Nonaqueous reversed-phase chromatography

Most small organic molecules are soluble in mixed organic-aqueous solvents and can be easily analyzed using RPLC. However, there are some polar compounds which are not soluble in typical RPLC solvent systems or are unstable in an aqueous mobile phase system. These compounds can be analyzed on an RPLC column with a nonaqueous solvent system. This technique is called "nonaqueous reversed phase chromatography" (NARP).20-21 The NARP technique is primarily used for the separation of lipophilic compounds having low to medium polarity and a molecular weight larger than... 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

WO Landen Jr, RR Eitenmiller. Application of gel permeation chromatography and nonaqueous reverse phase chromatography to high pressure liquid chromatographic determination of retinyl palmitate and /3-carotene in oil and margarine. J Assoc Off Anal Chem 62 283-289, 1979. 

Figure 7.3. HPLC analysis of triglycerides in olive oil using nonaqueous reversed-phase chromatography with refractive index detection. Reprinted with permission from reference 11.

Fnhanced-fluidity liquid reversed-phase chromatography has numerous applications including the separation of nonpolar and polar compounds. For example, EFLC and nonaqueous reversed-phase HPLC are the common means of achieving effective separations of high molecular weight homologous compounds. 

A nonaqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with refractive index (RI) detection was described and used for palm olein and its fractions obtained at 12.5°C for 12-24 h by Swe et al. (101). The objective of their research was to find the optimum separation for analysis of palm olein triglycerides by NARP-HPLC, and to find a correction factor to be used in calculating CN and fatty acid composition (FAC). The NARP-HPLC method used to determine the triglyceride composition was modified from the method of Dong DiCesare (88). Palm olein was melted completely at 70°C in an oven for 30 min prior to crystal-... 

HJCF Nelis, AP De Leenheer. Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids. Anal Chem 55 270-275, 1983. 

The mode of separation in the HPLC depends on the selection of the stationary and mobile phases. In HPLC of lipids, normal- and reversed-phase modes are primarily used, with the reverse phase being more common than the normal phase. Separation in the re-versed-phase mode is mainly by partition chromatography, whereas separation in the normal phase mode is primarily by adsorption chromatography. Normal-phase HPLC is used for the separation of the lipids into classes of Upids [1,F]. Reversed-phase HPLC (RP-HPLC), on the other hand, is mainly used to separate each lipid class into individual species [2,B1]. For example, several triglycerides were separated from each other via nonaqueous reversed-phase HPLC, involving an octadecyl (ODS) column and a nonpolar (non-aqueous) mobile phase. RP-HPLC alone can be used to separate the fat molecules into classes and species [2,B1]. 

However, nonaqueous eluents are needed for the reversed-phase chromatography of highly nonpolar analytes. 

RPC = reversed-phase chromatography, IEC = ion-exchange chromatography, NPC = normal-phase chromatography, SEC = size-exclusion chromatography, NARP = nonaqueous reversed-phase, PAD = pulsed amperometric detector, ELSD = evaporative light scattering detector. Pre-column or post-column derivatization required. 

The preparative separation of pneumocandins Aq and Bq (Fig. 22) proved to be fairly difficult. They were fairly well-separated on Cl8 reverse phase chromatography on an analytical scale, but this separation could not be scaled up at all. For example, 25 mg of the mixture was not separable on a 21.2-mm ID x 250-mm column. These compounds chromatographed poorly on silica gel until water was added to the mobile phase. As seen in Fig. 23, the lower phase of the biphasic system CH2Cl2/Me0H/H20 (65 35 10) gave radically improved resolution of the pneumocandins compared to its nonaqueous counterpart. 

Abbott, T, Peterson, R., McAlpine, J., Tjarks, L., and Bagby, M. (1989) Comparing centrifugal countercurrent chromatography, nonaqueous reversed phase HPLC and Ag ion exchange HPLC for the separation and characterization of triterpene acetates. J. Liquid Chromatogr. 12, 2281-2301. 

The detector should be compatible with both normal and reversed phase chromatography, nonaqueous and aqueous based solvents, some extent, we have chosen to explore new and potentially novel detection areas, because they seemed to offer promise of unusual success, and at times, because they seemed to offer solutions to existing problems of pressing need and concern. Hopefully, these and other criteria will prevail in the future as well. 

If the column is not used for a short period of time, for example, overnight or over the weekend, the column is preferentially stored in the mobile phase, in which it is used. This minimizes equilibration times on startup. However, for longer storage, the column should be stored in the solvent that the manufacturer recommends for a particular column. For bonded phases used in reversed-phase chromatography, the most common solvent is an organic solvent such as methanol or acetonitrile. Nonaqueous solvents minimize the hydrolysis of silica-based bonded phases. When the column is converted to such a solvent, care should be taken to remove any salt prior to the conversion. It is highly recommended to put a label on the column that describes the storage solvent. 

Reversed-phase chromatography is the method of choice for the final step of vitamin K assays. It can easily separate vitamin Ki(20) from lipids with closely related polarities and from structural analogs used as internal standard. Similar to the other fat-soluble vitamins nonaqueous reversed-phased systems are preferable because of the increased solubility of vitamin Ki(20) and co-extracted lipids in the eluents that can be used. 

Some separation problems (e.g., triglyceride separation [76]) require a hydrophobic stationary phase and a nonpolar (nonaqueous) mobile phase. This ehromatographic mode is called nonaqueous reversed-phase (NARP) chromatography. The most common commercially available reversed-phase materials are C2, C4, Cg, Cjg, and phenyl phases. 

Estimations in 1988-1989 about the portion of reversed-phase based LC assays for vitamin E ranged from > 70% to 90%. In approximately 70% of reversed-phase systems methanol-water mixtures were used as mobile phases. Occasionally, ethanol, acetonitrile, or isopropanol was substituted for methanol. Nonaque-ous reversed-phase chromatography occupied a marginal position and found application particularly for the simultaneous chromatography of retinol, a-to-... 

---