Solvent gradients

In most situations the eluent composition is chosen to minimize the effects of hydrophobic interaction, but these secondary effects can be used to advantage. By careful selection of a salt and its concentration, specific selectivities for analytes can be achieved without the use of organic solvents. Therefore, many separations usually run by solvent gradient reversed-phase methods can be completed with a purely aqueous isocratic eluent (13,14). 

Repetitive routine analysis of a specific sample (e.g., for Quality Control) will usually require a dedicated instrument. Therefore, the chromatograph and, in particular, the detector will be chosen for that specific analysis. Consequently, only one detector will be necessary and the purchase of an armory of detectors on the basis that they might be needed in the nebulous future is not advised. An alternative detector can always be obtained if and when the demand arises. The same argument applies to multi-solvent reservoirs and multi-solvent gradient programmers and other accessories that are not immediately required for the specific analysis in mind. 

The chromatographic conditions as mentioned above must in most cases be adapted to the matrix under study, i.e., to the predominant flavonoid classes. Paganga et al. studied aglycone flavonoids in apples, onions, and tomatoes, and were able to develop another solvent gradient enabling very good separation of the different aglycones (Table 2.3.2). 

Figure 1.17 Separation of large ring polycyclic aroaatic hydrocarbons extracted from carbon black on a 1.8 x 0.2 n I.D. fused silica capillary column packed with 3 micrometer spherical octadecylsllanized silica gel eluted with a stepwise solvent gradient at a flow rate of 1.1 mlcroliters/min with an inlet pressure of about 360 atmospheres. Under isocratic conditions this column yielded ca. 225,000 theoretical plates. (Reproduced with permission from ref. 238. Copyright Friedr. Vieweg t Sohn).

In this section a phenomenalogical approach to the description of solvent gradients will be adopted based largely on the linear solvent strength model prt osed by Snyder [552-555]. This is the least complicated of the models available and provides a reasonable approximation for typical experimental conditions. Mathematically more rigorous approaches have been developed by Jandera and Churacek [551,556], schoenmakers et al. [520,534,557] and Tomellini et al. [558]. 

Multiple development techniques using stepwise solvent gradients enable a subset of optimal separation conditions to be used to separate a mixture of wide polarity that cannot be separated using a single mobile phase (117,119,120,125). As an example of this approach the separation of 20 common protein amino acid PTH-derivatives is shorn in Figure 7.12 (126). Five... 

Figure 8.14 Senipreparative class separation of a diesel engine exhaust sample. Column 25 cm x 7.9 mm, lO micrometer Porasil. Solvent gradient hexane to 5% methylene chloride over 5 min., linear gradient to 100% methylene chloride over 25 min., isocratic for 10 min., linear gradient to 100% acetonitrile over 10 min., Isocratic for 5 min., step change to tetrahydrofuran for 10 min.,...

Figure 5.5 Two Bethods for generating binary solvent gradients.

Jandera, P, Predictive calcluation methods for optimization of gradient elution using binary and ternary solvent gradients, /. Chromatogr., 485, 113, 1989. 

The operating principles of three types of hplc pump are described, together with their advantages and limitations. Techniques for the production of solvent gradients and for the introduction of samples are considered. 

Fig. 4.3d and 4.3e show some of the other problems that can occur with two solvent gradients. 

Figure 4.7 shows the structures of important carotenoids (all-E) lutein, (all-E) zeaxanthin, (all-E) canthaxanthin, (all-E) p-carotene, and (all-E) lycopene. Employing a self-packed C30 capillary column, the carotenoids can be separated with a solvent gradient of acetone water=80 20 (v/v) to 99 1 (v/v) and a flow rate of 5 pL min, as shown in Figure 4.8 (Putzbach et al. 2005). The more polar carotenoids (all-E) lutein, (all-E) zeaxanthin, and (all-E) canthaxanthin elute first followed by the less polar (all-E) p-carotene and the nonpolar (all-E) lycopene. Figure 4.9 shows the stopped-flow II NMR spectra of these five carotenoids. The chromatographic run was stopped when the peak maximum of the compound of interest reached the NMR probe detection volume.

Molecular rotors with a dual emission band, such as DMABN or A/,A/-dimethyl-[4-(2-pyrimidin-4-yl-vinyl)-phenyl]-amine (DMA-2,4 38, Fig. 13) [64], allow to use the ratio between LE and TICT emission to eliminate instrument- and experiment-dependent factors analogous to (10). One example is the measurement of pH with the TICT probe p-A,A-dimethylaminobenzoic acid 39 [69]. The use of such an intensity ratio requires calibration with solvent gradients, and influences of solvent polarity may cause solvatochromic shifts and adversely influence the calibration. Probes with dual emission bands often have points in their emission spectra that are independent from the solvent properties, analogous to isosbestic points in absorption spectra. Emission at these wavelengths can be used as an internal calibration reference. 

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