Liquid chromatography , natural substances

As a result of its unique chemical and physical properties, silica gel is probably the most important single substance involved in liquid chromatography today. Without silica gel, it is doubtful whether HPLC could have evolved at all. Silica gel is an amorphous, highly porous, partially hydrated form of silica which is a substance made from the two most abundant elements in the earth s crust, silicon and oxygen. Silica, from which silica gel is manufactured, occurs naturally, either in conjunction with metal oxides in the form of silicates, such as clay or shale, or as free silica in the form of quartz, cristobalite or tridymite crystals. Quartz is sometimes found clear and colorless, but more often in an opaque form, frequently colored... 

Identification and quantification of natural dyes need high performance analytical techniques, appropriate for the analysis of materials of complicated matrices containing a small amount of coloured substances. This requirement perfectly fits coupling of modern separation modules (usually high performance liquid chromatography in reversed phase mode, RPLC, but also capillary electrophoresis, CE) with selective detection units (mainly mass spectrometer). 

Kirchoff, C., Bitar, Y., Ebel, S., and Holzgrabe, U. (2004). Analysis of atropine, its degradation products and related substances of natural origin by means of reversed phase high performance liquid chromatography./. Chromatogr. A 1046, 115-120. 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

This paper is the only one in the liquid chromatography portion of this symposium which will attempt to deal with chromatography specifically from the viewpoint of the pesticide metabolism chemist. A residue analyst knows what compound he must analyze for, and develops his method with the properties of that substance in mind. On the other hand, the pesticide metabolism chemist has a different problem. At the conclusion of the treatment, exposure, and harvest phases of a radiolabeled metabolism study, he divides his material into appropriate samples, and extracts each sample with selected solvents to obtain the radioactive materials in soluble form. Typically these extracts consist of low levels (ppm) of carbon-14 labeled metabolites in a complicated mixture of normal natural products from the plant, animal, or soil source. The identity of each metabolite is unknown, and each must be isolated from the natural background and from other labeled metabolites in sufficient quantity and in adequate purity for identification studies, usually by mass spectrometry. The situation is rather like looking for the proverbial "needle in the haystack" when one does not know the size, shape,or composition of the needle, or even how many needles there are in the stack. At this point a separation technique must be selected with certain important requirements in mind. 

Countercurrent chromatography (CCC) [1,2] is a process that avoids these difficulties. It is a form of liquid-liquid chromatography without a solid support, which separates soluble natural product substances on their partition, or differential solubility, between two immiscible solvents. The principle of separation (partition) is the same in both the laboratory and the production plant and is generic in that it can be applied to an extremely broad range of purification problems in many industries. Furthermore, because there is no solid support, there is 100% sample recovery and no need for any prepurification. 

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