Stationary phases and their applications

There are several stationary phases that can be used for separation of various compounds including lipids, natural products, biological compounds, drugs, etc. Examples are Celite (Supelco Inc., PA), cellulose powder, ion-exchange cellulose, starch, polyamides and Sephadex (Supelco Inc., PA), but the most popular ones for lipid separations include silica gel, alumina and kieselguhr. These adsorbents can also be modified by impregnation with other substances so as to achieve the desired separations. Based on their characteristics these phases can be classified as normal or reversed. 

Normal phases. Silica is generally the normal phase used in lipid separations. The average particle sizes in traditional TLC range between 10 pm and 50 pm, with a fairly wide size distribution. In normal phase TLC the stationary phase (such as silica) is polar and the mobile phase is relatively non-polar. Scott (1982) has extensively reviewed silica gel and its properties, the water absorption of the silica surface and its interaction with polar and non-polar solvents. 

High-performance silica is used in high-performance thin-layer chromatography (HPTLC). HPTLC differs from normal TLC in that the size of the absorbent (usually silica) is only 5 pm, with a narrow distribution. This enables HPTLC to give better separations compared with TLC, which uses a standard silica, and, moreover, HPTLC requires a smaller sample size and has a lower detection limit compared with conventional TLC. HPTLC plates of varying sizes are commercially available and of late have found considerable applications in the field of lipids. Weins and Hauck (1995), in their survey of TLC, conclude that the use of HPTLC plates increased by 30% over the period 1993-95. An excellent application of HPTLC is illustrated in Fig. 1.1 for the separation of neutral and complex lipids. Yao and Rastetter (1985) have achieved separation of more than 20 lipid classes of tissue lipids on HPTLC plates using four developing solvents. 

We have achieved good separation of polyunsaturated fatty acid esters from saturated, monosaturated and disaturated fatty acid esters and this has been used as a means of identifying the presence of polyunsaturates, especially fish oil, in samples (Fig. 1.2). However, if there is a considerable amount of trienoic fatty acids in the mixture, as in egg lipids, the separation is not very pronounced (Shantha and Ackman, 1991a). Nakamura, Fukuda and Tanaka (1996) have used similar TLC separation of marine lipids and were able to quantitate the amount of pol)amsaturated fatty acids by using scanning densitometry followed by Coomassie blue staining. 

We have used silica gel TLC to separate conjugated triglycerides and methyl esters from the non-conjugated species in the seeds of Momordica charantia and Trichosanthus anguina (unpublished results). These seeds contain between 50%-60% of their total fatty acids as a-eleostearic and punicic 

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Liu, X. and Pohl, CA. (2011) HlLlC behavior of reversed-phase/ion-exchange mixed-mode stationary phases and their applications, in Hydrophilic Interaction Liquid Chromatography (HILIC) and Advanced Applications (eds P.G. 

Thunberg L, Hashemi J, Andersson S (2008) Comparative study of coated and immobilized polysaccharide-based chiral stationary phases and their applicability in the resolution of enantiomers. J Chromatogr B 875 72-80... 

These is no formal method of categorizing the types of chiral stationary phases available for liquid chromatography (784). This diversity is both a strength, in providing a wide range of possibilities to solve a particular problem, and a weakness in that it is often difficult to predict which phase will be most useful for solving a particular problem. Compilations of commercially available chiral stationary phases and their proven applications are available (780-785). As new phases continue to be introduced at a hectic pace only a brief overview of their properties will be attempted here. 

This method allows the fractionation of citms oils based on the different polarity of terpene and aroma fraction ]20, 21]. Their different adsorption characteristics on stationary phases can be employed for the selective separation of these complex mixtures of natural substances. The method originates from analytical laboratory techniques and its application on various stationary phases is realised industrially today ]22, 23]. Adsorption on stationary phases and their subsequent desorption with various solvents is possible for different adsorption materials. Also partition chromatographic methods play an important role in citms flavour concentration processes, especially for aqueous citms essences. 

Stationary phases can be made highly selective by adding compounds to them which have affinities for certain chemical species. For example, silver nitrate, incorporated into a polar liquid preferentially retards the elution of alkenes by formation of weak -complexes. A selection of stationary phases with their maximum operating temperatures and useful applications is given in Table 4.10. 

A third area of development in carbohydrate l.c. analyses is in the combined techniques (see Section IV,3) and other methods that provide qualitative, as well as quantitative, information about sample constituents, such as high-performance liquid affinity chromatography. The use of specific lectin- and monoclonal antibody-based, stationary phases for analytical and preparative applications is now being considered. The basic concepts of these techniques have been reviewed - and their applications to carbohydrates have been discussed. 

Another popular and selective extraction technique widely used in bioanalysis is solid phase extraction (SPE). SPE is a separation process utilizing the affinity of the analytes to a solid stationary phase. By manipulating the polarity and pH of the mobile phase, the analytes of interest or undesired impurities pass through stationary phase sequentially according to their physical and chemical properties. For a SPE procedure, a wash step refers to the elution of the unwanted impurities which are discarded and the elution step refers to the elution of the analytes of interest which are collected. While the fundamental remains the same in decades, the continuing invention and introduction of new commercial stationary phases and accessory devices have boosted the application of SPE in bioanalysis and many other fields. 

Many reviews have been written on the preparation, physico-chemical properties and application of silica in modem separation science [5-7]. RP LC with silica based bonded stationary phases is utilised for the majority of LC separations in laboratories world-wide. Their ubiquity derives from their versatility, in that generally a wide range of both ionic and non-ionic analyte species can be separated with these columns by careful selection of the stationary phase and mobile phase properties. 

McCalley, D.V. Selection of suitable stationary phases and optimum conditions for their application in the separation of basic compounds by reversed-phase HPLC. J. Sep. ScL 2003, 26, 187-200. 

The term solid-phase extraction was introduced by personnel of the J. T. Baker Company in 1982. The method consists of retention of the analytes from a liquid or gaseous sample to a solid stationary phase and subsequent removal of analytes using an appropriate eluent. The main purpose of SPE is isolation and preconcentration of compounds of interest or sample clean-up and simplification of the matrix. Application of this sample preparation technique also allows extract fractionation. As a result of significant reduction in the volume of organic solvents used, high recovery, and the possibility of process automation, SPE is a good alternative for conventional liquid-liquid extraction. According to their affinity for the compound of interest, stationary phases are classified as follows ... 

Equation (10-1) is based on the assumption of simple additivity of all interactions and a competitive nature of analyte/eluent interactions with the stationary phase. The paradox is that these assumptions are usually acceptable only as a first approximation, and their application in HPLC sometimes allows the description and prediction of the analyte retention versus the variation in elution composition or temperature. For most demanding separations where discrimination of related components is necessary, the accuracy of such prediction is not acceptable. It is obvious from the exponential nature of equation (10-1) that any minor errors in the estimation of interaction energy, or simple underestimation of mutual influence of molecular fragments (neglected in this model), will generate significant deviation from predicted retention factors. 

The aim of this chapter is to highlight current developments in these various fields of preparative HPLC, with particular emphasis on applications that have been developed at Chemical Analytical Development at Novartis Pharma AG. Drug substance purifications from biological and synthetic sources are presented, along with the separation of chiral and/or achiral molecules on chiral stationary phases and typical isolations of by-products. Special attention is given to the determination of adsorption isotherms and their interplay with respect to the layout of chromatographic processes as well as the choice of... 

M. Lammerhofer and W. Lindner, Quinince and quinidine derivatives as chiral selectors. Beush type chiral stationary phases for high performance liquid chromatography based on chincona carbamates and their applications as chiral anion exchanger, J. Chromatogr. 741 (1966), 33. 

The first reported use of a vapor as the mobile phase is attributed to Martin and Synge in 1941. They used the principles of partition chromatography, whereas James and Martin, in 1952, described the first application of this method, gas-liquid chromatography (GLC), for the analysis of fatty acids and amines. Gas adsorption chromatography (GSC), on the other hand, involves the use of a solid stationary phase and separation is based on an adsorptive mechanism. This technique was first described in 1947 in a doctoral thesis by Prior,under the supervision of Professor Cremer, and subsequently in their 1951 publication.f ... 

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