Polar and Hydrophobic RP-Phases

What do these results mean The greater the difference in the corresponding numbers of the compounds, the more easily these compounds can be separated. For example, two substances with similar properties but different dipole moments and different log P values are probably easy to separate on polar phases, whereas a greater difference between the heats of formation of two compounds could indicate a good selectivity for their separation on hydrophobic phases. 

In the following, polar and apolar RP-phases are presented in brief and thereafter their suitability for the separation of specific classes of substances is discussed. Finally, some proposals with regard to column choice are given. 

For various reasons, the following types of phases show a more or less polar character  

Considering several criteria and a weighting, which need not be explained here in detail, the following phases can be denoted as some of the most polar types. These phases also show a low affinity for hydrophobic analytes and an enhanced selectivity for strong ionic pairs of analytes in buffered eluents. 

Fluofix INW Fluofix lEW Platinum EPS Hypersil ADVANCE SynergiPOLAR RP 

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Fig. 35. On the selectivity of polar and hydrophobic RP-phases for the separation of polar and apolar compounds for comments, see text.

Table 4. Polar and hydrophobic versions of RP-Phase based on the same silica gel.

Fig. 39. Separation of 4-hydroxy-/3-hydroxybenzoic acid on a polar (upper chromatogram) and on a hydrophobic RP-phase (lower chromatogram) for comments, see text.

Compound retention during RP-HPLC depends on the relative hydrophobicity of the sample compounds. As expected, the elution of phenolics for reversed-phase HPLC is in the order of decreasing polarity. Polarity is increased most by hydroxyls at the 4-position, followed by those at the 2- and 3-positions. Availability of the methoxy group and the acrylic substitution reduces polarity and increases retention times (4). Loss of polar hydroxy groups and/or addition of methoxy groups can decrease the polarity within each class of benzoic and cinnamic acid. Also, the presence of the ethylenic side chain in the cinnamic acids can reduce their polarity compared with similarly substituted benzoic acids (6). The elution order for benzoic acids is as follows (Table 1) gallic > a-resorcylic > protocatechuic > y-resorcylic > gentisic > p-hydroxyben-... 

In the RP CEC of neutral species selectivity is provided primarily by differences in the partition of the analytes between the hydrophobic stationary phase and the more polar mobile phase. There are also contributions from interactions with the silica support, the major one being polar interactions with ionised silanol groups. This is identical to the process in LC, albeit with the advantages of higher efficiencies in CEC resulting from the plug-flow profile. Additional selectivity is introduced in the case of charged species in CEC due to differences in the analytes electromobilities. 

Classical liquid chromatography is typically practiced in what is referred to as the normal-phase mode that is, the stationary phase is usually a polar sorbent such as silica and alumina and the mobile phase consists of a nonpolar constituent such as hexane modified with a somewhat more polar solvent such as chloroform or ethyl acetate. In this mode, the more polar compounds are preferentially retained. The reversed-phase (RP) mode utilizes the opposite approach for the separation of nonpolar analytes or compounds that have some hydrophobic character. In this case, the stationary phase must consist of sorbent that is nonpolar in nature and the mobile phase is composed of a primary polar solvent, usually water, that is modified by a more nonpolar constituent such as methanol, acetonitrile, or tetrahydrofuran. 

Typical applications of reversed-phase chromatography are shown in Table 2. Beyond analytical apphca-tions, RP-TLC on bonded phases is also a tool for physicochemical measurements, particularly for molecular hpophilicity determination of biologically active compounds. Hydrophobicity can be measured by partition between an immiscible polar and nonpolar solvent pair, particularly in the reference system n-oc-tanol-water. The partition coefficient, P, is frequently used to interpret quantitative structure-activity relationships (QSAR studies). 

Since the polarity range of adsorbents is bordered by silica on the polar side and RP-18 or hydrophobic polymeric phases on the non-polar side it is easy to assign these absorbents to normal or reversed phase systems. Medium polar packings (Chapter 3.2.1) possess polar properties because of the functional group as well as hydrophobic properties contributed by the spacer (Unger and Weber, 1999). Owing to this mixed nature these phases can not be directly assigned to a certain type of phase system. 

The separation of very polar compounds on stationary phases of type RP-18 requires the use of a mobile phase rich in water. Under these conditions the stationary phase, which is hydrophobic becomes suddenly waterproof and the capacity for separation weakens. For this reason phases presenting a residual polarity are preferred in order to maintain the interaction between the analytes and the water of the mobile phase (Figure 3.14). 

A nonpolar mobile phase passing through a packed column that contains a polar stationary phase defines normal-phase HPLC (NP-HPLC). For example, if -hexane comprises the mobile-phase and silica gel is used for the stationary phase, separations of nonpolar organic analytes as shown in Fig. 4.1 is accomplished. With respect to neutral organic compounds, the polar and ionic domains cannot be reached by NP- HPLC. NP-HPLC was the first high-pressure form of liquid chromatography to be developed. If the stationary phase could be made hydrophobic by chemical treatment and the mobile phase made more polar, a reversal of mobile/stationary-phase polarities could be achieved. Like it or not, we are stuck with this nomenclature RP-HPLC has certainly extended the range of analyte polarity that... 

Owing to the medium polarity of polyamide 6, the sorbent can be made more or less hydrophobic than the mobile phase by selecting appropriate polar and non-polar eluents therefore, normal- and reversed-phase (RP) chromatography and also two-dimensional techniques can be developed. 

The chemical structures of synthetic dyes show considerable variety. They generally contain more than one aromatic group, condensed aromatic substructures or heterocyclic rings (pyrazolone, thiazole, acridine, thiazine, oxazine) which are mainly hydrophobic, and, frequently, a polar basic or cationic group which is strongly hydrophilic. Due to these structural characteristics, they readily bind both to polar adsorptive and apolar reversed-phase (RP) chromatographic supports, making their successful separation difficult. As the synthetic dyes are not volatile... 

One of the most important parameters for weU separation of flavonoids is the composition of the eluent. Controlling the solubility of the flavonoids in the eluent is a cmcial factor for determining the combinatimi of solvents used. In RP-HPLC, analytes are retained on the stationary phase based on their hydrophobicity. Elution of flavonoids in RP-HPLC is therefore in the order of decreasing polarity. Polarity increases most by hydroxyls at the fourth position, followed by those at the second and third positions. Loss of polar hydroxyl groups or additions of methoxy groups reduce polarity and hence increase retention times. 

RP-HPLC (i.e., hydrophobic stationary phase/polar aqueous mobile phase) is the more usual mode to separate and analyze opium alkaloids. The most attractive advantage of RP HPLC is the ability to determine a wide variety of compounds, which have different molecular structures, polarity, and acidity/basicity. Analysis of opium alkaloids by NP-HPLC (i.e., polar stationary phase) is not common and no references have been found in the scientific literature. As it is reflected in Table 33.2, octadecyl colunms (C18) are the most commonly employed. When monolithic columns are used, high flow rates are allowed, which gives rise to very... 

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