Normal-phase liquid chromatography chromatograms

One column can be used for different types of liquid chromatography by changing the eluent components. As an example, a column packed with octadecyl-bonded silica gel has been used for size-exclusion liquid chromatography with tetrahydrofuran (THF), normal-phase liquid chromatography with n-hexane, and reversed-phase liquid chromatography with aqueous acetonitrile. Examples of the chromatograms are shown in Figure 1.4. 

Figure 10.9 Chromatograms of fortified coconut oil obtained by using (a) normal-phase HPLC and (b) GPC/normal-phase HPLC. Peak identification is as follows 1 (a,b), DL-a-toco-pheryl acetate, 2 (b), 2,6-di-terf-butyl-4-methylphenol 2 (a) and 3 (b), retinyl acetate 3 (a) and 4 (b), tocol 4 (a) and 5 (b), ergocalciferol. Reprinted from Analytical Chemistry, 60, J. M. Brown-Thomas et al., Determination of fat-soluble vitamins in oil matrices by multidimensional high-performance liquid chromatography , pp. 1929-1933, copyright 1988, with permission from the American Chemical Society.

In normal high pressure liquid chromatography, typical sample volumes are 20-200 p.L this can become as little as 1 nL in capillary HPLC. Pretreatment of the sample may be necessary in order to protect the stationary phase in the column from deactivation. By employing supercritical fluids such as carbon dioxide, pretreatment can be bypassed in many instances so that whole samples from industrial and environmental matrices can be introduced directly into the column. This is due to the fact that the fluid acts as both extraction solvent and mobile phase. Post-column electrochemistry has been demonstrated. For example, fast-scan cyclic voltammo-grams have been recorded as a function of time after injection of microgram samples of ferrocene and other compounds in dichloromethane solvent and which are eluted with carbon dioxide at pressures of the order of 100 atm and temperatures of 50°C the chromatogram is constructed as a plot of peak current vs. time [18]. 

Figure 1.4 Distribution isotherms, (a) The ideal situation corresponding to the invariance of the concentration isotherm, (b) Situation in which the stationary phase is saturated - as a result of which the ascent of the peak is faster than the descent (skewing factor greater than 1) (c) The inverse situation the constituent is retained too long by the stationary phase, the retention time is therefore extended and the ascent of the peak is slower than the descent apparently normal. For each type of column, the manufacturers indicate the capacity limit expressed in ng/compound, prior to a potential deformation of the corresponding peak. The situations (a), (b) and (c) are illustrated by authentic chromatograms taken out from liquid chromatography technique.

Typical chromatograms obtained by high-performance liquid chromatography (HPLC) of the retinyl esters from human RPE are illustrated in Fig. 5. When conditions are optimized for isomer separation (normal phase, uj r half of Fig. 5), there are two major peaks (2, 4), each with a shoulder on the leading edge. Peak 2 is 11-cw-retinyl palmitate the shoulder corresponds to 11-cij-retinyl stearate. Peak 4 is all-trans-retinyl palmitate the shoulder corresponds to all-trans retinyl stearate. Peak 5 is all-rra/u-retinyl stearate, and peak 3 (which is sometimes composite) is unidentified at present. Peaks 2 and 4 were collected separately and injected onto a reverse-phase column. Under these conditions the two component esters were completely resolved, as shown in the lower half of Fig. 5 (peaks 2.1, 2.2 and 4.1, 4.2). 

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