Isocratic normal phase chromatography

Product ester ee was determined by isocratic normal-phase high-performance liquid chromatography using a Chiralcel OD-H (250 mm x 4.6 mm) column and a 98 % hexanes/2 % isopropanol mobile phase at 1.75 mL min and 25 °C. The undesired (/ )-ester and desired (5)-ester were quantified using their characteristic retention times of 10.3 min and 21 min respectively during elution. 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

Normal-phase chromatography is still widely used for the determination of nonpolar additives in a variety of commercial products and pharmaceutical formulations, e.g. the separation of nonpolar components in the nonionic surfactant Triton X-100. Most of the NPLC analyses of polymer additives have been performed in isocratic mode [576]. However, isocratic HPLC methods are incapable of separating a substantial number of industrially used additives [605,608,612-616], Normal-phase chromatography of Irgafos 168, Irganox 1010/1076/3114 was shown [240]. NPLC-UV has been used for quantitative analysis of additives in PP/(Irganox 1010/1076, Irgafos 168) after Soxhlet extraction (88%... 

Reversed-phase chromatography also separates, isocratically, vitamin D2 or D3 from their respective previtamins and inactive isomers (207), but, unlike normal-phase chromatography, it can separate vitamin D2 from D3 using nonendcapped stationary phases (198). The 25-hydroxylated metabolites of vitamins D2 and D3 can be separated from one another using a Vydac 201 TP column (37). The separation of vitamin D2 from vitamin D3, and 25-hydroxyvitamin D2 from 25-hydroxy vitamin D3, allows the D2 form of the vitamin or its metabolite to be used as an internal standard for quantifying the corresponding D3 form. 

For flavones in citrus peel oils, separations were accomplished with isocratic mobile phases of 38% and 40% acetonitrile in H20 (1). The extracts of peel and cold-pressed peel oils were diluted in ethanol and analyzed by reversed-phase on various C18 columns with good results. For the dilute citrus oils, gradient elution was preferred, to prevent the accumulation of terpenes on the column. With normal-phase chromatography, the elution order is reversed terpenes elute with the solvent front and are not a problem. 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

The stationary phase is usually Cg or Cig, but the use of Spherisorb CN or Nucleosil columns was also reported. The mobile phase for reversed-phase chromatography is aqueous, while, for normal-phase chromatography, it generally contains pentane or hexane. The elution can be isocratic or with a gradient. The detection is usually UV in the range 116-254 nm. 

Jandera P, Petranek L, and Kucerova M (1997) Characterization and prediction of retention in isocratic and gradient-elution normal-phase high-performance liquid chromatography on polar bonded stationary phases with binary and ternary solvent systems. Journal of Chromatography A 791 1-19. 

Figure 3.8 Experimental van Deemter plot of H (cm) vs u (cm.s ) for isocratic elution (normal phase) of hexamethylben-zene with a mobile phase of 4.8 % (w/v) ethyl acetate in n-decane. The column was 25 cm long, 9 mm in diameter and packed with 8.5 pm silica gel. The curve fitting procedure gave values for the van Deemter constants, and thus the separate contributions to the curve from the multipath dispersion, longitudinal dispersion and the resistance to mass transfer were calculated as shown. Reproduced from Scott, http //www.chromatography-online.org/, with permission.

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