Isocratic gradient liquid chromatography

The remainder of this section deals primarily with selectivity optimization in isocratic liquid chromatography and with gradient elution Before entering these subjects proper, however, a discussion of the relevant chromatographic properties of solvents is in order as a framework for the intuitive selection of the preferred solvent or solvent mixtures for selectivity optimization. 

MacNair, J.E., Patel, K.D., and Jorgenson, J.W., Ultrahigh-pressure reversed-phase capillary liquid chromatography isocratic and gradient elution using columns packed with 1.0 pm particles, Anal. Chem., 71, 700, 1999. 

Jandera, P. and Churacek, J., Gradient elution in liquid chromatography. I. The influence of the composition of the mobile phase on the capacity ratio (retention volume, band width, and resolution) in isocratic elution — theoretical considerations, /. Chromatogr., 91, 207, 1974. 

Alexander IV, J.N., Poli J.B., and Markides K.E., Evaluation of automated isocratic and gradient nano-liquid chromatography and capillary electrochromatography, Anal. Chem. 71, 2398, 1999. 

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 (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

Romanyshyn, L. Tiller, P. R. Alvaro, R. Pereira, A. Hop, C. E. Ultra-fast gradient vs fast isocratic chromatography in bioanalytical quantification by liquid chromatography/ tandem mass spectrometry. Rapid Commun Mass Spectrom 2001, 15, 313-319. 

Tiller, P. R. Romanyshyn, L. A. Implications of matrix effects in ultrafast gradient or fast isocratic liquid chromatography with mass spectrometry in drug discovery. Rapid Commun Mass Spectrom 2002, 16, 92-98. 

Liquid chromatography can be operated under mild conditions in terms of pH, ionic strength, polarity of liquid, and temperature. The apparatus used is simple in construction and easily scaled up. Moreover, many types of interaction between the adsorbent (the stationary phase) and solutes to be separated can be utilized, as shown in Table 11.1. Liquid chromatography can be operated isocratically, stepwise, and with gradient changes in the mobile phase composition. Since the performance of chromatography columns was discussed, with use of several models and on the basis of retention time and the width of elution curves, in Chapter 11, we will at this point discuss some of the factors that affect the performance of chromatography columns. 

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). 

Donovan SF, Pescatore MC (2002) Method for measuring the logarithm of the octanol-water partition coefficient by using short octadecyl-poly(vinyl alcohol) high performance liquid chromatography columns. J Chrom A 952 47-61 Du CM, Valko K, Bevan C et al. (1998) Rapid gradient RP-HPLC method for lipophilicity determination s solvation equation based comparison with isocratic methods. Anal Chem 70 4228-4234... 

For metabolite isolation, 1.5 liters of pooled urine were applied to a XAD-2 resin column first. The ethyl acetate extract obtained containing 85 % of the radioactivity was applied upon evaporation to semipreparative HPLC on a Zorbax RX C18 column (9.4 x 250 mm, 5 pm) using gradient elution. Fractions obtained were further separated by isocratic elution on the semipreparative column. The metabolite fractions obtained were finally purified by preparative thin-layer chromatography. Liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis was applied to the isolated metabolite fractions for structure elucidation. 

Reverse phase liquid chromatography has typically been used for the separation of PFCs, employing either Cg or Cig columns [96], although the use of perfluoroalkyl columns has also been reported [115]. Mobile phases are typically mixtures of methanol-water or acetonitrile-water and are often modified with ammonium acetate to improve chromatographic separation and MS sensitivity. Both isocratic and gradient elution methodologies have been employed [96]. LC-MS/MS methods [116, 117] have also been developed for the separation of PFSA and PFCA isomers and generally employ linear perfluorooctyl stationary phases and acidified mobile phases. 

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