Reversed-phase organic modifiers

Mobile phases commonly used in reversed-phase HPLC are hydro-organic mixtures. The most common reversed-phase organic modifiers include methanol and acetonitrile and/or combinations of these two modifiers. Other mobile-phase modifiers such as tetrahydrofuran, IPA, and DMSO [32] have been also used for minor selectivity adjustment however, they are not common due to their high backpressure limitations and/or high background UV absorbance. 

Figure 2.17. Examples illustrating the effect of solvent selectivity on resolution using different organic modifiers under RPLC conditions. The inset shows that the three typical reversed phase organic modifiers (ACN, MeOH, and THF) have very dissimilar solvent properties. Diagram courtesy of Academy Savant.

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

P. Zhuang, R. Thompson, and T. O Brien, A retention model for polar selectivity in reversed phase chromatography as a function of mobile phase organic modifier type, /. Liq. Chrom. Rel. Technol. 28 (2005), 1345-1356. 

Highly hydrophilic compounds belonging to biogenic amines were analyzed in a reversed phase system, modified with the addition of ionic liquids l-Ethyl-3-methylimidazolium hexafluoro-phosphate and the chaotropic salt NaPp6 on a Discovery HS CIS column under acidic conditions. The effect of the additives concentration and the presence of organic solvent on the analytes chromatographic parameters, such as retention factor, tailing factor and theoretical plate numbers, were determined (24). 

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 macrocyclic glycopeptides CSPs arc capable of operating in three different mobile phase systems reversed phase, normal phase, and the new polar organic mode. The new polar organic mode refers to the approach when methanol is used as the mobile phase with small amounts of acid and/or base as the modifier to control... 

In addition to temperature and flow rate, the retention and selectivity in reversed phase are controlled by (i) the concentration and type of organic modifier and (ii) the type, concentration and pH of the buffer. 

Comparisons of LC and SFC have also been performed on naphthylethylcar-bamoylated-(3-cyclodextrin CSPs. These multimodal CSPs can be used in conjunction with normal phase, reversed phase, and polar organic eluents. Discrete sets of chiral compounds tend to be resolved in each of the three mobile phase modes in LC. As demonstrated by Williams et al., separations obtained in each of the different mobile phase modes in LC could be replicated with a simple CO,-methanol eluent in SFC [54]. Separation of tropicamide enantiomers on a Cyclobond I SN CSP with a modified CO, eluent is illustrated in Fig. 12-4. An aqueous-organic mobile phase was required for enantioresolution of the same compound on the Cyclobond I SN CSP in LC. In this case, SFC offered a means of simplifying method development for the derivatized cyclodextrin CSPs. Higher resolution was also achieved in SFC. 

Baczek, T., Markuszewski, M., Kaliszan, R. Linear and quadratic relationships between retention and organic modifier content in eluent in reversed phase high-performance liquid chromatography a systematic comparative statistical study. 

Reversed-Phase BPC Linear increase in tB (Organic modifier) 5-7% B/t,... 

Antia, F. D. and Horvath, Cs., Dependence of retention of the organic modifier concentration and multicomponent adsorptive behavior in reversed-phase chromatography, /. Chromatogr., 550, 411, 1991. 

McCalley, D. V., Effect of organic solvent modifier and nature of solute on the performance of bonded silica reversed-phase columns for the analysis of strongly basic compounds by high-performance liquid chromatography, /. Chromatogr A, 738(2), 169, 1996. 

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