Modes of Separation

Lower phase Upper phase Mode of separation catalyst ... 

General Description. Liquid chromatography encompasses any chromatographic method in which the mobile phase is a liquid (c.f. gas chromatography). A variety of stationary phases and retention mechanisms are available such that a broad range of modes of separation are possible. It is worthwhile to briefly describe the important modes that find use in clinical chemistry. 

Antidrcular development is very rarely applied in planar chromatographic practice for preparative separation. This mode of separation was introduced by Kaiser [40]. Studer and Traitler adapted the antidrcular U-chamber from CAMAG to preparative separations on 20 x20 cm plates [22], as mentioned earher. The sample mixture was spotted at the circumference of the plate, and the mobile phase was moved from the circumference to the center of the plate. The bands obtained are elongated, but their diameter measured perpendicular to the direction of mobile phase movement... 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

Bioactivity-guided separation has in recent years become the prevalent mode of separation. Typically, it involves extraction of the whole animal with aqueous... 

Discussed below are various modes of separations in HPLC. Included here is brief coverage of mobile-phase selection for various modes of chromatography and elementary information on mechanism, choice of solvents and columns, and other practical considerations. It should come as no surprise that reversed-phase HPLC is discussed at greater length in this section because it is the most commonly used technique in HPLC (more detailed discussion is provided in Section 15.8). Clearly,... 

The columns commonly used in HPLC can be classified based on mode of separation, selected backbone, particle size, and functionalities (Table 15.14). 

It is clearly not possible to cover all these applications within the scope of this chapter. Detailed applications can be found in the literature [1-3,5,7]. Reproduced below is a case study to show how one may select a mode of separation in HPLC and at the same time develop a better understanding of separation mechanism. 

Non-silica-based RP-HPLC stationary phases have also been developed and their separation capacity has been compared with those of silica-based ones. The porous structure of crosslinked polymer gels may be responsible for the markedly different selectivity and retention characteristics. Up till now, the mode of separation on polymer stationary phases is not entirely understood at the molecular level. It has been established that the size-exclusion effect may influence the retention of analyses on polymer gels. 

The concept of the four different modes of separation is an oversimplification. In reality there are no distinct boundaries, and several different mechanisms often operate simultaneously. 

Other than selecting the column and mobile phase for the correct mode of separation, optimizing different HPLC parameters (injection volume, run time, wavelength, and detector) is equally important for achieving acceptable capacity factor (k ), resolution ( R), and tailing factor (T). 

Successful application of chromatographic techniques relies on resolution, or the resolving power of the particular technique used. Resolution is defined by the relation of selectivity and efficiency of the chromatographic gel media (i). Selectivity is a function of the mode of separation of the gel (i.e., gel filtration, ion exchange, etc.) and efficiency is a function of the support matrix (Le., particle shape, size distribution, mechanical stability, density of interactive chemical groups, etc.). Each of the various modes of chromatographic separation have unique advantages that dictate where and when in a purification process these techniques should be used. 

P Lundahl, Q Yang. Liposome chromatography a new mode of separation using lipid bilayer. Protein Nucleic acid Enzyme (Tokyo) 35 1983-1998, 1990 (in Japanese). 

This chapter illustrates basic concepts, instrumental aspects, and modes of separation of electromigration techniques performed in capillary format. It shonld be noted that most of the fundamental and practical aspects of the electromigration techniqnes performed in capillary tubes also apply when the techniques are carried out in microchannels fabricated on plates of reduced dimensions, communally referred to as chips. 

Gas chromatographic separation has not gained wide acceptance in spite of being quite sensitive and specific. This mode of separation is complicated by the need for derivatization of sulfonamide residues before gas chromatographic analysis. These drugs are subjected to derivatization via methylation with diazomethane (223, 224, 253, 254, 271), or double derivatization via methylation followed either by silylation with Ai-methyl-Ai-trimethylsilytrifluoroacetamide (261) or by acylation with A-methyl-bis(trifluoroacetamide) (256). This derivatization step is required not only to form the volatile derivatives of the sulfonamides but also to improve their chromatographic properties (thermal stability and decreased polarity). 

Some Available Data. A brief list of extractive distillation processes of actual or potential commercial value is in Table 13.7 the column of remarks explains why this mode of separation is adopted. The leading applications are to the separation of close-boiling aromatic, naphthenic, and aliphatic hydrocarbons and of olefins from diolefins such as butadiene and isoprene. Miscellaneous separations include propane from propylene with acrylonitrile as solvent (DuPont, U.S. Pat. 2,980,727) and ethanol from propanol with water as solvent [Fig. 13.24(b)],... 

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