Separation by HPLC

Direct resolution by HPLC is performed using sdica gel stationary phases coated with optically active tetranitrofluorenyhdene (TAPA) derivatives. This is a peculiar method for polyaromatic systems, in that it is based on 

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A mixture of methyl paraben, ethyl paraben, propyl paraben, diethyl phthalate, and butyl paraben is separated by HPLC. This experiment emphasizes the development of a mobile-phase composition capable of separating the mixture. A photodiode array detector demonstrates the coelution of the two compounds. 

Physical Methods. Vitamins D2 and D exhibit uv absorption curves that have a maximum at 264 nm and an (absorbance) of 450—490 at 1% concentration (Table 8). The various isomers of vitamin D exhibit characteristically different uv absorption curves. Mixtures of the isomers are difficult to distinguish. However, when chromatographicaHy separated by hplc, the peaks can be identified by stop-flow techniques based on uv absorption scanning or by photodiodearray spectroscopy. The combination of elution time and characteristic uv absorption curves can be used to identify the isomers present in a sample of vitamin D. 

V. A. Davankov, Ligand-exchange phases in Chiral Separations by HPLC, A. M. Krstulovic, Ellis Horwood Ltd., Chichester (1989) Chapter 15. 

Alcohol sulfates and alcohol ether sulfates separated by HPLC on a styrene-divinylbenzene copolymer column with 4 1 (v/v) methanol and 0.05 M ammonium acetate aqueous solution as the mobile phase were analyzed by simultaneous inductively coupled argon plasma vacuum emission spectroscopy (IPC), monitoring the 180.7-nm sulfur line as a sulfur-specific detector [294]. This method was applied to the analysis of these surfactants in untreated wastewaters. 

For monographs on the use of liquid chromatography to effect resolutions, see Lough, W.J. Chiral Liquid Chromatography, Blackie and Sons London, 1989 Krstulovic, A.M. Chiral Separations by HPLC Ellis Horwood Chichester, 1989 Zief, M. Crane, L.J. Ref. 122. For a review, see Karger, B.L. Anal. Chem., 1967, 39 (8), 24A. 

Arguably the ultimate LC-MS interface would be one that provides El spectra, i.e. a spectrum from which structural information can be extracted by using famihar methodology, and this was one of the great advantages of the moving-belt interface. There is, however, an incompatibility between the types of compound separated by HPLC and the way in which electron ionization is achieved and therefore such an interface has restricted capability, as previously discussed with respect to the moving-belt interface (see Section 4.2 above). 

For betaxanthins, partial synthesis is quite common and presents a viable tool for identification by co-injection experiments. - Starting from a red beet extract or semi-purified betanin-isobetanin blend, alkaline hydrolysis by addition of 32% ammonia is initiated. Spectrophotometric monitoring at 424 nm allows the release of betalamic acid to be followed. Betaxanthins are obtained through the addition of the respective amino acid or amine in at least 20-fold molar excess followed by careful evaporation. Since the starting material most often consists of a racemic betacyanin mixture, the resulting betaxanthin will also consist of two stereoisomers that may not easily be separated by HPLC. ... 

A disadvantage of the first-generation device was that reaction and detection units were separated by HPLC tubing, which caused delay in analysis ]72-74],... 

Aldoses, for example mannose, fucose, arabinose, and others, have been detected at the subpicomole level by their reduction to aldites, which are then transformed into pemaphthoates by means of naphthoylimidazole. These pemaphthoates show strong fluorescence at 374 nm, and can be easily separated by HPLC.[46]... 

The reductive demercuration was marred by the loss of about half of the peroxide due to competing deoxymercuration which afforded 4-cycloocten-l-ol. An additional complication was the formation of a small amount of trans-1,2-epoxy-cw-cyclooct-5-ene. The bicyclic peroxide 50 was readily separated from the unsaturated alcohol by silica chromatography, but complete removal of the epoxide was more difficult. Preservation of the peroxide linkage was markedly higher in the bromodemercuration. The diastereoisomeric dibromoperoxides 51 were separated by HPLC, although only one isomer was fully characterised. 

An alternative approach for the synthesis of 2, 3 -0,0-cyclic //-phosphonate 20a was based on the condensation of a mixture of uridine 3 - and 2 -//-phosphonates (23 and 24 respectively) induced by pivaloyl chloride (Scheme 9) [24], Its reaction with elemental sulfur in carbon disulfide gave 5 -0-DMT-uridine 2, 3 -cyclic phosphorothioate (21a) which after final deprotection afforded the desired 2, 3 -cyclic phosphorothioate 22a (Scheme 9). Its Sp and Rp diastereomers were separated by HPLC [24],... 

The formed 5 -protected 2, 3 -(9,(9-cyclic boranophosphate 26 was finally deprotected by acid treatment and isolated in 70% overall yield by ion-exchange chromatography (Scheme 10) [24], The two pure P-diasteromers of 27 were separated by HPLC. 

Nishiyama and Kuninori [65] described a combination method of assay for penicillamine using HPLC and postcolumn reaction with 6,6 -dithiodi(nicotinic acid). Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil 08-10, with 33 mM KH2PO4 (adjusted to pH 2.2 with H3PO4) or 33 mM sodium phosphate (pH 6.8) as the mobile phase. Detection was by postcolumn derivatization with 6,6 -dithiodi(nicotinic acid), and measurement of the absorbance of the released 6-mercaptonicotinic acid was made at 344 nm. The detection limit for penicillamine was 0.1 nmol. A comparison was made with a... 

Some luminol derivatives have been developed as CL labeling reagents. Analytes prelabeled with luminol derivatives are separated by HPLC, mixed with postcolumn reagents such as hydrogen peroxide and an alkaline solution of potassium hexacyanoferrate (III), and then detected by a CL detector. Highly sensitive determination is possible by optimizing the conditions to increase the CL reaction efficiency for each analyte. 

System C is used when an immobilized enzyme reactor (IMER) is introduced into system B. The analyte(s) separated by HPLC is converted to a suitable species for CL detection with an IMER, and then mixed with the CL reagent. In this system, a buffer solution as a mobile phase and an ion-exchange-type column are preferable for an enzyme reaction. 

Two compounds were separated by HPLC with an Rs value of 0.75, the plate number for the second compound being 4500. Calculate the number of plates required to obtain resolutions of (a) 1.0 and (b) 1.5. 

Tautomerism. Owing to the presence of the guanidine moiety in the structure of tegaserod, the three tautomers shown below are possible. Tautomer 6a is assigned to the solid state of tegaserod, as confirmed by X-ray structure analysis of the 5-0-benzyl derivative. In solution, 6b and 6c also exist in equilibrium depending on the experimental conditions. For instance in DMSO, the solvent used to measure NMR spectra, 6b is preferred. Tautomer 6c is formed in methanol after exposure to xenon light, and from this solution the tautomer can even be separated by HPLC. 

In HPLC, the mobile phase is a liquid in which the sample must be soluble, and detection is most often accomplished by ultraviolet (UV) absorption. It is generally a slower process than GC however, its advantage is that the compounds to be separated are not limited by their boiling point, although low-boiling compounds are almost never separated by HPLC. Solid mixtures, as long as they are soluble in the mobile phase, can be analyzed by HPLC, whereas the solids that are typically encountered in soil analysis are not usually volatile enough to analyze via GC. 

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