From Two Gradient Runs

More accurate values of S and log few could be obtained from two gradient runs. 

For this purpose, two gradients with a different tc are necessary (20 and 60 min are acceptable for a conventional LC column geometry) [9]. This methodology was 

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In 1996, Snyder and Dolan elaborated the linear solvent strength (LSS) approach allowing the evaluation of log few from a single gradient run and its precise determination from two gradient runs [48]. From a practical point of view, LSS is the most useful approach to describe theoretical aspects and LSS gradients are convenient for optimization studies. Several commercially available optimization software packages which are able to predict resolution or retention in RPLC are currently based on this approach. 

Values of Kand Z derived in this fashion are shown in Table VI. We see that values of K and Z obtained in these two ways agree closely, and are also in good agreement with experimental values obtained from isobratic data (Table VI). We can also obtain values of and c from two gradient runs. 

Fio. 2. Isocratic retention (k ) venus mobile-phase composition (<(>) as derived from le-versed-pbase gradient runs ((, values) for curved plots of log k versus p. (a) Results for two gradient runs (igi and toj), — best-fit linear-curve approximation to log A versus, (b) Smilar results for three gradient runs (foi fo>)- Quarry a/. (17). 

Figure 18.14 represents an example of the computer-assisted optimization of linear gradient run time and temperature. It was necessary to perform four preliminary experiments which included two different temperatures (50 and 60 °C) and two gradient run times (17 and 51 min). From the data of these four runs the optimum conditions were calculated by the computer to be 57 °C and 80 min. The simulated chromatogram of this proposal is shown on top of Fig. 18.14. Some peak pairs are not resolved better than with R = 0.7. Since not all peaks are of equal scientific importance, it was decided that peaks 8, 9, 12, 13, 15, and 16 did not need to be resolved. With this facilitation the computer proposed 55 °C and 54 nun (nuddle). The real chromatogram is almost identical with the simulated one (bottom).

Based on chemical shifts and peak multiplicities, the on-flow HPLC-NMR characterisation of the majority of the components in the mixture of 27 tripeptides was achieved and demonstrated that this approach is likely to be an effective method for compound mixtures. The elution positions of all of the alanyl-containing peptides were determined, with the exception of A-M-M-NH2, which may have co-eluted with another peptide or may have been synthesised in a much smaller quantity. The only other tripeptides for which assignments have not been obtained are the MY2-NH2 isomers and two of the three M2Y-NH2 isomers. These eluted towards the end of the gradient run and are not as well resolved under these HPLC conditions. Additionally, with changes in the relative chemical shifts of the solvent signals, the intensities of the non-TV-terminal a-CH protons and the methionyl [3-methylene signals from these peptides may have been reduced by the effects of the solvent suppression irradiation of the water and acetonitrile resonances, respectively. With further optimisation of the elution conditions, it is possible that all 27 analytes could have been resolved and characterised. 

M. A. Quarry, R. L. Grob, and L. R. Snyder, Prediction of isocratic retention data from two or more gradient runs. Analysis of some associated errors, Anal. Chem., 58 901 (1986). 

The differential equation Eq. 2 can be solved after introducing the dependence of k on the volume of the eluate passed through the column from the start of the gradient run V. Any dependence of fe on U can be divided into two parts 1) a dependence of k on the concentration of a strong eluting component in the mobile phase (p controlled by the thermodynamics of the distribution process (the retention equation) and 2) the parameters of Eq. 1 describing the gradient profile, adjusted by the operator. 

Figure 11.10. Two-dimensional separation of E. coli protein mixture, using IEF with carrier ampholytes, pH range 3-10, in the first (horizontal) direction, and SDS-PAGE was mn from top to bottom in the second dimension on a 9-14% polyacrylamide gradient running gel cast with 0.1% SDS.12 [Reprinted, with permission, from P. H. O Farrell, The Journal of Biological Chemistry 250 (No. 10 May 25), 1975, 4007 4021. High Resolution Two-Dimensional Electrophoresis of Proteins . Copyright 1975 by the American Society for Biochemistry and Molecular Biology, Inc.]...

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. 

Figure 8.10. Computer screen of DryLab 2000 Plus simulation software showing the manual entry of retention data from results of four gradient runs at two different T and ts using the 150-mm-long, 5-pm Symmetry C8 column.

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