Mobility polar components

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

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. 

Since the separation depends on the relative solubilities of the components in the two phases, the polarities of the components and of the stationary and mobile phases are important to consider. If the stationary phase is somewhat polar, it will retain polar components more than it will nonpolar components, and thus... 

There are two types of partition chromatography that are distinguishable based upon the relative polarities of the mobile and stationary phases. In normal phase chromatography, a highly polar stationary phase is used with a relatively nonpolar mobile phase. As a general principle in normal phase chromatography, the least polar components are eluted first and, increasing the polarity of the mobile phase decreases the elution time. 

In reverse-phase chromatography, the stationary phase is nonpolar (often a hydrocarbon) and the mobile phase is relatively polar (e.g., water, methanol, and acetonitrile). The most polar components elute first, and increasing the mobile phase polarity (i.e., decreasing the organic solvent concentration) increases elution time. 

Similar to other coupled methods of polymer HPLC, for example, LC CC (Section 16.5.2), the choice of the column packing and the mobile phase components for EG-LC depends on the retention mechanism to be used. Adsorption is preferred for polar polymers applying polar column packings, usually bare silica or silica bonded with the polar groups. The eluent strength controls polymer retention (Sections 16.3.2 and 16.3.5). The enthalpic partition is the retention mechanism of choice for the non polar polymers or polymers of low polarity. In this case, similar to the phase separation mechanism, mainly the solvent quality governs the extent of retention (Sections 16.2.2, 16.3.3, and 16.3.7). It is to be reminded that even the nonpolar polymers such as poly(butadiene) may adsorb on the surface of bare silica gel from the very weak mobile phases and vice versa, the polymers of medium polarity such as poly(methyl methacrylate) can be retained from their poor solvents (eluents) due to enthalpic partition within the nonpolar alkyl-bonded phases. 

With binary and ternary supercritical mixtures as chromatographic mobile phases, solute retention mechanisms are unclear. Polar modifiers produce a nonlinear relationship between the log of solute partition ratios (k ) and the percentage of modifier in the mobile phase. The only form of liquid chromatography (LC) that produces non-linear retention is liquid-solid adsorption chromatography (LSC) where the retention of solutes follows the adsorption isotherm of the polar modifier (6). Recent measurements confirm that extensive adsorption of both carbon dioxide (7,8) and methanol (8,9) occurs from supercritical methanol/carbon dioxide mixtures. Although extensive adsorption of mobile phase components clearly occurs, a classic adsorption mechanism does not appear to describe chromatographic behavior of polar solutes in packed column SFC. 

One weakness of the dominant reverse phase separations mechanism has been the poor retention of highly polar analytes, and hydrophilic interaction liquid chromatography (HILIC) has emerged as an alternative. In HILIC, a polar stationary phase such as silica gel is used to retain highly polar analytes. Mobile phases components similar to those described above for reverse phase separations are used, but the proportions of aqueous vs. organic are changed. Analytes are retained under conditions of relatively low water content, and eluted using increased water content. 

A similar separation occurs in the HPLC column. Either the mobile or stationary phase is polar and attracts the more polar component in the injected mixture. Let us assume that our column packing is polar and we are pumping a nonpolar mobile phase down the column. Both components have a partition affinity for the packing and will be retained. But the more polar of the two will be retained longer. Since equilibration is continuously being upset in favor of the moving liquid phase, the less polar component washes out faster and is eluted first from the column (see Fig. 1.3). Eventually, both compounds will wash off the column into the detector. 

The same purple mixture separated in our separatory funnel example is dissolved in the methylene chloride and shot onto the column through the injector. The two compounds to be separated are swept together onto the column. As fresh mobile phase causes them to pass down the column, the more polar component (the red dye) is more highly attracted to the polar column surface... 

For example, for a polar silica column equilibrated with a mobile phase of methylene chloride in hexane (nonpolar), you would dilute with more hexane to increase the k of relatively polar components. Adding methylene chloride, the more polar of the two solvents, would decrease k s causing all components to wash off faster. With k changes, peak position changes are proportional and in the same direction. The order of resolved peaks will remain the same unresolved peaks should begin to pull apart. 

The most common variable used to control a is the stronger solvent in the mobile phase. The stronger solvent is the mobile phase component most like the column in polarity. Changing the chemical nature of this stronger solvent will produce shifts in the relative peak positions. For instance, if we are unable to achieve the desired separation on a C18 column using acetonitrile in water, we can produce an a effect by shifting to methanol in water an opposite effect occurs on switching to tetrahydrofuran in water (Fig. 4.8). 

Eqn.(3.73) suggests that any mixture of two solvents with the same ° value (iso-eluotro-pic solvents) will also have the same eluotropic strength. This would allow the application of a similar strategy for the definition of iso-eluotropic multicomponent mobile phase mixtures as was used for RPLC in section 3.2.2.1. In practice, the situation in LSC has proved to be more complicated, because an effect described as solvent localization limits the validity of eqns.(3.72) and (3.73) if polar components (such as acetonitrile or methyl t-butyl ether) are present in the mobile phase. This makes it difficult to calculate the composition of iso-eluotropic mixtures for LSC with sufficient accuracy for optimization purposes [360-363]. 

Table 3.10b lists the optimization parameters that may be used in LLC. Clearly, the polarities of the two phases largely determine retention and selectivity. The exact composition of (preferably) the mobile phase may be varied to optimize the separation (i.e. variations in the nature and the concentration of mobile phase components, without substantial variations in the polarity). Even if the temperature is not a major optimization parameter, adequate temperature control is required in all LLC experiments. Therefore it may be experimentally straightforward to exploit temperature as a secondary optimization parameter. 

Table 3.10d lists the parameters for LSC. Again, most separations may be optimized by optimizing the eluotropic strength (primary parameter) and the nature (secondary parameter) of the mobile phase. The latter parameter involves the preparation of different iso-eluotropic mixtures containing different solvents, or small quantities of very polar components ( modulators ). As in the case of RPLC, there are several additional parameters that are not frequently exploited. 

Since the mobile phase is moving on a dry bed, several other undesirable effects occur. The adsorption of the first liquid (at the front) on the stationary phase is exothermic, causing the front to have a higher temperature than the rest of the system. Since the temperature of the system is not usually controlled but is allowed to assume the ambient value, some evaporation may occur at the solvent front. If the solvent is composed of a mixture of liquids, preferential evaporation of the most volatile one will cause a slight change in the solvent composition. In fact, the adsorption of a mixed mobile phase will probably also cause some changes in composition because the most polar component will be preferentially sorbed. The situation can become so severe that solvent demixing can occur. At best, a mixed solvent mobile phase is probably not uniform across the planar bed, and some temperature differentials probably exist as well. 

The silica gel used in TLC has the same properties as that used in LSC in columns, and the discussion about silica in Chapter 9 is relevant. In brief, the silica has a heterogeneous energy surface and many very active silanol groups. It picks up water from the atmosphere very readily and will preferentially adsorb the most polar component in a mobile phase mixture, as just described. Most of the discussion about LSC is also applicable to TLC. 

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