Ternary mobile phases

Optimizing an HPLC separation using five ternary mobile phases. Peaks ... 

Normal-phase separation of carotenoids Ternary mobile phase composition 80 ... 

Isocratic R-P separation of substituted Ternary mobile phase composition 74)... 

Rapid R-P isocratic separation of Ternary mobile phase composition 75)... 

Most cases of isocratic HPLC reported were carried out on reversed-phase chromatography on silica-based ODS (C,8 bonded phase) columns (1) for citrus juices. For mobile phases utilizing C,8 columns, isocratic solvents of 80-82% aqueous acetic acid with acetonitrile were commonly used. A ternary mobile-phase system of water-acetonitrile-glacial acetic acid... 

In this work correlations between mobile phase solvent strength and chromatographic retention of a number of different solute families will be presented. The first solvent strength measurements on ternary mobile phases will also be presented. Finally, a retention mechanism for packed column SFC is proposed. 

The same group demonstrated the use of the step gradient to improve speed of the CEC analysis of DNA adducts derived from syn benzo[g]chrysene-11,12-dihydrodiol-13,14-epoxide. They used a three-step gradient, which started with a quaternary mobile phase followed by two ternary mobile phases. The analysis was completed in... 

Polar group selectivity also occurs in ternary solvent systems (5.10). For example, the addition of 5% to 25% of a third solvent to a water-acetonitrile mixture can alter the relative retention of peaks, and often resolve overlapping peaks. Dolan et al (11) have employed ternary mobile phases of water, methanol and tetrahydrofuran to analyze vitamin tablets where interfering peaks could not be resolved with binary mixtures. See Figure 4. 

Figure 4. Separation using ternary mobile phase (Conditions (A) 95% methanol in water at 4 mL/min on 30-cm column (B) 95% methanol in water at 0.5 mL/ min on four 30-cm columns (increased number of plates) (C) 12% water/25% THF/63% methanol at 6 mL/min on 30-cm column (increased selectivity) column was Microbondapak C, , 10 ym)( 11)...

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. 

Figure 5.8 Illustration of a two-dimensional optimization using a modified Simplex algorithm. A ternary mobile phase for RPLC is being optimized. The third component is acetonitrile. Figure taken from ref. [505]. Reprinted with permission.

If only mixtures of a given eluotropic strength are considered as the result of a gradient scan, then a further optimization of the primary parameter (solvent eluotropic strength) is not contemplated and the number of parameters involved in the optimization process is effectively reduced by one. In the optimization of a ternary mobile phase composition one of the three volume fractions is defined by the two others, as their sum must equal one. 

The latter was also the case for the optimization of the composition of a ternary mobile phase in RPLC by Issaq et al. [554]. The ternary mixture was formed by mixing two limiting (non iso-eluotropic) binary mixtures and a fourth order polynomial equation was fitted through five equally spaced data points. 

Figure 5.18 (a) Figure showing the retention surfaces for some aromatic solutes in RPLC. Critical bands have beat constructed according to eqn.(5.16) below each solute. The dashed line indicates the optimum ternary mobile phase composition, (b) Chromatogram obtained at the predicted optimum composition. Figures taken from ref. [555], Reprinted with permission. 

Weyland et al. [560,561] used this method to optimize ternary mobile phase compositions for the separation of sulfonamides by RPLC. They fitted the retention surfaces to a quadratic model similar to eqn.(3.39), and also used a combination of a threshold resolution and minimum analysis time (min tm fl / vmin> 1.25 eqn.4.24) [560]. This criterion may yield a good optimum if the optimization is performed on the final analytical column (see table 4.11). 

A more or less opposite goal was pursued by de Smet et al. (574], who attempted to reduce the number of stationary phases to a single one, by choosing a cyanopropyl bonded phase of intermediate polarity, which can be used in both the normal phase and the reversed phase mode (see figure 3.8). Furthermore, because of a clever choice of modifiers, the total number of solvents required was restricted to six n-hexane, dichloromethane, acetonitrile and THF for NPLC and the latter two plus methanol and water for RPLC. A variety of drug samples could be separated with a selected number of binary and ternary mobile phase mixtures. 

Figure 5.32 Initial phase selection diagrams for three possible ternary mobile phase systems applied to the separation of five diphenyl amines. Top (Initial) retention lines. Bottom (initial) response line. Criterion normalized resolution product (r eqn.4.19 drawn line) Also shown is the response surface using the product resolution criterion (IIeqn.4.18 dashed line). The required chromatograms are shown in figure 5.33 (a, b and c). Figure taken from ref. [576]. Reprinted with permission.

The predictive method of Jandera et al. [628] requires knowledge of the isocratic retention data of all solute components in binary and (preferably) ternary mobile phase mixtures. Once these data are available, the method may be very helpful in obtaining an adequate (but not an optimum) separation with a ternary gradient. Unfortunately, the data required for the application of this predictive method are almost never available, and hence a large number of experiments need to be performed before any predictions can take place. When this is the case the method is of very little practical use. 

Figure 9.30. Statistical design of mixtures of ternary mobile phases. Reprinted with permission from ref. 65.

The use of an acetonitrile/methanol/water eluent has been shown to improve resolution of PAH chromatographed on a Vydac reverse phase column. This improved resolution should result in greater precision and accuracy in the quantitation of individual PAH, Determination of optimal solvent concentration is simplified through application of statistical design techniques. These techniques can be further utilized to investigate various ternary mobile phases in combination with different stationary phases. 

Figure 11 Separation of an adduct mixture formed from the in-vitro reactions of a/7f/-5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxide with calf thymus DNA with a ternary mobile phase consisting of 41% MeOH, 16% CAN, and 6 mM NH40Ac, (A) standards, (B) adducts from DNA reacted in vitro. (Reproduced from Am. Lab., 1999 30 15-29. Reprinted from Ref. 63, with permission.)...

Kazoka, H. and Shatz, V. 1996. Dynamically generated stationary hqnid phase systems with silica and ternary mobile phases containing ethylene glycol and formamide. Journal of Chromatography A, 732 231-8. 

Figure 4.34 Optimizing an hplc separation using five ternary mobile phases. Peaks (t) benzyl alcohol, (2) phenol, (3) 3-phenylpropanol, (4) 2,4-dimethylphenol, (3) ben zene, and (6) diethyl o phthalate. (After R. D. Conlon, The Perkin-Elmer Solvent Optimization System, Instrumentation Research, p. 95 (March 1985). Courtesy of...

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