Non-aqueous reversed phase chromatography

The benzoylated derivatives of both hydroxy and non-hydroxy ceramides have been resolved using non-aqueous reversed phase chromatography with a variety of mobile phases including either 0.05% methanol in n-pentane or mixtures of hexane-ethyl acetate (94 6, 95 5, 97 3) (Sugita et al., 1979 Iwamori and Moser, 1975 Iwamori et al., 1979). 

Fig. 10.2 Reversed-phase separation of alkene homologues. (Reproduced by permission of Du Pont.) Conditions stationary phase, Zorbax CDS mobile phase, 0.75 ml min tetrahydrofuran-acetonitrile (10 90) (this is an example of non-aqueous reversed-phase chromatography ) IR detector, 3.4 im.

Try gradient with dioxane-water or non-aqueous reversed-phase chromatography with acetonitrile-dichloromethane... 

Parris NA (1978) Non-aqueous reversed-phase chromatography of glycerides using infrared detection. J Chromatogr 149 615-624... 

M. Weissenberg, I. Schaeffler, E. Menagem, M. Barzilai and A. Levy, Isocratic non-aqueous reversed-phase high-performance liquid chromatography separation of capsanthin and cap-sorubin in red peppers (Capsicum annuum L.), paprika and oleoresin. J. Chromatogr.A 757... 

The elution volumes of polystyrene and benzene in the size-exclusion mode were 0.98 and 1.78 ml, respectively (Figure 1.4A). This means that separations by molecular size can be achieved between 0.98 and 1.78 ml in this system. In the normal phase mode the elution volumes of octylbenzene and benzene were 1.98 and 2.08 ml, respectively, in n-hexane solution (Figure 1.4B). This type of chromatography is called adsorption or non-aqueous reversed-phase liquid chromatography. These are adsorption liquid chromatography and non-aqueous reversed-phase liquid chromatography. The elution order of the alkylbenzenes in the reversed-phase mode using acetonitrile was reversed... 

Lin, J. T., Woodruff, C. L., and McKeon, T. A. 1997. Non-aqueous reversed-phase high-performance liquid chromatography of synthetic triacylglycerols and diacylg-lycerols. J. Chromatogr. A, 782,41-48. 

Some very hydrophobic samples, e.g., lipids, are strongly retained and not eluted in an acceptable time even with pure methanol or acetonitrile as the mobile phase. Such samples are usually adequately resolved by normal-phase chromatography, but they can be often equally well or even better separated by non-aqueous reversed-phase (NARP) chromatography in mixed mobile phases containing a more polar (e.g.. acetonitrile or methanol) and a less polar (e.g., tetrahydrofuran. dichloromethane. methyl-r-butyI ether) organic solvent. Ternary non-aqueous mobile phases may contain even hexane or heptane. The retention decreases with increasing concentration of the less-polar... 

Jandera, P., Guiochon, G. Effect of the sample solvent on band profiles in preparative liquid chromatography using non-aqueous reversed-phase high-performance liquid chromatography, J. Chromatogr., 1991, 588, 1-14. 

Alternatively, where samples are extracted into organic solvents, normal phase HPLC can also be used. This option is particularly valuable for the resolution of optical isomers of the A vitamins and, for example, 13-cw-retinol can be resolved using this system (Egberg et al., 1977). Retinoyl species may be converted to retinol prior to chromatography but the subsequent profile is difficult to interpret because of the number of possible intermediates that may also be generated. A computer-assisted analysis of vitamin A extracts from two species of tobacco leaf using non-aqueous reversed phase is shown in Fig. 11.8.2. 

Lin, J.T., Snyder, L.R., and McKeon, T.A. (1998) Prediction of Relative Retention Times of Triacylglycerols in Non-Aqueous Reversed-Phase High-Performance Liquid Chromatography, J. Chromatogr. A 808,43-49. 

Stubiger, G., Pittenauer, E. and Allmaier, G. (2003) Characterisation of castor oil by on-line and off-line non-aqueous reverse-phase high-performance liquid chromatography-mass spectrometry (APCI and UV/MALDI). Phytochem. Anal. 14, 337-346. 

Leveque NL, Heron S, Tchapla A. Regioisomer characterization of triacylglycerols by non-aqueous reversed-phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent. J Mass Spec 2010 45 284-96. 

Deye JR, Shiveley AN, Oehrle SA, Walters KA. Separation of substituted fullerenes using non-aqueous reversed-phase liquid chromatography—mass spectrometry. J Chromatogr A 2008 1181(1-2) 159-61. 

Parris NA. Non-aqueous reversed-phase liquid chromatography a neglected approach to the analysis of low polarity samples. J Chromatogr A 1978 157 161—70. 

Bushway, R.J., Determination of a- and p-carotene in some raw fmits and vegetables by high-performance liquid chromatography, J. Agric. Food Chem., 34, 409, 1986. LeseUier, E. et al.. Optimization of the isocratic non-aqueous reverse phase (NARP) HPLC separation of trans/cis a- and p-carotenes, J. High Res. Chromatogr., 12,447,1989. Saleh, M.H. and Tan, B., Separation and identification of cis/trans carotenoids isomers, J. Agric. Food Chem., 39, 1438, 1991. 

Parris NA (1983) Isocratic non-aqueous reversed phase liquid chromatography of carotenoids. Anal Chem 55 270-275... 

Figure 2 (right). Reverse-phase HPLC elution profile of the tumor-localizing fraction of HPD isolated by non-aqueous gel exclusion chromatography. (B) Hydrolysis of this material in 50% aqueous THF containing IM HCl, at 37 °C for 24 hours. (C) After hydrolysis in IM NaOH, 50% aqueous THF under the same condition. The major porphyrins resulted are hematoporphyrin (HP), hydroxyvinyl deutero-porphyrin (HVD), and protoporphyrin (PP). 

RA Williams, R Macrae, MJ Shepherd. Non-aqueous size-exclusion chromatography coupled online to reversed phase high performance liquid chromatography. Interface development and applications to the analysis of low-molecular weight contaminants andadditives in foods. J Chromatogr 477 315-325, 1989. 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

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]. 

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