Optimization of the mobile phase

In the absence of a suitable method of optimization of the mobile phase for AMD (19), the procedure generally used is to start with 100% methanol (5t = 5.1, Sy = 2.18) and to reduce its concentration in 15 stages, during which procedure the amount of solvent B (diethyl ether or dichloromethane) will increase from 0 to 100%. From the 15th to the 25th step, the concentration of solvent B is reduced and the concentration of -hexane increased, until it reaches 100% (Figure 8.12(a)). 

For a snccessfnl PLC separation, the selection and the optimization of the mobile phase are essential becanse the separation is generally inferior to that of analytical TLC owing to the larger particle size and particle size distribntion of the adsorbent and the overloading of the plate with sample. On thinner layers (0.5 to 1 nun) higher resolntion can generally be achieved, whereas on thicker layers (1.5 to 2 mm) resolution is more limited. PLC is suitable for samples containing no more than live compounds [1]. 

The most important aspects of the selection and optimization of the mobile phase are presented by Touchstone and Dobbins [17], Gocan [13], Siouffi and Abbou [18], Kowalska et al. [19], and Cimpoiu [20]. 

The PRISMA model is a system for the optimization of two- to five-eomponent mobile phases, developed by Nyiredy et al. to simplify the optimization proeess in different planar and column chromatographic systems [66]. This model for the seleetion of solvents and optimization of the mobile phase was developed first for TEC and high-performanee liquid ehromatography (HPLC) [38,67]. 

FIGURE 4.11 The PRISMA model. (Adapted from Siouffi, A.-M. and Abbou, M., Optimization of the mobile phase, in Planar Chromatography, A Retrospective View for the Third Millennium, Nyiredy, Sz., Ed., Springer Scientific, Budapest, 2001, chap. 3. With permission.)... 

Morita et al. [69] optimized the mobile phase composition using the PRISMA model for rapid and economic determination of synthetic red pigments in cosmetics and medicines. The PRISMA model has been effective in combination with a super modihed simplex method for fadhtating optimization of the mobile phase in high performance thin layer chromatography (HPTLC). 

Cimpoiu et al. [72] made a comparative study of the use of the Simplex and PRISMA methods for optimization of the mobile phase used for the separation of a group of drugs (1,4-benzodiazepines). They showed that the optimum mobile phase compositions by using the two methods were very similar, and in the case of polar compounds the composition of the mobile phase could be modified more precisely with the Simplex method than with the PRISMA. 

Pelander et al. [81] developed a computer program for optimization of the mobile phase composition in TLC. They used the desirability function technique combined with the PRISMA model to enhance the quahty of TLC separation. They apphed the statistical models for prediction of retardation and band broadening at different mobile phase compositions they obtained using the PRISMA method the optimum mobile phase mixtures and a good separation for cyanobacterial hepatotoxins on a normal phase TLC plate and for phenolic compound on reversed-phase layers. 

All the advantages of these methods for the optimization of the mobile phase by means of preassays in TLC can be exploited at the preparative scale. Finally, the separated zones may be easily removed from PLC plate and eluted in order to isolate quantities of the expected compounds. In PLC selection of the mobile phase, the subsequent recovery of the separated zone should be taken into consideration also. 

K. Morita, S. Koike and T. Aishima, Optimization of the mobile phase by the prisma and simplex methods for the HPTLC of synthetic red pigments. J. Planar Chromatogr.-Mod. TEC, 11 (1998) 94-99. 

Farre et al. (14) described the determination of 5-nitrofurylacrylic acid (5-NFA) in wines from different areas in Spain. Determination of (5-NFA) was achieved by optimalization of the mobile phase by re versed-phase HPLC. The mobile phases studied were 25% methanol or 23% acetonitrile, deionized water, alone or together with acetic acid-acetate buffer (pH 4.4) or glacial acetic acid. The 5-NFA was separated onaLiChrosorbRP-18 (150 X 3.2-mm-ID) column eluted with acetonitrile water glacial acetic acid mixture (25 75 1.5) at the rate of 0.6 ml/min. The experiment was carried out at room temperature, and the detection wavelength was 360 nm. 

Resolution is optimized by adjusting the buffer pH and the amount of organic modifiers. The most commonly used buffers are perchlorate, acetate, and phosphate. The protocol of the selection and optimization of the mobile phase for the enantiomeric resolution of drugs on polysaccharide-based CSPs in reversed-phase mode is presented in Scheme 2. Table 4 correlates the effects of separation conditions for neutral, acidic, and basic drugs on polysaccharide-based CSPs. From Table 4, it may be concluded that a simple mixture of water and an organic modifier will produce chiral separation of a neutral molecule because there is no... 

Hence, optimization of the mobile phase offers advantages mainly during the optimization process, while optimization of the stationary phase offers its main advantages after the optimization. So far, researchers have been more concerned with the optimization process... 

For the important case of the optimization of the mobile phase composition in reversed phase LC (RPLC), a typical two-dimensional response surface tends to be much less rugged, especially if the number of sample components is relatively small (n<10). A typical example is shown in figure 5.5. The selection of the normalized resolution product (r, eqn.4.19) as the criterion has also contributed to the smoother appearance of figure 5.5 relative to figure 5.1. Note that the criterion r has been recommended in chapter 4 for optimization processes in which the dimensions of the column are to be optimized after completion of the procedure (table 4.11). Therefore, the grid search approach is more appropriate for this kind of optimization than for optimization processes on the final analytical column. 

Figure 5.5 Example of a response surface for the optimization of the mobile phase in RPLC. Horizontal axis ternary mobile phase composition. Drawn line response surface using the resolution product as the criterion. Dashed lines retention surfaces for individual solutes (In k). For further details see section 5.5.2. Figure taken from ref. [504], Reprinted with permission. 

The two applications shown here concern the optimization of the mobile phase composition in RPLC. However, the method may easily be adapted to other problems. It is most practical if straight retention lines can be obtained. It should be noted that this is not usually the case for retention as a function of mobile phase composition in RPLC. In fact, Colin et al. [555] adapted the value of the hold-up time (t0) such as to obtain straight lines. The fact that they succeeded in doing so for all of 11 solutes considered at the same time is remarkable, but it may not always be possible. In any case, adapting t0 in order to linearize the retention lines will be an awkward practice. 

This equation is used in Chapter 9 in the discussion regarding the optimization of the mobile phase in LC. 

Jiang et al. studied optimization of the mobile phase composition for the HPLC separation of dihydropyridine drugs, including nimodipine [32]. These workers used a stainless steel column (15 cm x 4.6 mm) 10 pm operated at 30°C. The mobile phase was methanol-acetonitrile-water (24.8 27 48.2). 

The development of most ion-pairing chromatography (IPC) methods started with optimization of the mobile phase composition after an appropriate column was selected. The theory described in Chapter 3 assists the chromatographer to perform educated guesses. Chapters 7 through 10 discuss the main qualitative and quantitative attributes of tunable mobile phase parameters to illustrate their influence on the global performance of the method. 

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