Acetonitrile, methanol, and tetrahydrofuran

Figure 4.4 Polarity, proton acceptor and dipole properties of water and organic modifier mixtures.----------------, acetonitrile, --, methanol, and---tetrahydrofuran.

Steps in method development (1) determine the goal of the analysis, (2) select a method of sample preparation, (3) choose a detector, and (4) use a systematic procedure to select solvent for isocratic or gradient elution. Aqueous acetonitrile, methanol, and tetrahydrofuran are customary solvents for reversed-phase separations. A separation can be optimized by varying several solvents or by using one solvent and temperature as the principal variables. If further resolution is required, flow rate can be decreased and you can use a longer column with smaller particle size. Criteria for a successful separation are 0.5 < < 20, resolution >2.0, operating... 

Note that the actual bonded moiety is typically a dimethylalkylsilane the Cig column is really precisely defined as having a dimethyloctadecylsilane bonded phase.) Those solvents used in conjunction with RP columns are called reversed-phase solvents. The most common RP solvents are mixtures of water with water-soluble solvents such as acetonitrile, methanol, and tetrahydrofuran. Uncommon cases are the use of nonaqueous solvents in reversed-phase separations. These are classified as NARP (nonaqueous reversed-phase) separations. An example of an NARP mobile phase would be a 50/50 v/v methanol/acetonitrile mixture. 

Katz, Lochmuller and Scott also examined acetonitrile/water, and tetrahydrofuran (THF)/water mixtures in the same way and showed that there was significant association between the water and both solvents but not nearly to the same extent as methanol/water. At the point of maximum association for methanol, the solvent mixture contained nearly 60% of the methanol/water associate. In contrast the maximum amount of THF associate that was formed amounted to only about 17%, and for acetonitrile the maximum amount of associate that was formed was as little as 8%. It follows that acetonitrile/water mixtures would be expected to behave more nearly as binary mixtures than methanol/water or THF/water mixtures. 

In gradient elution of weak acids or bases, gradients of organic solvent (acetonitrile, methanol, or tetrahydrofuran) in buffered aqueous-organic mobile phases are most frequently used. The solvent affects the retention in similar way as in RPC of nonionic compounds, except for some influence on the dissociation constants, but Equations 5.8 and 5.9 usually are accurate enough for calculations of gradient retention volumes and bandwidths, respectively. 

C18 column from different suppliers (3-5 pm of particle size) are commonly used for amino acid determination, with mobile phases consisting of an acetate or phosphate buffer and an organic solvent such as acetonitrile, methanol, or tetrahydrofuran. To avoid peak tailing for the basic amino acids (in the presence of uncapped silanols), triethylamine is often added as a modifier. Depending on the complexity of the sample, complicated tertiary gradients can be necessary to separate all the researched amino acids [196],... 

Solvent systems encompass a dizzying array of permutations of organic solvents, buffers, and other mobile-phase additives. However, the most commonly employed solvent systems involve acetonitrile, methanol, and/or tetrahydrofuran. Buffers are typically acetate (pKa 4.8) or phosphate (pKa 1.3 and 6.7) at approximately 100 mM. For the analysis of a small number of free amino acids, isocratic elution is often possible. For the determination of an overall amino acid profile from a hydrolysate sample, complicated ternary gradients are often necessary. 

If this information is not available, I will try to separate the mixture using a Ci8 column in acetonitrile and water. Something like 70% of the separations in the literature are now made on a C18 silica-based column. Acetonitrile is my solvent of choice because of its low wavelength transparency, its polarity, and its intermediate position between methanol and tetrahydrofuran. Generally, I will use 254nm for the detector because the majority of the literature separations can be made at this wavelength (see the Separations Guide in Appendix A). 

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. 

TABLE 11. Second-order rate constants (in units of 108 M 1 s 1) for reaction of (l-sila)hexatrienes 21a-c with acetone, methanol (MeOH) and 2,2,2-trifluoroethanol (TEE) in isooctane (OCT), acetonitrile (MeCN) and tetrahydrofuran (THF) solution at 23 °C47,48... 

The infrared spectrum (Nujol mull) of Na2[ReH9] has URell) at 1835(s), (br) and 5(ReH) at 745(s), ca. 720(sh), and 630(sh) cm.-1. In aqueous alkali rRe-H = 19.1. The compound is soluble in water and methanol, slightly soluble in ethanol, and insoluble in 2-propanol, acetonitrile, ether, and tetrahydrofuran. Alkali stabilizes the water and methanol solutions. With acids... 

The mobile phase in RPC contains water and one or more water-soluble organic solvents. The most useful are, in order of decreasing polarities, acetonitrile, methanol, dioxane, tetrahydrofuran and propanol. By the choice of the type of the organic solvent, selective polar interactions, dipole-dipole, proton-donor or proton-acceptor, with analytes can be either enhanced or suppressed and the selectivity of the separation adjusted. For simplicity, binary mobile pha.ses are used more often than those containing more than one organic solvent in water, as they often make possible an adequate separation of various samples. However, ternary or less often quaternary mobile phases offer advantage of fine-tuning the optimum selectivity of more difficult separations. This is discussed in more detail in Section 1.4.6. 

It should be noted that most bonded-phase silica-based columns are less stable outside the pH range 2 to 8. A buffer suitable for HPLC should be transparent at the detection wavelength if UV-detection is to be used, should be stable, inert to the HPLC system and should not chemically react with the sample or with the mobile phase. The retention is adjusted by the addition of a moderate concentration of organic solvent to the mobile phase (up to 30-4098 acetonitrile, methanol or tetrahydrofuran. according to the solubility limits). 

As in normal phase (see section 3.5.3), the first step in mobile-phase optimization is the determination of the solvent strength that will elute the analytes with a A value between 2 and 10 from the chosen stationary phase. It is not important which modifier is chosen to determine the initial conditions, and methanol-water (50 50, v/v) is a convenient starting place. Once the initial conditions have been established, a variety of techniques may be employed to obtain the optimum separation. Most optimization strategies involve the establishment of the isoelutropic concentrations of methanol-water, acetonitrile-water and tetrahydrofuran-water. The isoelutropic concentrations can be determined by experiment or from tables of isoelutropic mixtures (e.g. Table 3.5) (Wells, 1988). The binary solvent systems A, B, C (Table 3.5, Figure 3.7) define the isoelutropic plane, which is then explored to obtain the optimum combination of water, methanol, tetrahydrofuran, and acetonitrile required for the separation. 

In mobile phases containing 10-50 mM phosphate or acetate buffers of pH 2-8.5, the ionization of weak acids (at pH <7) or bases (at pH >7) can be more or less suppressed to improve separation and peak symmetry. By adjusting the pH in the range 1.5 U around the differences in the degree of ionization of the individual sample components can often be utilized to control the separation selectivity. The retention is usually adjusted by the addition of up to 30-40% acetonitrile, methanol, or tetrahydrofuran to the mobile phase. 

Polar solvents are normally used alternatives to water include, acetonitrile, propylenecarbonate, methanol, and tetrahydrofuran. Dimethylformamide is not so good but has been used despite some... 

Over 500 HPLC packings have been described in the Hterature. Nevertheless, as the result of years of development, only a limited number of types of stationary phases remain on the market. Most of the conventional HPLC separations today are performed using monodisperse silica gel 3 or 5 Xm microbeads, especially those grafted with C4, C8, or C18 alkyl chains, as well as with cyano-propyl or amino-propyl groups. The last two bonded silicas and bare silica are used in normal phase (NP) HPLC, where the mobile phase (usually hexane with small amounts of isopropyl alcohol) is less polar than the stationary phase. Even more popular is the reversed phase (RP) mode, which uses polar eluents (mosdy water or methanol with such additives as acetonitrile, methanol, or tetrahydrofuran (THE)) in combination with nonpolar alkyl-bonded stationary phases. 

The effect of organic modifiers such as acetonitrile (ACN), ethanol, isopropanol, methanol, and tetrahydrofuran on CEC separations is quite complicated. Organic modifiers added to an aqueous buffer can increase the solubility of neutral and nonpolar compounds, but at the same time yield some EOF effects in concert with changes in retention factors. 

This thinking has carried through to the present day and is reflected in our choices of mobile-phase fluids in LC water, acetonitrile, methanol, tetrahydrofuran, hexane, etc., are still among our popular choices. However, these particular materials are completely dependent on the conditions of column temperature and outlet pressure. Tswett s original conditions at his column outlet, actually the earth-bound defaults we call ambient temperature and pressure, determined his solvent choices and continue to dominate our thinking today. 

Bonded phases are the most useful types of stationary phase in LC and have a very broad range of application. Of the bonded phases, the reverse phase is by far the most widely used and has been applied successfully to an extensive range of solute types. The reverse phases are commonly used with mobile phases consisting of acetonitrile and water, methanol and water, mixtures of both acetonitrile and methanol and water, and finally under very special circumstances tetrahydrofuran may also be added. Nevertheless, the majority of separations can be accomplished using simple binary mixtures. 

The mobile phases that are most effective for use with reverse phases are aqueous mixtures of methanol or acetonitrile and for subtle adjustments, ternary mixtures of water, methanol and acetonitrile or tetrahydrofuran can be used. The greater the water content the more the solutes with dispersive groups will be retained and in fact, in pure water, many substances are irreversibly held on a reverse phase. As already discussed, this characteristic make reverse phases very useful for solute extraction and concentration from aqueous solutions prior to analysis. 

On the assumption that = 2, the theoretical values of the ion solvation energy were shown to agree well with the experimental values for univalent cations and anions in various solvents (e.g., 1,1- and 1,2-dichloroethane, tetrahydrofuran, 1,2-dimethoxyethane, ammonia, acetone, acetonitrile, nitromethane, 1-propanol, ethanol, methanol, and water). Abraham et al. [16,17] proposed an extended model in which the local solvent layer was further divided into two layers of different dielectric constants. The nonlocal electrostatic theory [9,11,12] was also presented, in which the permittivity of a medium was assumed to change continuously with the electric field around an ion. Combined with the above-mentioned Uhlig formula, it was successfully employed to elucidate the ion transfer energy at the nitrobenzene-water and 1,2-dichloroethane-water interfaces. 

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