High-pressure liquid chromatography normal phase

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]. 

Robbins, R.J. Leonczak, J. Johnson, J.C. Li, J. Kwik-Uribe, C. Prior, R.L. Gu, L. 2009. Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography-fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples. J. Chromatogra. A 1216 4831 840. 

Figure 4. Normal phase high-pressure liquid chromatography of cholesterol esters isolated from atherosclerotic lesions of human aorta (I), free cholesterol (II), oxygenated cholesterol esters (III), cholesteryl arachidonate (IV), cholesteryl linoleate (V), cholesteryl oleate and cholesteryl palmitate.

Isolation of potential anticancer compounds from bioactive extracts involves bioactivity-guided fractionation. The DNA-damaging natural products encountered in our studies were extracted by MEK and/or methanol, and the general methodology which we have employed in our bioassay-directed fractionation of these extracts is schematically presented in Fig. 7. These fractionations involved solvent-solvent partition, Sephadex LH-20 gel filtration, normal phase and reversed-phase (RP) column, preparative thin-layer and high pressure liquid chromatography (HPLC). Silica gel chromatography was employed only if bioactive compounds were found to be stable under these mildly acidic conditions. 

Wittwer, J.D., Jr. Application of high pressure liquid chromatography to the forensic analysis of several benzodiazepines. J.Liq.Chromatogr., 1980, 3, 1713-1724 [simultaneous chlordiazepoxide, cloraze-pate, ( razepam, demoxapam, desmethyldiazepam, diazepam, flurazepam, medazepam, nitrazepam, oxazepam, prazepam normal phase]... 

Normal-phase liquid chromatography (NPLC) is the oldest chromatographic mode, discovered by M.S. Tswett more than 100 years ago. It has been the predominant mode in thin-layer chromatography (TLC) and low-pressure dry-column liquid chromatography before the introduction of reversed-phase technique, which is a preferred mode in high-performance liquid chromatography (HPLC). 

De la Puente ML (2004) Highly sensitive and rapid normal-phase chiral screen using high-performance liquid chromatography-atmospheric pressure ionization tandem mass spectrometry (HPLC/MS). J Chromatogr A 1055 55-62... 

The mass spectrometer is a very sensitive and selective instrument. However, the introduction of the eluent into the vacuum chamber and the resulting significant pressure drop reduces the sensitivity. The gas exhaust power of a normal vacuum pump is some 10 ml min-1 so high capacity or turbo vacuum pumps are usually needed. The gas-phase volume corresponding to 1 ml of liquid is 176 ml for -hexane, 384 ml for ethanol, 429 ml for acetonitrile, 554 ml for methanol, and 1245 ml for water under standard conditions (0°C, 1 atmosphere). The elimination of the mobile phase solvent is therefore important, otherwise the expanding eluent will destroy the vacuum in the detector. Several methods to accomplish this have been developed. The commercialized interfaces are thermo-spray, moving-belt, electrospray ionization, ion-spray, and atmospheric pressure ionization. The influence of the eluent is very complex, and the modification of eluent components and the selection of an interface are therefore important. Micro-liquid chromatography is suitable for this detector, due to its very small flow rate (usually only 10 p min - ). 

Kagan M, Chlenov M, Kraml CM. 2004. Normal-phase high-performance liquid chromatographic separations using ethoxynonafluorobutane as hexane alternative. II. Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry applications with methanol gradients. J Chromatogr A 1033 321. 

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