Separation and Assay

Physical Methods.—Separation and Assay. A range of isomers of astaxanthin (8) diacetate (9-cis, 13-cis, 15-cis, 9,9 -di-cis, 9,13-di-cw, 9,13 -di-cw, 13,13 -di-cw, 13,15-di-cw), prepared by thermal and iodine-catalysed isomerization of irans-(S) have been separated by h.p.l.c.126 A procedure has been developed for separation of bean leaf etioplast pigments, including carotenoids,127 by h.p.l.c. H.p.l.c. separations of esters of all-trans-, 9-cis-, 11-cw-, and 13-cw-retinol,128-130 and determinations of retinol in serum,131 retinol and 13-cw-retinoic acid,132 and the aromatic retinoid (195)133 in plasma have been described. A reversed-phase ion-pair 

Visser and H. Cerfontain, Reel. Trav. Chim. Pays-Bas, 1981, 100, 153. 

Leclercq and M. Bourgeay-Causse, Rev. Inst. Pasteur Lyon, 1981, 14, 475. 

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Armstrong WG (1968) A method for the simultaneous separation and assays of peptides and attached carbohydrate and fluorescent components. Automat Anal Chem (Technicon Symp 3rd, 1967) 1, 295-299. 

Physical Methods and Physical Chemistry.—Separation and Assay Methods. A procedure for h.p.l.c. of plant pigments has been used to separate the carotenoids of spinach and of a diatom. H.p.l.c. separations of citrus carotenoids, and of retinal (67) isomers have also been reported. Carotenoid mixtures have also been separated efficiently and rapidly by centrifugal chromatography. ... 

Cell walls, total membrane-bound components, and ribosomes were separated and assayed for cellulase activity to study the subcellular localization of the enzymes as follows. Segments (approx. 5 g fresh wt) were ground in two volumes of extraction medium containing 0.4M sucrose (ribonuclease-free), 5mM Mg acetate, lOmM Tris-HCl (pH 7.5 at 22°C), 20mM KC1 and 5mM / -mercaptoethanol. The brei was filtered and the filtrate centrifuged at 500 Xg for 20 min. The post-500 Xg supernatant was fractionated essentially as previously described (28). Aliquots (7 mL) of the supernatant were layered on a discontinuous gradient composed of 2 mL 70% (w/v) sucrose and 3 mL 15% (w/v) sucrose both in lOmM Tris-HCl (pH 7.5 at 22°C), lOmM KC1, 2.5mM Mg acetate and ImM / -mercaptoethanol. The tubes were centrifuged at... 

These final diversity elements were spatially encoded, i.e., the resin pools are kept separated and assayed separately, to allow for identification of the final monomers without the need for a further chemical encoding step. As a result no further pooling was necessary after the quaternization reaction and we were able to tailor the most favorable reaction conditions for incorporation of the required alkylating agents in each reaction. 

Many methods have been developed for the quantitative determination of each class of surfactants. The analysis of commercial surfactants is much more complicated since they may be comprised of a range of compounds within a given structural class, may contain surface-active impurities, may be formulated to contain several different surfactant classes, and may be dissolved in mixed organic solvents or complex aqueous salt solutions. Each of these components has the potential to interfere with a given analytical method so surfactant assays are sometimes preceded by surfactant separation techniques. Both the separation and assay techniques can be highly specific to a given surfactant/solution system. Table 3.4 shows some typical kinds of analysis methods that are applied to the different surfactant classes. 

As mentioned earlier, many investigators have used detergents instead of diethyl ether in the phospholipase C assay however, these compounds are to be avoided if the intention is to analyze the phosphorylated bases liberated in this reaction. Interference in the separation and assay procedures is a problem. 

Separation and Assay Methods. The g.l.c. behaviour of the hydrogenation products of about sixty carotenoids, carotenoid acetates and trimethylsilyl ethers, and triterpenoid carotenoids has been studied extensively. The relative retention times of these compounds in three systems at different temperatures and with temperature... 

The example clearly demonstrates the scale of difficulty involved in speciation analysis, and the fact that it requires a case-by-case approach. A helpful tool in the process is a list of typical errors (Table 12.3). Each time, however, one should be aware that the procedures are extremely complex, the stability of compounds is a source of multiple problems, and the quality of separation and assaying depends to a... 

Separation and Assay. H.p.l.c. methods have been described for the determination of chloroplast pigments,phytoplankton pigments,and provitamin A carotenoids in tomatoes.Other chromatographic procedures have been devised for the separation of Capsicum carotenoids and chloroplast pigments from tobacco mutants, " and methods for the high-speed video-densitometric determination of carotenoids separated by t.l.c., and for the dual assay of carotenoids and vitamin A in human liver have been reported. H.p.l.c. has been used for the separation of cis-trans-isomers of retinal, retinol and retinyl... 

Separation and Assay. Methods have been described for the h.p.l.c. and m.s. determination of ubiquinone homologues in animal tissues,h.p.l.c. analysis of ubiquinone and phylloquinone in blood,of menaquinone-4 (230), 2,3-epoxymenaquinone-4 (231), and 4-demethylmenaquinone-4 (232) and of phylloquinone and menaquinone-4. The polarographic determination of phylloquinone and menaquinone-4 has been reported. 

Degraded Carotenoids Physical Methods Separation and Assay N.M.R. Spectroscopy Mass Spectrometry Chiroptical Methods Electronic Absorption Spectroscopy Infrared and Resonance Raman Spectroscopy Other Spectroscopic Techniques Miscellaneous Physical Chemistry Photoreceptor Pigments Biosynthesis and Metabolism Stereochemistry Enzyme Systems Inhibition and Regulation... 

THE FACTORS AND THEIR LEVELS. EXAMINED IN THE ROBUSTNESS TEST OF (82) ON A CHROMATOGRAPHIC METHOD FOR THE SEPARATION AND ASSAY OF A DRUG SUBSTANCE AND TWO RELATED COMPOUNDS IN TABLETS... 

Most of the SIA-HPLC systems have been applied for the separation and assay of radionuclides. The reason for selecting such a system is the potential radiation and contamination of an operator during the sampling process. By using SIA-HPLC systems, all steps are automated and the contact of the operator with... 

Solid and liquid phases in equilibrium at -2° were separated and assayed. Imidazole and penicillin (presumably Grant et al, used penicillin G) concentrations changed by less than 10% from their initial values. Subsequent storage of the solid at -2° led to as much as 77% hydrolysis while storage of tl e liquid at both -2° (unfrozen) and +22° gave no hydrolysis. 

Physical Methods. H.p.l.c. procedures for the separation and assay of ubiquinone and homologues278-280 and of menaquinone cis- and fra/ts-isomers, 2,3-epoxides, and chain-length homologues281 282 have been described. A XH n.m.r. study has been reported283 of the location and motion of ubiquinones in perdeuteriated phosphatidylcholine bilayers. Other aspects of the interaction of ubiquinone with phospholipid monolayers have been studied.284... 

One of the problems of barbiturate analysis is the isolation of the compound from biological material.427,428,437,449,513 Extraction is the most accepted method, but some assays take advantage of omitting the isolation and purification step. Special attention was devoted to separation and assay of particular barbiturate drugs in combination with other compounds. For example, many methods were devised for the determination of phenobarbital and hydantoin used together in anticonvulsive therapy.514 Several procedures were proposed for the determination of barbiturates along with their metabolites in body fluids.437... 

Physical Methods.—Separation and Assay. Increasing use is being made of h.p.l.c. in the carotenoid field. This technique will clearly become the method of choice for carotenoid separation, purification, and analysis. The first papers on carotenoid h.p.l.c. have recently been published. These include a systematic study of the chromatography of model mixtures of carotenes, carotenediols, cis trans isomers, and diastereoisomers. ° Many papers report the use of h.p.l.c. 

More recently, Moine-Ledoux et al. (1996) used the remarkable resolution of capillary electrophoresis (CE) to separate and assay proteins in must and wine. The wine was first dialyzed with... 

In addition to the tannin concentration of a wine, enologists also need information on the structures that govern the properties of the various compounds. Of course, the ideal solution would be to separate and assay the various molecular units included in the concept of tannin. Thanks to high-performance techniques, these analyses are now possible, although they are still difficult to implement. 

Brocks, D.R. Pasutto, F.M. Jamali, F. Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers. J. Chromatogr. 1992, 581, 83-92. 

Separation and Assay. Procedures for the separation, purification, and assay of carotenoids and retinoids by h.p.l.c., g.c., and g.c.-m.s. are given in an extensive article." Another, general, review includes information on the h.p.l.c. separation of retinoids.A particularly useful method has been developed for resolution and analysis of some carotenoid optical isomers.For example, (3R,3 R)-, (3S,3 S)-, and (3/ ,3 5)-astaxanthin were converted into the diastereomeric (-)-camphanic acid diesters, which were separated by h.p.l.c. This procedure has been used to analyse the isomeric composition of a natural astaxanthin sample. An h.p.l.c. procedure for separation of a-, P-, and y-carotenes (173)—(175) and lycopene (176) has been described." Several papers describe methods for the h.p.l.c. separation and purification of various retinal and retinol isomers and derivatives.A procedure for the preparative t.l.c. of oxidation products of retinyl acetate has been described,and a competitive protein-binding radioassay for retinoic has been reported. 

The average composition of copolymers can be determined most readily for the case where the monomeric units can be isolated and identified by suitable scission or degradation reactions. This is the usual method for the elucidation of protein structure. The proteins are hydrolyzed by acids and / or bases in an automated apparatus. The resulting amino acids are chromato-graphically separated and assayed quantitatively via the color reaction with ninhydrine in so-called amino acid analyzers. 

HIBA/sodium octanesuIfonate-a-HIBA systems were employed for separation and assay of lanthanide fission products. In one of the studies, the HPLC method was used to separate and estimate a lanthanide fission product, e.g., lanthanum and a heavy element, e.g., uranium. The bum-up obtained was found to be in good agreement with the conventional mass spectrometric method. In another study. 

Anhydro-4-0-(a-L-idopyranosyluronic acid)-D-[l- H]mannitol is cleaved by a-L-iduronidase to give, as one of the products, 2,5-anhydro-D-[l- H]mannitol, which can be separated and assayed. This procedure can be used in the diagnosis of mucopolysaccharidosis type I (Hurler s syndrome). 

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