Mobile phase polarity

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. 

The major parameter for column selection is the intended application. A balance of mobile-phase polarity in comparison with the polarity of the stationary... 

Table 7.89 lists the main characteristics of MDHPLC (see also Table 7.86). In MDHPLC the mobile-phase polarity can be adjusted in order to obtain adequate resolution, and a wide range of selectivity differences can be employed when using the various available separation modes [906]. Some LC modes have incompatible mobile phases, e.g. normal-phase and ion-exchange separations. Potential problems arise with liquid-phase immiscibility precipitation of buffer salts and incompatibilities between the mobile phase from one column and the stationary phase of another (e.g. swelling of some polymeric stationary-phase supports by changes in solvents or deactivation of silica by small amounts of water).

Mobile phase polarity Low-medium Medium-high... 

To increase retention of solutes Decrease mobile phase polarity Increase mobile phase polarity... 

Eor the analysis of petroleum hydrocarbons, a moderately polar material stationary phase works well. The plate is placed in a sealed chamber with a solvent (mobile phase). The solvent travels up the plate, carrying compounds present in the sample. The distance a compound travels is a function of the affinity of the compound to the stationary phase relative to the mobile phase. Compounds with chemical structure and polarity similar to those of the solvent travel well in the mobile phase. For example, the saturated hydrocarbons seen in diesel fuel travel readily up a plate in a hexane mobile phase. Polar compounds such as ketones or alcohols travel a smaller distance in hexane than do saturated hydrocarbons. 

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. 

An important challenge for pumping systems represent the frequent changes of mobile phase polarity and viscosity, especially in the course of the coupled polymer HPLC experiments. If possible, a separate SEC instrument should be under operation for each particular eluent. 

T.A. Berger, and J.F. Deye, "Effect of Mobile Phase Polarity." accepted by J.Chromatogr. Sci. 

The reverse is true for the line drawn in figure 3.7 with a negative slope (Ss < Sm), and for this reason this system is called a reversed phase (RP) system. The particular line in figure 3-7 connects a typical non-polar (alkane-like) phase with Ss=7 to a polar mobile phase with Sm = 18. Such a mobile phase could for instance be created by mixing methanol (5 16) with water (5w 25) in the correct proportions. Since a very wide range of mobile phase polarities can be covered with mixtures of methanol and water, or even tetrahydro-furan (THF Sx 10) and water, the reversed phase system is a very flexible one. Without changing the column (stationary phase), it can be applied for the elution of a wide variety of solutes. 

Alongside the two lines drawn in figure 3.7 as examples for phase systems, dotted areas are indicated towards the mobile phase axis. This is done to indicate that, when eqn.(3.30) is strictly obeyed, a small capacity factor is expected to result. By choosing the mobile phase polarity slightly further away from that of the solute, the capacity factor can be moved into the optimum range. However, the margin thus created is usually very small and... 

Clearly, this is an impossible proposition. Practical mobile phase polarities will be restricted to the range between 7 (for alkanes) and Sm = 25.5 (for water). 

However, the polar adsorbent may also be combined with an even more polar mobile phase up to a mobile phase polarity of 25.5 and a solute polarity of about 21. Hence, this line with positive slope in figure 3.8 represents the use of polar adsorbents in the reversed phase mode for the separation of very polar solutes. 

The ionic strength of the eluent will affect the retention of both neutral and ionized species. For non-charged molecules the effects of increasing the ionic strength of the eluent can be understood as an increase in the mobile phase polarity, leading to an increase in retention ( salting-out effect ). 

M c Mobile phase polarity (modifier content) In k vs.

[Pg.108]

RPLC Mobile phase polarity Solvent programming (gradient elution)... 

RPLC Mobile phase polarity pH Nature of modifier(s) stationary phase... 

Bonded phases may be used in both normal and reverse phase chromatography. When normal phase chromatography is done on bonded phase packings, the packing is more polar than the mobile phase. Polar bonded phases such as the cyanopropyl and aminopropyl functionalities are popular for this use. These bonded phases are less subject to changing retention times of compounds because water is adsorbed from the mobile phase onto the stationary phase, a frequent concern when using bare silica packings for normal-phase separations. 

FIGURE 5-51. Effect of mobile phase polarity on retention of phthalate plasticizers. Column juPorasil (silica, 10 p,m), 3.9 mm ID x 30 cm flow rate 2 mL/min detector UV at 254 nm. Mobile Phase (a) is 5% ethyl acetate in isooctane, (b) is 5% butyl acetate in isooctane. 

An example of the influence of mobile-phase polarity upon the retention and selectivity of sample molecules is shown in Figure 5-51. In normal phase, the most polar compound is retained the longest. This is reflected by the observation that the dimethyl phthalate is the most polar and is retained the longest. By changing from ethyl acetate to butyl acetate, the overall mobile phase is less polar and, hence, all of the compounds increase in retention. However, in addition, there is one change in selectivity between the diethyl and the diphenyl molecules. 

Columns For most analyses, separation is achieved by partition of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and nonpolar mobile phases are described as normal phase, while the opposite arrangement, polar mobile phases and nonpolar stationary phases, is called reversed-phase chromatography. Partition chromatography is almost always used for hydrocarbon-soluble compounds of a molecular weight that is less than 1000. The affinity of a compound for the stationary phase, and thus its retention time on the column, is controlled by making the mobile phase more or less polar. Mobile phase polarity can be varied by the addition of a second, and sometimes a third or even a fourth, component. 

In common with other polar solutes, peptide-nonpolar stationary phase interactions can be discussed in terms of a solvophobic model. In this treatment solute retention is considered to arise due to the exclusion of the solute molecules from a more polar mobile phase with concomitant adsorption to the hydrocarbonaceous bonded ligand, where they are held by relatively weak dispersion forces until an appropriate decrease in mobile-phase polarity occurs. This process can be regarded as being en-tropically driven and endothermic, i.e., both AS and AH are positive. 

In the case of ion-pair complexes between the chiral additive and the enantiomeric analytes, their interaction should be maximized by adjusting the mobile-phase polarity. Solvents of lower dielectric constant favor ion-pair formation. 

When increasing the aqueous content in the mobile phase polar templates usually become less retained on MIPs, whereas templates of moderate to low polarity become more retained. The latter increase in retention is due to the hydrophobic effect [32, 145]. Thus, in contrast to the behaviour of other types of... 

The introduction of the chemically bonded apolar reversed phase materials as stationary phases is responsible for the common use of HPLC in routine laboratory analysis, because the sample can be applied directly from the aqueous solution into the HPLC-system. For the mobile phase, polar solvents such as water or aqueous buffer solutions or gradient mixes of aqueous solvents with methanol or acetonitrile are used mainly. 

In normal-phase chromatography, the least polar component is eluted first increasing the polarity of the mobile phase decreases the elution time. In contrast, with reversed-phase chromatography, the most polar component elutes first, and increasing the mobile-phase polarity increases the elution time. 

Most chiral HPLC analyses are performed on CSPs. General classification of CSPs and rules for which columns may be most appropriate for a given separation, based on solute structure, have been described in detail elsewhere. Nominally, CSPs fall into four primary categories (there are additional lesser used approaches) donor-acceptor (Pirkle) type, polymer-based carbohydrates, inclusion complexation type, and protein based. Examples of each CSP type, along with the proposed chiral recognition mechanism, analyte requirement(s), and mode of operation, are given in Table 3. Normal-phase operation indicates that solute elution is promoted by the addition of polar solvent, whereas in reversed-phase operation elution is promoted by a decrease in mobile-phase polarity. 

The capillary format of SFC is attractive because of the potential of interfacing with a wide array of detectors available when carbon dioxide is used as the mobile phase. Several advances, beyond the issue of mobile-phase polarity, are, however, required prior to the technique becoming viable for pharmaceutical analyses. Capillary SFC instrumentation has lacked the requisite analytical performance for pharmaceutical analyses, and difficulties are encountered due to the acidic nature of fused silica and the problem of measuring impurities while, at the same time, not overloading the stationary phase with the main component. ... 

The best mixture (with respect to retention) for separating ten phenols by reversed-phase chromatography was found to be water-methanol (60 40), represented by chromatogram (J). The mobile phase polarity can be calculated as foUows ... 

In practice, separation of enantiomers by the use of chiral stationary phases is not free from problems. Chiral stationary phases are difficult to prepare reproducibly, are sometimes of lower chromatographic efficiency than expected, and optimization of separation conditions is restricted by the fixed nature of the chiral centres. Chiral mobile phases are free from many of these problems, optimization of the separation is more convenient, and conventional reversed-phase columns may be used. Thus N-(2, 4-dinitrophenyl)-L-alanine-n-dodecyl ester has been used as a non-ionic chiral mobile phase additive for the resolution of 1-azahexahelicenes by reversed-phase chromatography. The resolution obtained was found to be a function of the mobile phase polarity and the concentration of chiral additive used. 

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