Precision column chromatography

Instead, the idea of Coupled-Column Chromatography was employed (1. Here, this means the manual coll don of fractions from one chromatograph and their reinjection into another. Coll tion of GPC fractions and their analysis by other instruments or re-injection has often been utilized qualitatively in GPC. However, precise quantitative analysis is much less often reported (6). 

The column chromatography technique using Dowex 50 ion-exchange resin, introduced in 1951 (M2) and improved in 1954 (M3) by Moore and Stein, first made possible the precise quantitative analysis of amino acids liberated in the course of acid hydrolysis of urine. Similar results were also obtained by Muting in 1954 (M4), who used paper chromatography methods. In this procedure amino acids were quantitatively determined after staining on the paper and elution of the resulting spots. 

Heptachlor and heptachlor epoxide have been measured in samples of human milk using GC/ECD and GC/MS (Mussalo-Rauhamaa et al. 1988 Polishuk et al. 1977b Ritcey et al. 1972). Sample preparation steps for milk involve homogenization with chloroform/methanol, lipid extraction with petroleum ether, hexane or acetone-hexane, clean-up by column chromatography, and elution with acetonitrile, hexane, methylene chloride, or dichloromethane-petroleum ether. Precision, accuracy, and sensitivity were not reported for most of the studies however, one study reported a sensitivity in the low-ppb range (Ritcey et al. 1972). 

In principle, h.p.Lc. arose from conventional liquid column chromatography, following the development of g.l.c. and realisation that it was a rapid and accurate analytical method. This led to a reappraisal of the liquid column chromatographic system, which in turn resulted in research developments in instrument design and in the manufacture of column-packing materials. These now have precise specifications to make them suitable for adsorption, normal and reversed phase partition, ion exchange, gel permeation, and more recently affinity chromatography. 

Separation is required when (1) a mixture is too complex for a direct analytical measurement (e.g., spectroscopy), (2) the materials to be analyzed are very similar, such as isomers, (3) it is necessary to prepare highly purified materials, and (4) a measurement of the amount of a particular material is needed. Filtration, open-column chromatography, and thin-layer chromatography are used for relatively easy separations. Modern HPLC is a technique for making precision separations of complex mixtures and offers high-resolution separating capability to solve problems faster and better. 

The recent development of commercial HPLC systems has provided a powerful instrumentation for the separation, characterization, identification, and quantitation of minute amounts of essential dietary components (68,69). Developments in hardware and packings for HPLC have overcome the problems of nonreproducible behavior and low efficiency separations previously associated with column chromatography (70). HPLC has already been applied to the quantitative analysis of analgesics, pesticides, and fat-soluble vitamins with precision and accuracy and a minimum of sample clean-up. Such instrumentation provides a rapid, accurate, and sensitive technique for the separation and analysis of subnanomole quantities of a wide range of complex high-molecular-weight, nonvolatile, thermally labile, compounds that are vital for metabolic and nutritional studies. 

Cleanup is performed by column chromatography. Silica/allumina or Florisil (synthetic magnesium silicate salt), deactivated or suitably activated (for instance 130 °C for 12 h ) are the most frequently used stationary phases. Their performances are checked by standard solution in order to find out the best solvent or mixture of solvents and the optimum volume to be used for selectively eluting PCBs and leaving interferents in the column. V-Hexane and dichloromethane are the most widely used solvents. Better precision on cleanup results has been observed when Florisil cartridges are used instead of silica gel ones. In some cases, in order to minimize interference from other non-PCB organic compounds, additional fractionations are performed. 

The concentration profiles of the reactant and the products were monitored by means of high precision liquid chromatography (HPLC), equipped with a Biorad Aminex HPX-87C carbohydrate column according to the method described in [4]. 

Various analytical techniques used for hydrocarbon analyses of sea water samples, ultraviolet and fluorescence spectrophotometry (Keizer and Gordon, 1973), infrared spectrometry (Carlberg and Skarstedt, 1972), liquid column chromatography together with the determination of heat of adsorption (Zsolnay, 1977b) provide only data about a fraction of the hydrocarbons or yield a value equivalent to the total hydrocarbon concentration. Thus, we will only consider studies where hydrocarbons are precisely determined by gas—liquid chromatography and mass spectrometry. 

Quantitation may be done crudely on the spots separated by planar chromatographic techniques such as TLC or slab gel electrophoresis (see Chapter 13). One might compare the optical density, the fluoresence, or the degree of stationary phase fluoresence suppression by the unknown spot to a series of standards of known concentration. In contrast, the electrical signals from the variety of detectors used in various column chromatography instruments can be precisely, reproducibly, and linearly related to the amount of analyte passing through the detector cell. If all parameters of injection, separation, and detection are carefully controlled from run to run, and especially if appropriate quantitative internal standards are incorporated in the procedure, accuracy and precision better than +1 % may be attained. 

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