300 pi/ min and 40 °C. Detection was done by positive ESI MS/MS in MRM mode...

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.

The power of fluorescence detection was illustrated on the separation of PAH s by Yan et al. [64]. The 16 U.S. EPA priority PAHs were separated in isocratic mode in less then 10 min using 100 pm I.D. columns packed with 1.5 pm nonporous octadecyl silica particles. Separation efficiencies of 750,000 plates/m were obtained when the PAHs were detected by ICFD while 300,000-400,000 plates/m were found for OCFD. 

For isocratic mode of CEC separations, standard CE instrumentation is sufficient. This applies particularly for equipment that has the provision of column pressurization. In practice this is achieved by applying a gas under a pressure of 2-12 bar to both inlet and outlet vials. Column thermostating in CEC is regarded mandatory to avoid excessive radial temperature gradients within the capillary. In such instruments, sample is typically injected electrokinetically and alternatively by applying the external gas pressure to the sample vial. Detection occurs on-column i.e. directly through a non-packed section of the capillary following immediately the end of the bed. 

The analytical separation was obtained at low flow velocity (0.2 cm/sec) and in the isocratic mode to optimize resolution. Absorbance detectors set at 245 and 280nm respectively, were used in series for effluent monitoring. 215nm detection, though optimum for the cannabinoids, was precluded due to co-elution of en-... 

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