HPLC separation products

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

Popova and colleagues47 carried out TLC of oxidation products of 4,4 -dinitrodiphenyl sulphide (the sulphoxide and sulphone) on silica gel + a fluorescent indicator, using hexane-acetone-benzene-methanol(60 36 10 l) as solvent mixture. Morris130 performed GLC and TLC of dimethyl sulphoxide. For the latter, he applied a 6% solution of the sample in methanol to silica gel and developed with methanol-ammonia solution(200 3), visualizing with 2% aqueous Co11 thiocyanate-methanol(2 1). HPLC separations of chiral mixtures of sulphoxides have been carried out. Thus Pirkle and coworkers131-132 reported separations of alkyl 2,4-dinitrophenyl sulphoxides and some others on a silica-gel (Porosil)-bonded chiral fluoroalcoholic stationary phase, with the structure ... 

The initial route to taranabant relied on a late stage amide bond coupling between racemic amine rac-2 and pyridine acid 3 mediated by (benzotriazol-l-yloxy)tripyr-rolidinophosphonium hexafluorophosphate (Py-Bop), followed by chiral HPLC separation of the product to afford a single enantiomer (Scheme 9.1). 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Size-exclusion HPLC (SE-HPLC) separates proteins on the basis of size and shape. As most soluble proteins are globular (i.e. roughly spherical in shape), separation is essentially achieved on the basis of molecular mass in most instances. Commonly used SE-HPLC stationary phases include silica-based supports and cross-linked agarose of defined pore size. Size-exclusion systems are most often used to analyse product for the presence of dimers or higher molecular mass aggregates of itself, as well as proteolysed product variants. 

An HPLC separation of a two-component pharmaceutical product yielded the following data ... 

Formation of several colored products during reaction of nitrosoarenes with thiols has been repeatedly observed12,26,68. Two different orange-colored conjugates were found during HPLC separation of mixtures of 4-nitrosophenetol and GSH. The UV spectra were indicative of a quinoid structure, and further studies revealed these adducts to be a monocyclic and a bicyclic conjugate. In both cases the reactive quinoid structure gives rise to formation of secondary, stable end products. 

The performance of HPLC separations can be increased not only by the application of two- or multidimensional techniques but also by the use of simulated moving bed (SMB) [100-102] or true moving bed (TMB) techniques [103,104], SMB is a multicolumn separation technique allowing the continuous separation of analyses with higher productivity and smaller eluent consumption than the traditional single-column procedures. TMB... 

Traditional methodologies for structural identification of trace level impurities in drng substances/products usually involve fractionation of each impurities using a scaled-np analytical chromatographic method, followed by off-line spectroscopic analysis. Coupling of HPLC separation and electrospray mass spectrometry allows on-line acquisition of full scan mass spectra and generation of tandem mass spectrometric data. LC/ESI MS has revolntionized trace analysis for qnalitative and quantitative studies in pharmaceutical analysis. 

Another classical reaction which has been reinvestigated by the batch method (with quick HPLC separation of samples withdrawn at various times) is the acid-catalyzed substitution of Co(CN)5N - by SCN. The products are Co(CN)5H202-, Co(CN)5SCN5- and Co(CN)5NCS . The kinetics of these reactions are difficult to disentangle by in situ spectral examination alone, and indeed this procedure has led to erroneous conclusions. Care must be observed that the separation procedure used in the batch method does not alter the conditions of the system. ... 

Table 1.3 summarizes all commercialized monolithic columns that are currently available for HPLC separation. The steadily growing number of commercially available products, based on monolithic packings, express the potential of this kind of stationary phases in particular fields of chromatography and the increasing demand from customer side. 

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