Assays column chromatography

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

A series of diastereomerically pure 5 -0-DMT-nucleoside 3 -0-(2-thio-l,3,2-oxathiaphospholanes) and their oxathiaphospholane ring-substituted analogues 283-294 were isolated in 80-83% yield by column chromatography on silica gel of the appropriate diastereomeric mixtures [the ratio ca 55 45 (31P NMR assay)] obtained from the reaction of 2 - A, IV- dii so p rop y I a m i n o- 1,3,2-oxathiaphospholane 279-281 with 5 - 0 -D M T- n uc I cosides 282a-d in the presence of tetrazole (phosphi-tylation), followed by addition of sulfur (Scheme 67) [105-107]. 

Evidently, due to the cross-reactivity of both mono- and di-desethyl metabolites, a specific assay of flurazepam could not be developed successfully without first separating it from its metabolites effectively by the help of column chromatography. 

Bartlett GR. Phosphorous assay in column chromatography. J Biol Chem 1959 234 466. 

Column Chromatography of Crude Toxin. The WSAP obtained from culture 350F was retained in the crude state for assay. The 266.2 mg of WSAP obtained from 350G was treated on successive columns of silicic acid, DEAE cellulose, and Sephadex G-15 and yielded a single semipurified toxic product (GT-4) of 23.1 mg or 9% of the starting crude extract (Table I). 

Salicylic acid and Its metabolite were separated by two methods. The first was thin layer chromatography on cellulose with BAW solvent as for the In vivo metabolism studies. A quicker separation was achieved with a polyamide column. The entire 400 pL from an individual assay was placed on top of a 0.8 x 2.0 cm column packed with Polyamide-6 (Accurate Chemical and Scientific Corp.). The salicylic acid metabolite was eluted with 6 mL water but salicylic acid was retained. 3a70B scintillation fluid (Research Product International Corp.) was used to determine the radioactive content of the entire 6 mL of eluant. Separation of salicylic acid and its metabolite by polyamide column chromatography was verified by thin layer chromatography. 

Hoffmann GF, Brendel SU, et al. (1992) Mevalonate kinase assay using DEAE-cellulose column chromatography for first-trimester prenatal diagnosis and complementation analysis in mevalonic aciduria. J Inherit Metab Dis 15 738-746... 

Purity can be assayed by chromatography (on thin-layer plates, Kieselguhr paper or columns), by UV or NMR procedures. 

Cleanup or fractionation procedures that have been used in the more recent fat-soluble vitamin assays include sterol precipitation, open-column chromatography, solid-phase extraction, and high-pressure gel permeation chromatography. High-performance LC has been used on a semipreparative scale in vitamin D and vitamin K assays to obtain purified fractions of sample extracts. This technique is discussed in Sec. V.B.3. 

The more recent applications of open-column chromatography in fat-soluble vitamin assays utilize liquid-solid (adsorption) chromatography using gravity-flow glass columns dry-packed with magnesia, alumina, or silica gel. Such columns enable separations directly comparable with those obtained by thin-layer chromatography to be carried out rapidly on a preparative scale. 

Three recent reviews specifically cover HPLC methods for quantitating riboflavin in foods. In addition to HPLC methods, Nielsen (81) summarized paper chromatography, TLC, and open-column chromatography procedures for quantitating total riboflavin and the individual vitamers in foods, pharmaceuticals, and biological samples. Russell (44) included a brief discussion of the standard methods, along with HPLC and flow injection analyses published between 1990 and 1994 for total riboflavin and the individual vitamers in foods. Ball (45) reviewed HPLC methods for quantitation of riboflavin, as well as chemical and microbiological riboflavin assays for foods. 

Although classical column chromatography over chiral stationary phases has been used for several years, HPLC has until recently been regarded mainly as an analytical tool. However, methods have now been developed and many papers have been published on the various applications of particular stationary phases in preparative HPLC, which may be the method of choice when the preparation of small amounts of material are required for screening purposes, for use in biological assays, or as standards in purity assays. The use of chromatographic techniques may be quicker and less risky than custom synthesis and resolution and can guarantee material to a defined specification. 

Assay (See Chromatography, Appendix IIA.) Determine the content of propan-2-ol and volatile impurities using a suitable gas chromatograph equipped with flame-ionization detector and a 1.8-m x 6-mm (id) steel column, or equivalent, packed with 10% P.E.G. 400 on 60- to 80-mesh Chromosorb W (or equivalent). Maintain the column at 90°, and set both the injection port temperature and the detector temperature to 150°. Use helium as the carrier gas, with a flow rate of 45 mL/min. Inject between l-p,L and 5-p,L samples, and from the chromatograms so obtained, determine the content of each constituent by the method of area normalization. 

Assay (See Chromatography, Appendix BA.) Inject a 10-p,L portion of sample into a suitable gas chromatograph in which the detector is the thermal conductivity type and the column is 1-m x 8-mm (id) stainless steel tubing (Perkin Elmer Instruments, or equivalent) packed with 4% Carbowax compound 20 M on 40- to 60-mesh Chromosorb T, or equivalent materials. The carrier gas is helium flowing at 75 mL/ min. The injection port temperature is 240° the column temperature is 120° to 200°, programmed at a rate of 5°/min and the block temperature is 250°. Under the conditions described, the approximate retention time for Propylene Glycol is 5.7 min, and 8.2, 9.0, and 10.2 min for the three isomers of... 

Extensive use of column chromatography has been necessary to separate the Rauwolfia alkaloids, and in this connection attention is drawn to a publication which concerned itself with the more refined technique of gradient elution chromatography (123). Paper chromatography has been used extensively for analytical, fractionation, and identification purposes (124, 15). This tool is not particularly useful for the assay of crude extracts, since certain alkaloids show up clearly whereas others cannot be resolved. More information as to specific alkaloidal composition is better obtained from more highly purified fractions. 

L-Kynurenine was separated from 3-hydroxykynurenine (observed in some assays) by chromatography on a Beckman Ultrasphere ODS column (4.6 mm x 150 mm). The mobile phase (1.5 m/min) was a 5 235 mixture of acetonitrile and 0.1 M ammonium acetate buffer (pH 4.65). The column effluent was monitored at 365 nm. 

G. R. Bartlett, J. Biol. Chem., 234 466-469 (1959). Phosphorus Assay in Column Chromatography. 

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