Normal-Phase Solvents

Four frequently used normal-phase (NP) solvents will be discussed in detail hexane, dichloromethane, isopropyl alcohol, and ethyl acetate. Hexane is one of the most frequently used NP solvents. As in the case of acetonitrile, the low UV cutoff wavelength of 195 nm and the low background absorbance at and above 210nm make hexane an excellent choice for the majority of compounds analyzed in the NP mode (see Fig. 1.2). (Note that cyclopentane, cyclohexane, and heptane have similar UV characteristics.) In addition, under normal circumstances hexane has excellent long-term chemical stability. Because of the limited solubility of hexane, and of related hydrocarbons, in water and acetonitrile, their use in reversed-phase separations is rare. 

The nonpolar character of hexane and the alkanes severely limits their ability to solubilize polar compounds. Since the NP support material typically has a polar surface (such as silica), some polar mobile phase component is needed to elute the solute. Therefore, a low level (typically 5C5% v/v) of a more polar component, such as dichloromethane or ethyl acetate, often needs to be included as part of the mobile 

Dichloromethane has a UV cutoff of 233 nm and is effectively used for compounds with chromophores with 250 nm. However, since it is used at such low levels in NP work, absoifoance is rarely an issue. Dichloromethane (and chloroform) is unstable and degrades through a free-radical process. Amylene and cyclohexene are commonly used chloroalkane preservatives. Although commonly used as a low-volume mobile phase component in NP separations, dichloromethane has found only limited use in RP work because of its low water solubility. Conversely, solubility with alkanes and good sample-solubilizing characteristics make dichloromethane a very useful component in NP mobile phases. 

Ethyl acetate is of intermediate solvent strength to dichloromethane and IPA. A major drawback is the very high UV cutoff of 256 nm. An advantage, though, is that it is stable and unreactive in normal HPLC use. It is immiscible with water and has limited use in RP methods. 

Acetone is included in this discussion not because it is frequently used in either RP or NP separations but because (1) it has excelloit solubilizing characteristics, evident through the fact that it is often referred to as a universal solvent, and (2). it exemplifies a spectral characteristic for ketones that may be of particular impcHtance, albeit for a limited number of separations. 

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While partially concurrent eluent evaporation is easier to use, and is preferred for the transfer of normal phase solvents, concurrent eluent evaporation with co-solvent trapping is the technique of choice for transfer of water-containing solvents, because wettability is not required. 

This phase is also highly loadable and has been used in SFC where some exceptional loadings have been achieved. In LC conditions, normal-phase solvents, such as hexane-ethanol or isopropanol, can be used. 

In the other method (h), the effluent from the HPLC column was mixed with a mixture of concentrated H2S04, glacial acetic acid, and KI solution, and the mixture was passed through a Teflon reaction coil kept immersed in a 70-80°C water bath. As before, the carrier gas, introduced after denitrosation, carried the liberated NO through a series of cold traps into the TEA. The system worked well with aqueous mobile phases but not with normal-phase solvents. Response for NPRO was linear from 3.5 to 900 ng injection, with a coefficient of variation of 3-5%. The temperature of the reaction coil seemed to be critical for the determination of A-nitro-samines, which are difficult to denitrosate with HI. While a temperature of 23°C was adequate for the denitrosation of NPRO and several A-nitrosoureas, a much higher temperature (up to 70°C) was required for comparable response from NDMA or NPYR. 

One advantage of supercritical solvents when compared to conventional organic normal-phase solvents is that the eqnilibration rate after changing conditions is mnch more rapid even on interactive snrfaces, such as silica (Steuer et al., 1988). The solvent power of an SCF is strongly linked to its density (controlled by pressnre and temperatnre) and can also be adjnsted by addition of an organic solvent (referred to... 

Commonly used reversed-phase LC solvents, including water, acetonitrile, and methanol, are ideal for LC/MS. All reversed-phase solvents need to be degassed prior to LC/MS analysis to maintain the stability of ion signals. This can be achieved by sonihcation, helium sparging, or vacuum membrane degassing. When solvents of high aqueous content are to be used, the source and probe temperatures should be raised to assist desolvation in the ion source. Normal-phase solvents such as dichloromethane, hexane, toluene, and other hydrocarbons are not suitable for ESI-MS because a polar mobile phase for ionization is needed in ESI. These normal-phase solvents and their typical solutes are sufficiently volatile to be analyzed by APCI and work well with APCI-MS. 

When using NP the chromatographer must remember to convert then-system to NP mode if RP mode was used previously. Any aqueous/buffer left in the system could precipitate out when the normal-phase solvents are pumped than the system. Water contamination in the mobile phase lines can also lead to water absorption on the column and change the chromatography significantly. It is generally recommended, that if the system was previously in RP mode, to flush the system with pure water for about 15 minutes at 2 mL/min. Then use IPA to flush the system for an additional 10 minutes at 2 mL/min. The system should then be flushed with the desired NP mobile phase for 5 minute at 2mL/min. Then the NP column can be installed and equilibrated with the NP mobile phase. 

The constants a, y can be determined from three experimental values of retention factors, k, A/ and Two of these values can be. selected to represent the data in binary mobile phases with the concentrations

ternary mobile phase, A at X = 0 and Ai at = 1. Only one experimental value. A , should be determined experimentally in a single ternary mobile phase at a concentration ratio X,. From A], At and Aj the constants a, /3, y can be calculated using Eiqs. (1.27)-( 1.29) and introduced into Eq. (1.26) to make possible prediction of retention in temaiy normal-phase solvent systems ... 

TLC is a good technique to use when normal-phase solvents provide optimum separation. Typical thin-layer separations are performed on glass plates that are coated with a thin layer of stationary phase. The stationary phases used in TLC encompass all modes of chromatography including adsorption, normal- and reverse-phase, ion-exchange, and size-exclusion." The equipment required is simple and inexpensive. TLC is an ideal technique for the isolation of compounds because of its simplicity. However, for TLC to be successful, the impurity and/or degradant level should be at or above 1%. Any component present below this level is very difficult to isolate on a TLC plate because of higher detection limits. 

The choice of solvent directly influences the retention of the analyte on the sorbent and its subsequent elution, whereas the solvent polarity determines the solvent strength (or ability to elute the analyte from the sorbent in a smaller volume than a weaker solvent). The relative solvent strengths for normal- and reversed-phase sorbents are illustrated in Table 8.2. Obviously, this is the ideal. In some situations, it may be that no individual solvent will perform its function adequately so it is possible to resort to a mixed-solvent system. It should also be noted that for a normal-phase solvent, both solvent polarity and solvent strength are coincident, whereas this is not the case for a reversed-phase sorbent. In practice, however, the solvents normally used for reversed-phase sorbents are restricted to water, methanol, isopropyl alcohol and acetonitrile. 

Fig. (1). HPLC profile [normal phase solvent (B)] of the Raumuria hirtella extract.

One significant commercial development which has been taking place is that it has been demonstrated that macrocyclic antibiotic CSP may be used successfully with normal phase solvents, mixtures of n-hexane and ethanol being the most frequently used. Most of the illustrative examples cited in the commercial literature feature non-polar analytes not all of which are of pharmaceutical interest (Fig. 3.6) but nonetheless it is a good selling point that these CSP may be used with the complete range of mobile phase polarities. 

Normal-phase solvents are especially volatile. This is an advantage for preparative chromatography, but also can cause trouble, if this is not taken into consideration in analytical chromatography. Retention times can shift as a result of selective evaporation of some components of the mobile phase. Also, safety should be considered. Some normal-phase solvents, especially hydrocarbons, are highly flanunable others have adverse health effects. Consult the applicable matoial safety data sheets. All containers including waste containers should be closed. When not in use, the mobile-phase container should be sealed. 

This type of material, obviously, can be somewhat labile in aqueous solvents and would only be usable under certain buffered conditions. However, when this stationary phase is used with normal phase solvents (dispersive or hydrophobic solvents) the system is stable. The obvious next stage was to couple the protected glycine directly to the amino propyl group using dicyclohexyldiimide as the coupling agent. This reaction proceeds as follows. 

As a rule of thumb, the diffusion coefficients of small molecules in normal-phase solvents can be assumed to be 2.5 x 10 cm min , in reversed-phase... 

FIGURE 1.2 Typical absorbance-wavelength curves for common normal-phase solvents hexane, ethyl acetate, and dichloromethane. 

This is not the case when low levels of volatile modifiers are used. The greatest problems arise with the use of extremely volatile low-level mobile phase modifiers (MPMs), such as trifluoroacetic acid and triethylamine in reversed-phase solvent systems and ethyl acetate or dichloromethane components in normal-phase solvent systems. These low-level MPMs are typically used at the 0.1-1.0% v/v level. A continuous sparge over the course of the day will greatly reduce their concentrations via volatilization, and dramatic changes in peak retention and peak shape can result (see Fig. 1.13),... 

Absorbance vs. wavelength Dissolved O2 in solvents Effects of gradient work, 7,18 General, 3-9 Normal-phase solvents... 

These derivatives have demonstrated selectivity in a variety of mobile phase conditions. They offer some unique capabilities, especially in typical normal phase solvents. In a study of a variety of chiral pterocarpans, all enantiomers could be... 

The CB-DMP has the ability to operate in a typical normal phase solvent but the majority of mobile phases found successful in the literature for this phase have been the polar organic mode followed closely by the reversed-phase mode. The same has been true for the CB-DNP. The number of successful enantioseparation in a typical normal phase conditions has been reported but limited. It appears again that the utility of steric hindrance places a very substantial and successful role for these new derivatives. 

Gradient from 90 5 5 to 65 17,5 17,5 n-heptane/CH2Cl2/ MeOH in 190 min Various isocratic mixtures of n-hexane/ethyl acetate Various isocratic mixtures of -hexane/ethyl acetate other normal phase solvents also investigated... 

Park, J.H. Carr, P.W. (1989). Interpretation of normal-phase solvent strength scales based on linear solvation energy relationships using the solvatochromic parameters n, a and p. /, Chromatogr.,3, 465, 123-136. 

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