Normal-phase system

This is because the increased turbulence from higher flow rates decreases the possibility for inclusion complexation, a necessary event for chiral recognition in reversed phase. Some effect has also been observed in the new polar organic mode when (capacity factor) is small (< 1). Flow rate has no effect on selectivity in the typic normal-phase system, even at flow rates up to 3 inL miir (see Fig. 2-11). 

A uniform film of analyte, which is required for the production of good quality spectra, can usually be obtained from mobile phases which contain predominantly organic solvents (normal-phase systems). As the percentage of water in the mobile phase increases, however, droplets tend to form on the belt, irrespective of the belt speed. If the belt is not exactly horizontal, and this is often the case, especially after it has been in use for some time, the droplets are likely to roll off the belt and be lost, thus reducing the overall sensitivity of the analysis dramatically. 

There are two commonly used ways to elute a given compound in HPLC the normal-phase mode (t)s><5m) and the reversed-phase mode (<5m><5s). Reversed-phase systems offer superior general selectivity. Solutes are eluted in ascending order of polarity in normal-phase systems and in descending order of polarity in reversed-phase systems. 

There are a number of HPLC methods available for determining AES. Generally, normal-phase systems are used for the determination of EO... 

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

Based on the high peak capacity of CE, the separation speed, and the availability of numerous chiral selectors and the simplicity of the systems, chiral CE is superior to chiral HPLC separations. This is as well reflected by the high number of publications on chiral CE in recent years. Chiral HPLC is suffering from low peak capacity (broad peaks), system stability (often normal phase systems), pressure sensitivity of columns (often cellulose-based column materials), and as a consequence long separation times. 

The mobile phases used for chiral separations on CSPs differ depending on the type of column and range from normal-phase systems containing high amounts of nonpolar solvent (e.g., hexane) to... 

I Liquid-liquid partition chromatography, where the sample components are partitioned between a moving liquid phase and a stationary liquid phase deposited on an inert solid. The two solvent phases must be immiscible. The stationary phase may he a large molecule chemically bonded lo the surface of a solid (bonded liquid phase) lo prevent loss by solubility in the moving phase. This method can also be subdivided into normal-phase systems, in which Ihe moving phase is less polar than the stationary phase, and reverse-phase systems, in which it is more polar. 

Retention characteristics and elution order of carotenoid cis-trans isomers with C30-bonded phases are strikingly similar to those obtained with normal-phase systems using calcium hydroxide columns (190). Different carotenoids exhibit varying retention behavior in response to temperature changes for C30 and C34 polymeric stationary phases as compared with a Cl8 polymeric phase (179). These behaviors are believed to be related to conformational changes in the longer stationary phases with temperature. The slot model proposed for the retention of planar... 

Another property that is useful in selecting the proper chromatographic conditions (column, mobile phase, etc.) is water solubility or distribution coefficient between a polar (e.g., acetonitrile) and a nonpolar (e.g., -heptane) solvent. Data on water solubility and UV absorption maxima for a large number of NOC can be obtained from Druckrey et al. (25). Eisenbrand et al. (26) reported the distribution coefficients between acetonitrile and n-heptane for several NOC. Those for V-nitroso-dioctylamine, NDMA, and V-nitrosomethyl-2-hydroxyethylamine were reported to be 0.5,17.3, and 32.0, respectively, in this system. This would suggest that in a normal-phase system, using a silica column, these compounds would elute in the same order in which they are mentioned, but the elution order would be reversed in a reversed-phase (C18 column) system. 

The choice of the proper stationary and mobile phases for the foregoing purpose would depend on several factors, such as the nature (polarity, stability in mobile phase) of the NOC analyzed and the availability/compatibility of the detector used. For example, if only a TEA is available as a detector, the use of an ion-exchange or a reversed-phase system is ruled out, because both require aqueous mobile phase for proper operation. Moisture in the mobile phase causes freeze-up of the cold traps in the TEA and also results in noisy response due to interference during chemiluminescence detection. Similarly, if one is using, as the detector the newly developed Hi-catalyzed denitrosation-TEA (62) or the photolytic cleavage-TEA (58), a reversed-phase system using aqueous mobile phase would be the method of choice. These detectors, however, have not been demonstrated to work in the normal-phase system. The use of an electrochemical detector will also be incompatible with an organic solvent as the mobile phase. 

NDPHA, A-nitrosoatrazine, A-nitrosobenzylphenylamine, A-nitrosocarbazole, and A-nitroso-carbaryl in foods. In another study, Sen et al. (67) used a normal-phase system for the determination of NDBZA in cured pork products packaged in elastic rubber nettings (Fig. 4). As can be seen from the figure, appropriate solvent programming allowed the simultaneous determination of six nonpolar and three polar NOC. 

By definition, the relative retention is larger than (or equal to) 1. Thus, for a normal phase system, where 8S > 5m, it follows from eqn.(3.31) that 5- > Sp and hence the more polar solute will elute last. Again, the reverse is true for a reversed phase system. Because the signs of the two factors in eqn.(3.31) which involve solubility parameters will always be thesame, we may state that it is the absolute difference between the polarities of the two phases that should be maximized. Therefore, the selectivity of a phase system (V) may be defined as... 

In LLC systems there is not a substantial difference between the selectivity characteristics in the normal phase and the reversed phase mode. The choice of either will mainly be determined by the sample. Polar samples (in polar solvents) will preferably be injected in a reversed phase system and non-polar samples in a normal phase system. 

A major advantage of the use of normal phase systems may be the possibility to use UV-absorbing (or even fluorescent) pairing ions for the separation of non-UV absorbing solutes. If a UV absorbing pairing ion is in the (aqueous) stationary phase and if this is subsequently eluted from the column as an ion-pair in the presence of sample ions, then a very sensitive detection may be possible (see e.g. ref. [379]). 

Since the samples run in prep LC are often very crude, it is to be expected that the column packing will become contaminated with chemicals that did not elute. Cleaning can be accomplished with polar solvents like propanol or ethyl acetate (for normal phase systems), or the packing can simply be discarded. The latter alternative may be cheaper, considering the cost of solvents and waste disposal. 

Before applying this strategy to a normal phase system, we need to consider some additional procedures that have become part of the optimization process. 

Frequently the mobile phase is a mixture of liquids chosen and optimized by trial and error because results can be obtained so quickly and easily. The principles of mobile phase selection discussed in the last chapter are, of course, relevant. For normal phase systems on silica gel, a nonpolar... 

With polar liquid-liquid adsorption chromatography, based on chemically bonded normal-phase systems, the distribution coefficient can be equated with the solubility parameter 8j of a solute such that retention is given by... 

Loro, K. A., Orlan, R., Zhang, R, Usherwood, P. N. R., and Nakanishi, K. (1993). Anal. Biochem. 215, 38-44. Separation of the sticky pqjtides from membrane proteins by HPLC in a normal-phase system. Author s note. We were unable to solubilize either DGK3M or any of the polyamino acids in the solvent systems reported in their work, suggesting their method may not be completely general. 

A single solvent only rarely provides suitable separation selectivity and retention in normal-phase systems, which should be adjusted by selecting an appropriate composition of a two- or a multi-component mobile phase. The dependence of retention on the composition of the mobile phase can be described using theoretical models of adsorption. With some simplification, both the Snyder and the Soczewinski models lead to identical equation describing the retention (retention factor. A) as a function of the concentration of the stronger (more polar) solvent, (p. in binary mobile phases comprised of two solvents of different polarities [,121 ... 

Reproducibility of gradient-elution retention data in normal-phase systems with mobile phases comprised of two organic solvents — a polar and a non-polar one — depends on a number of experimental factors that should be controlled. To get reproducible results it is necessary to keep a constant adsorbent activity and to control the water content in the mobile phase [24]. The best way is to use dehydrated solvents kept dry over activated molecular sieves and filtered just before use [88]. It is very important to work at a constant temperature (using a thermostatted column). 

On the other hand, different approaches should be used in reversed-phase and in normal-phase systems for the prediction of retention with the ternary selectivity gradients where the concentration of solvent 1 is increased and the concentration of solvent 2 simultaneously decreased at a constant (pj. In reversed-phase systems where Eq. (1.18) describes the retention both in binary mobile phases comprised of water and organic solvent 1 and in mobile phases containing water and organic solvent 2 ... 

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