Single gradient run

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. 

Theoretical peak capacity, P, is much larger than the number of peaks that can be really separated in a single gradient run in structurally non-correlated samples with random distribution of retention. According to statistical treatment, a 90% probability that a compound of interest will be found as a pure peak in the chromatogram requires real peak capacity exceeding the number of sample compounds by a factor of 20, and that the peak capacity required to separate all sample compounds dramatically increases with the second power of the number of compounds [163-166]. 

Tile possibility of using short wavelengths will depend on the nature and the purity of the solvents. From this point of view, acetonitrile may be preferred to methanol for RPLC. However, as discussed above, methanol-water gradients offer the possibility to estimate the isocratic retention behaviour fairly accurately from a single gradient run, because of the validity of eqn.(3.46). In mixtures of THF and water eqn.(3.46) is only approximately observed, whereas it is completely invalid in mixtures of acetonitrile and water (see table 3.1). 

The CHI can be obtained without preliminary method development direcdy from a single fast-gradient run with a cycle time less than 15 min with a 150-mm column [40] or 5 min with 50-mm column [42]. In this case, the obtained retention time, tr, is expressed within an organic phase concentration (< o) scale using a calibration set of compounds. CHI value can be obtained from ... 

A clean-up process-scale RP-HPLC step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 80 1 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 11.4). Separation is achieved using an acidic (often acetic-acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30 per cent acetonitrile). 

The second approach using gradient mode to assess log P values first proposed by Valko et al. is the use of the chromatographic hydro phobicity index (CHI) obtained from a single fast gradient run (<15 min) [103]. Since CHI is considered as a relevant parameter for QSAR studies, it was demonstrated using LSER that differences occur between CHI parameters and true log P because C HI (and log k) are sensitive to H-bond acidity ability whereas log P is not [104]. Thus, CHIs have to be used with precaution. 

In addition, parallel approaches that apply a single-spray mass spectrometer have been established. They address the fact that in serial LC, a considerable portion of the cycle time has to be devoted to sample loading or column re-equili-bration after a gradient run, thereby pausing data acquisition. This problem may be avoided by a switching procedure between two columns. In a setup described by Janiszewski et al., one sample is eluted and analyzed through the first column, while the second column is regenerated by an auxiliary pump and loaded with... 

The absolute magnitude of the CHI parameter depends on the values assigned to the set of standards. The method has the advantage that, once the calibration equation has been established, the retention parameter is obtained from a single fast gradient run, thus saving time and solvents. The CHI parameter has been reported to correlate satisfactorily with log P. 

Equipped with such a method to estimate the gradient of the energy with respect to the parameters, we can describe a simple steepest descent algorithm that tries to optimize the parameters in a single VMC run. 

The main focus of our work is photosynthetic pigments (chlorophylls, bacteriochlorophylls, and carotenoids). The important absorptions cover the spectral range from 300 to around 850 nm. Single HPLC runs last typically 30 min and use gradient elution, and 10-15 compounds are not uncommon. No dedicated machine was available at the time when the system was installed we therefore use a general purpose spectrometer with a semimicro flow cell (30 fil) and without beam focusing. Since the... 

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