Chromatography resolution technique

Racemic mixtures of sulfoxides have often been separated completely or partially into the enantiomers. Various resolution techniques have been used, but the most important method has been via diastereomeric salt formation. Recently, resolution via complex formation between sulfoxides and homochiral compounds has been demonstrated and will likely prove of increasing importance as a method of separating enantiomers. Preparative liquid chromatography on chiral columns may also prove increasingly important it already is very useful on an analytical scale for the determination of enantiomeric purity. 

Hall et al. (127) compared free solution capillary electrophoresis (FSCE) and micellar elec-trokinetic capillary chromatography (MEKC) techniques with HPLC analysis. Four major food-grade antioxidants, propyl gallate (PG), BHA, BHT, and TBHQ, were separated. Resolution of the 4 antioxidants was not successful with FSCE, but was with MEKC. Separation was completed with excellent resolution and efficiency within 6 min and picomole amounts of the antioxidants were detectable using UV absorption. In contrast, reversed-phase HPLC separation was not as efficient and required larger sample amounts and longer separation time. 

With the development of the chiral ligand exchange chromatography by Davan-kov, this technique has been used frequently for the chiral resolution of racemic compounds containing electron-donating atoms. It is useful for providing the basic information on the chiral resolution and, hence, is still in use. In spite of this, there are some limitations with this chiral resolution technique. The most... 

Application of high performance liquid chromatography to the resolution of complex mixtures of fatty acids in water [9,10] has provided an alternative to the high temperature separation obtained by gas chromatography. Both techniques have similar limits of detection, but lack the ability to analyse environmental samples directly. Analysis requires that the fatty acids be separated from the organic and inorganic matrices, followed by concentration. 

Kumpalume P, Ghose S (2003), Chromatography the high-resolution technique for protein separation, In Hatti-Kaul R, Mattiasson B (Eds), Isolation and Purification of Proteins, Marcel Dekker, New York, pp. 29-56. 

Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations 51]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography. 

As mentioned, asymmetrically pure compounds are important for many applications, and many different strategies are pursued. However, in spite of many methods being developed, the classic resolution technique of diastereomeric crystallization is still preferentially used to prepare optically active pure compounds in bulk quantity. Crystallization is commonly used in the last purification steps for solid compounds because it is the most economic technique for purification and resolution. Attempts to achieve crystallization after completed reaction without workup and extraction is called a direct isolation process. This technique can be cost-effective even though the product yield obtained is lower. Special conditions may be needed in this case, and the diastereomers can be classified into two types diastereomeric salts and covalent diastereomeric compounds, respectively. Diastereomeric salts can, for example, be used in the crystallization of a desired amine from its racemic mixture using a chiral acid. Covalent diastereomers can, on the other hand, be separated by chromatography, but are more difficult to prepare. Another advantage of crystallization is the possibility of combining in situ racemi-zation reactions and diastereomeric formation reactions to get the desired pure compounds. This crystallization-induced resolution technique is still under development because of its requirements for optimized conditions [55, 56],... 

FFF is a separation technique that encompasses a range of procedures based on theory and subsequent instrumentation originally developed and advanced by Gid-dings.75-76 FFF is a high-resolution chromatography-like technique applicable to the separation of macromolecules, colloids and particles encompassing a size range of... 

Both the quantity and properties of cellulases produced by microorganisms depend on the culture conditions. Commonly, cellulases are produced by culture of the organism either (a) in a liquid medium, which may be stationary, shaken, or submerged with aeration, or (b) by a Koji process on a solid substrate such as wheat bran (7). The complexity of the crude cellulosic carbon source usually leads to the production of a mixture of hydrolytic enzymes which may include amylases, proteases, chitinases, etc., in addition to the cellulases. Separation of proteins from culture filtrates by high resolution techniques such as chromatography, electrophoresis, or electrofocusing often reveals a number of enzyme species which may differ in specificity toward cellulosic substrates. These forms may represent ... 

The functional requirements of fraction collectors have changed as liquid chromatography evolved from low-pressure (low-resolution) to high-pressure (high-resolution) techniques. This evolution was led by developments in column technologies and packing materials, and followed by adaptations to pumps, detectors, injectors, and fraction collectors. The following subsections briefly describe some of the key operational considerations of fraction collection devices. 

Furthermore, in some cases, these detectors may be used in conjunction with a higher chromatographic resolution/selectivity to minimize matrix interference. Comprehensive two-dimensional gas chromatography (GCxGC) has been under development for more than ten years [10(and references contained therein)] as an enhanced resolution technique. The technique involves continuously transferring or modulating short time periods from a primary column, the first dimension, to a second column of different polarity or selectivity, which serves as the second dimension. The modulation period is typically less than the peak widths observed on the... 

Proteolytic digestion, good chromatography, and a mass analyzer with high resolution help to filter through subpopulations of peptides present. Ion mobility has also been shown to help reduce sample complexity [52] Complements and constraints both high- and low-resolution techniques for structural analysis, such as NMR, X-ray crystallography, molecular dynamics simulations, CD,... 

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