Nonaqueous reversed-phase

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... 

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. 

Temperature variation was more influential on methylene selectivity for the mixtures with high percentages of acetonitrile, while the reverse was true for methanol/C02 mixtures. Like in nonaqueous reversed-phase HPLC, a temperature increase lowered the methylene selectivity. The main conclusion from this work is that acetonitrile/C02 mixtures would be preferred over methanol/C02 mixtures as a flrst attempt to separate homologs. 

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). 

Earlier work in the HPLC analysis of TGs used a differential refractometer as the detector a number of papers have detailed isocratic systems combined with refractive index (RI) detectors, often with acetonitrile/acetone mobile phases. Although aqueous mobile phases were generally used with alkyl-bonded phase columns, due to the lipophilicity of TGs, water could not be used in the mobile phase for this particular application therefore the mobile phases generally employed consisted of mixtures of acetone and acetonitrile and occasionally tetrahydrofuran, methylene chloride, or hexane (the conspicuous absence of water in the mobile phase prompted the term nonaqueous reverse phase, or NARP, to describe these systems). 

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-... 

FW Quackenbush, RL Smallidge. Nonaqueous reverse phase liquid chromatographic system for separation and quantitation of provitamins A. J Assoc Off Anal Chem 69 767-772, 1986. 

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. 

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

Since these ligates are bonded to the surface of the silica and will not wash off, it is no longer necessary to choose a mobile phase with a polarity opposite to that of the bonded phase. That is to say, a reverse phase support like ODS could be used with a relatively nonpolar organic mobile phase, thus establishing a system that does not fit into either category— reverse phase or normal phase. Some workers have given such a system the name nonaqueous reverse phase, or NARP. 

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]. 

Figure 10.2 Reversed-phase separation of alkene homologues (reproduced with permission of Du Pont). Conditions stationary phase, Zorbax ODS mobile phase, 0.75 ml min" tetrahydrofuran-acetonitrile (10 90) (this is an example of nonaqueous reversed-phase chromatographyl) IR detector, 3.4 im.

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. 

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.

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. 

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. 

Note that the actual bonded moiety is typically a dimethylalkylsilane the Cig column is really precisely defined as having a dimethyloctadecylsilane bonded phase.) Those solvents used in conjunction with RP columns are called reversed-phase solvents. The most common RP solvents are mixtures of water with water-soluble solvents such as acetonitrile, methanol, and tetrahydrofuran. Uncommon cases are the use of nonaqueous solvents in reversed-phase separations. These are classified as NARP (nonaqueous reversed-phase) separations. An example of an NARP mobile phase would be a 50/50 v/v methanol/acetonitrile mixture. 

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. 

---