Reversed-phase chromatographic

Macrocyclic glycopeptides. The first of these CSPs - based on the cavity of the antibiotic vancomycin bound to silica - was introduced by Armstrong [25]. Two more polycyclic antibiotics teicoplanin and ristocetin A, were also demonstrated later. These selectors are quite rugged and operate adequately in both normal-phase and reversed-phase chromatographic modes. However, only a limited number of such selectors is available, and their cost is rather high. 

Cyclodextrins can solubilize hydrophobic molecules in aqueous media through complex formation (5-8). A nonpolar species prefers the protective environment of the CDx cavity to the hulk aqueous solvent. In addition, cyclodextrins create a degree of structural rigidity and molecular organization for the included species. As a result of these characteristics, these macrocycles are used in studies of fluorescence and phosphorescence enhancement (9-11), stereoselective catalysis (.12,13), and reverse-phase chromatographic separations of structurally similar molecules (14,15). These same complexing abilities make cyclodextrins useful in solvent extraction. 

Welerowicz, T., Buszewski, B. The effect of stationary phase on lipophilicity determination of 3-blockers using reverse-phase chromatographic systems. Eiomed. Chromatogr. 2005, 39, 725-736. 

Cohen, K. A., Schellenberg, K., Benedek, K., Karger, B. L., Grego, B., and Hearn, M. T. W., Mobile-phase and temperature effects in the reversed phase chromatographic separation of proteins, Anal. Biochem., 140, 223, 1984. 

Li, J. and Carr, P.W., A study of the efficiency of polybutadiene-coated zirconia as a reversed-phase chromatographic support, Anal. Chem., 69(11), 2193,1997. 

Farine, S., Villard, C., Moulin, A., Mouren, G. M., and Puigserver, A., Comparative quantitative analysis of sucrose and related compounds using ion exchange and reverse phase chromatographic methods, Int. ]. Biol. Macromol., 21, 109, 1997. 

Eadsforth, C.V., Moser, P. (1983) Assessments of reversed phase chromatographic methods for determining partition coefficients. Chemosphere 12, 1459-1475. 

Figure 4.8 Regions of reversed-phase chromatographic mechanisms. (Reproduced from K.K. Unger, R. Janzen, and G. Jilge, Chromatographia, 24 144 [1987]. With permission of Friedr. Vieweg Sohn.)...

Henry, M.C., Wolf, L.K., and Messmer, M.C., In situ examination of the structure of model reversed-phase chromatographic interfaces by sum-frequency generation spectroscopy, J. Phys. Chem. B, 107, 2765, 2003. 

Tanaka E, Terada M, Nakamura T, Misawa S, Wakasugi C. 1997. Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 micron porous microsphe-rical silica gel. J Chromatogr B Biomed Sci Appl 692(2) 405-412. 

Riggin and Howard (1979, 1982), Matsui et al. (1983), Fabre et al. (1984), and Ahuja et al. (1988) reported that High Performance Liquid Chromatography (HPLC) with UV or electrochemical detection is capable of analyzing 1,2-diphenylhydrazine. Reversed phase chromatographic columns have been used most often (Ahuja et al. 1988 Fabre et al. 1984 Riggin and Howard 1979, 1982). Cyano-amino polar bonded phase columns also have been used (Matsui et al. 1983). Using a reversed phase and UV detection, the minimum amount detected (on column amounts) is approximately 6-7 ng and the minimum amount quantifiable is less than 1 pg (Ahuja et al. 1988 ... 

It can be seen from Figure 3 that the molar mass distribution of the hydrophilic compounds (fraction I) is broad although they are eluted by RPC in a narrow zone (Fig. 2). Their reversed-phase chromatographic fractionation is thus based almost exclusively on their polarity, molecular size having no effect on the process. 

Eadsforth, C. V., and P. Moser, Assessment of Reverse-Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere, 1983 12, 1459-1475. 

In reversed-phase HPLC on a Cl8 column, only six peaks are usually found, and their order of elution is 5-tocotrienol, (3- and y-to-cotrienols, a-tocotrienol, 5-tocopherol, (3- and y-tocopherol, and a-tocopherol. Figure D 1.5.4 is an example of a reversed-phase chromatograph of tocopherols and tocotrienols in rice bran oil. The tocols with unsaturated side chain have shorter retention time than those with saturated side chain. The methyl substituents on the chromanol ring also affect the retention times of tocopherols and tocotrienols. However, the effect is reversed, compared with the normal-phase HPLC method. 

Various applications of PITC in analyses of acid hydrolysates of purified proteins and peptides (91) and of hydrolysates of feeds (92) have been described. Bidlingmeyer et al. (93) showed reversed-phase chromatographic analysis of free amino acids and hydrolysates of foods (soybean flour, mozzarella cheese, beer, and soy sauce) with formation of PITC derivatives to be a fast, reproducible method that presented a very good correlation with the results obtained by ion-exchange chromatography. 

Table 4 Reverse Phase Chromatographic Methods for Determination of Organic Acids ...

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

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