Mobile phase ternary solvent mixtures

Herslof and Kindmark (105) report the use of the mass detector (light-scattering detector) in this sense. The column used (250 X 4.6-mm ID) was packed with LICHROSPHER RP 100 and thermostated at 22°C. The mass detector oven temperature was 40°C, and the inlet gas pressure was 15 psi. The mobile phase consisted of mixtures of acetonitrile, ethanol, and hexane. Gradients from 0 to 100 of ternary premixtures of the three solvents were used at a flow rate of 1 ml/ min. The sample was dissolved in hexane-isopropanol (1 1), and 12 /A were injected (approx. 150 fj.g total sample). The chromatogram of the standard mixture of TGs is shown in Fig. 35. The separations of soybean oil and a mixture of soybean and coconut oils illustrate the resolution of vegetable oils into TG species (Fig. 36). 

The popularity of the poly(saccharide) derivatives as chiral stationary phases is explained by the high success rate in resolving low molecular mass enantiomers. It has been estimated that more than 85% of all diversely structured enantiomers can be separated on poly(saccharide) chiral stationary phases, and of these, about 80% can be separated on just four stationary phases. These are cellulose tris(3,5-dimethylphenyl carbamate), cellulose tris(4-methylbenzoate), amylose tris(3,5-dimethylphenyl carbamate), and amylose tris(l-phenylethyl carbamate). Typically, n-hexane and propan-2-ol or ethanol mixtures are used as the mobile phase [111]. Both the type and concentration of aliphatic alcohols can affect enantioselectivity. Further mobile phase optimization is restricted to solvents compatible with the stationary phase, such as ethers and acetonitrile, as binary or ternary solvent mixtures, but generally not chloroform, dichloromethane, ethyl acetate, or tetrahydrofuran. Small volumes of acidic (e.g. tri-fluoroacetic acid) or basic (n-butylamine, diethylamine) additives may be added to the mobile phase to minimize band broadening and peak tailing [112]. These additives, however, may be difficult to remove from the column by solvent rinsing to restore it to its original condition. 

Solvent triangle for optimizing reverse-phase HPLC separations. Binary and ternary mixtures contain equal volumes of each of the aqueous mobile phases making up the vertices of the triangle. 

Where there are multi-layers of solvent, the most polar is the solvent that interacts directly with the silica surface and, consequently, constitutes part of the first layer the second solvent covering the remainder of the surface. Depending on the concentration of the polar solvent, the next layer may be a second layer of the same polar solvent as in the case of ethyl acetate. If, however, the quantity of polar solvent is limited, then the second layer might consist of the less polar component of the solvent mixture. If the mobile phase consists of a ternary mixture of solvents, then the nature of the surface and the solute interactions with the surface can become very complex indeed. In general, the stronger the forces between the solute and the stationary phase itself, the more likely it is to interact by displacement even to the extent of displacing both layers of solvent (one of the alternative processes that is not depicted in Figure 11). Solutes that exhibit weaker forces with the stationary phase are more likely to interact with the surface by sorption. 

The minimum operating temperatures for various solvent mixtures used in the reversed phase HPLC are shown in Table 9.4. Values for acetonitrile were experimentally determined based on the temperature at which the system could no longer pump the mobile phase [56]. These values are approximate and will vary somewhat with pressure. Values are not shown for THF-water systems. While THF freezes at -65°C, work in our laboratory [56] has shown that water-THF mixtnres separate and the water component freezes at the freezing point of water, making these mixtnres nnnsable below 0°C [96]. No data is available for ternary mixtures, though the addition of another solvent may eliminate the separation of THF and water. 

The operating conditions range from an excitation wavelength of 360-365 nm and an emission wavelength 425-435 nm. The intensity of aflatoxin fluorescence depends strongly on the injected solvent, with a higher response if the sample is injected in the mobile phase (usually a ternary mixture of methanol/acetonitrile/water) and a lower one if injected in the methanol or acetonitrile only. 

Usually, when an HPLC method is developed, an acceptable degree of separation for all the components of interest in our sample is required in a reasonable time. The mobile phases more frequently used are the classical mixtures of methanol-water and acetonitrile-water in different proportions. If a satisfactory separation cannot be achieved using a binary solvent mixture as mobile phase, a ternary composition may be used. 

Tertiary systems. With methanol/carbon dioxide mixtures the addition of even the most polar additives has only a small impact on the mobile phase solvent strength as measured with Nile Red. With TFA concentrations below 1 to 2 % in methanol, ternary mixtures of TFA/methanol/carbon dioxide produce the same apparent solvent strength as binary methanol/carbon dioxide mixtures. As much as 5 or 10 % TFA in methanol is required to noticeably increase the solvent strength of TFA/methanol/carbon dioxide mixtures above those for binary methanol/carbon dioxide mixtures, as shown in Figure 4. 

Ternary mixtures of additives, less polar modifiers (like methylene chloride), and non-polar supercritical fluids have not been extensively used as chromatographic mobile phases. A few measurements suggest that they do not produce the dramatic decrease in solute retention (compared to the additive in methanol) implied by the increases in solvent strength in Figure 8. 

Mobile phases are usually binary or ternary mixtures of solvents. Selectivity is affected mostly by mobile phase composition rather than strength, and peak shape and retention are both influenced by the addition of organic modifiers.101 Some compounds naturally have 77-donor or 77-acceptor groups and can be resolved directly. In many cases, however, introduction of 77-donating groups by derivatization steps is necessary. Figure 2.20 shows the proposed three-point interaction of 3-aminobenzo[a]pyrene, a polycyclic aromatic hydrocarbon (PAH), with a Pirkle-type stationary phase.111 Two possible interactions are illustrated, showing the best orientations for maximum interaction. 

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. 

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. 

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. 

Occasionally, a binary mixture will not enable the separation to be attained. In this case a ternary mixture may be helpful. There is much in the literature about using more than one organic solvent in the mobile phase for attaining selectivity. Not only do chromatographers share insights about how and why solvents contribute to a separation, but there are also available software routines for computer-assisted development of mobile phases using... 

Case 3 involves the use of mobile-phase mixtures B/C, where B and C are polar, but only C can localize. The solvent B can also be a blend of a nonpolar solvent A with a pure solvent B (A/B), in which case we deal with ternary-solvent mobile phases A/B/C. The effect of restricted-access delocalization on values of e in mixtures B/C is illustrated in Fig. 12 (solid circles) for mobile phases composed of acetone (C) and benzene (B), with silica as adsorbent. The open circles are comparative data for acetone/hexane (A/C) mixtures. The same tendency ofec to increase for smaller Oc is found for acetone in mixtures B/C as is found for mixtures A/C. This is further confirmed by the data of Fig. 13, where %tc for several mobile phases B/C (B is benzene, C varying) are plotted versus dc- The precision of these experimental values of is much reduced in the case of mobile phases B/C in comparison to those of type A/C, as can be seen in... 

Binary mixtures, however, have only limited abilities for controlling mobile-phase selectivity. Therefore, ternary and even quaternary mobile phases that contain two or more different polar solvents along with a nonpolar solvent are often used to achieve the required selectivity. If the ratio of the concentration of two polar solvents is constant but the sum of the their concentration is being changed with respect to that of the nonpolar solvent, the effect on retention is much the same as when the concentration of the single strong solvent... 

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