Solvent program

The effect of solvent programming can be simulated by means of a computer in much the same way as temperature programming can be simulated. Reiterating equation (13) from chapter 4... 

In addition, if the program is run for the second enantiomer, then the ratio of the retention times of the two isomers can be calculated (the separation ratio) for a range of different solvent program rates. 

The peak capacity is not pertinent as the separation was developed by a solvent program. The expected efficiency of the column when operated at the optimum velocity would be about 5,500 theoretical plates. This is not a particularly high efficiency and so the separation depended heavily on the phases selected and the gradient employed. The separation was achieved by a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. Initially the tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase. 

FIGURE 4 Table showing the solvent programming steps used for verifying pump compositional accuracy. This program is used specifically for the Waters Alliance 2695 system. 

Kutter, J. P., Jacobson, S. C., Matsubara, N., and Ramsey, J. M. (1998). Solvent-programmed microchip open-channel electrochromatography. Anal. Chem. 70, 3291-3297. 

The choice of solvents for gradient elution is still somewhat empirical however, using the data from Table 3.5 narrows the choices. Modern HPLC instruments are equipped with solvent programming units that control gradient elution in a stepwise or continuous manner. 

Olsson et al. (69), as well as Arnoldsson and Kaufmann (70), further improved the mobile-phase composition. Experimental design revealed that besides THF, n-butanol as well as ammonium acetate were appropriate modifiers too. The contribution of THF and n-butanol was kept constant throughout the whole solvent program and was about 5% for each solvent each mobile-phase mixture also contained 180 mg of ammonium acetate per liter. As a typical example, the chromatogram of the ILPS-proposed mixed soybean phospholipids standard, which is also referred to as the Spectralipid SN standard mixture, is shown in Fig. 5. 

NDPHA, A-nitrosoatrazine, A-nitrosobenzylphenylamine, A-nitrosocarbazole, and A-nitroso-carbaryl in foods. In another study, Sen et al. (67) used a normal-phase system for the determination of NDBZA in cured pork products packaged in elastic rubber nettings (Fig. 4). As can be seen from the figure, appropriate solvent programming allowed the simultaneous determination of six nonpolar and three polar NOC. 

A variety of detectors have been used for the HPLC determination of NOC in foods. These include UV, fluorescence, electrochemical, TEA, and various postcolumn denitrosation detectors. To be applicable for the low-ppb detection of these compounds in foods, an HPLC detector should meet the following criteria high sensitivity and specificity, responsive to all classes of NOC, linearity over a fair range of concentration, compatibility with both normal- and reversed-phase mobile phases, and minimal interference from changes in solvent composition, thereby making it amenable to solvent programming. As will be seen from the following discussion, none of the detectors currently available meet all these criteria. 

Horizontal and descending TLC are other forms of chromatography which require special equipment. The advantages of these systems are not clear. They are usually more expensive than ascending equipment, but often have other features for stepwise gradient elution or continuous solvent programming. 

F1g. 1. Solvent program (methanol-H O) for HPLC separation of BaP metabolites at 21°C. 

The steady movement of the baseline either up or down the scale is referred to as drift. Drift is often indicative of variations in chromatographic conditions, such as temperature or solvent programming. It can also be indicative of instrument instability owing to temperature effects on the detector. 

In some cases we may speed up the selection of appropriate primary parameters with the help of programmed analysis, i.e. temperature programming in GC or solvent programming in LC. Another useful scouting technique may be thin layer chromatography (TLC). Possibilities for establishing the appropriate values of the primary parameters will be discussed in section S.4. 

To achieve this goal, method developers should ideally find themselves in the opposite situation, being equipped with flexible, advanced instrumentation, including a variety of possible injectors and detectors, facilities for temperature or solvent programming, etc.. Multichannel detectors are very useful, as they may be of assistance in recognizing the different sample components when they move about in the chromatogram during the selectivity optimization process (section 5.6). 

RPLC Mobile phase polarity Solvent programming (gradient elution)... 

By analogy with the term programmed temperature GC [605] we will use the term programmed solvent LC , although solvent programmed LC is also commonly used. 

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