Column separation, modes

Analytical column (separation-mode reverse-phase)... 

A more detailed discussion of the stationary phase types and mechanism of interaction and separation theory in relation to chiral compounds is given in Chapter 22. A large number of chiral stationary phases are currently available to meet the needs of the pharmaceutical industry for determination of the enantiomeric purity of active pharmaceutical ingredients, raw materials, and metabolites. As a consequence, there are a multitude of options in terms of columns, separation mode, and separation conditions to explore in achieving an enantioseparation. 

The hydrophobicity of the gel surface differs depending on the type of gel. In polymer-based series, the GPC KF series has the most hydrophobic gel surface whereas the SB-HQ series has the most hydrophilic gel surface. In order to obtain an ideal GFC separation mode, a suitable column should be chosen whose gel surface has hydrophobicity similar to that of the sample. 

Efforts have been made, however, to extend the range or extent of samples that can be analysed by using a two-dimensional separation when used in heart-cut mode. This has been reported to include the use of numerous parallel micro-traps to essentially store the primary column eluent fractions ready for second-column separation, and the use of parallel second-dimension columns. 

Table 7.89 lists the main characteristics of MDHPLC (see also Table 7.86). In MDHPLC the mobile-phase polarity can be adjusted in order to obtain adequate resolution, and a wide range of selectivity differences can be employed when using the various available separation modes [906]. Some LC modes have incompatible mobile phases, e.g. normal-phase and ion-exchange separations. Potential problems arise with liquid-phase immiscibility precipitation of buffer salts and incompatibilities between the mobile phase from one column and the stationary phase of another (e.g. swelling of some polymeric stationary-phase supports by changes in solvents or deactivation of silica by small amounts of water).

Principles and Characteristics Multidimensionality in planar chromatography takes a broader context than is common for column separations. For TLC, various modes of multidimensional separations are used ... 

Like plasma and urine, matrixes from plant or environmental sources contain a vast diversity of components. Thus, achiral-chiral LC-LC is also useful for analysis involving samples from these sources. Stalcup et al. (1991) studied the enantiomeric purity of scopolamine extracted from Datura sanguinea in both homogenized plant leaves and commercial extracts. A reverse-phase separation on a C j g column separated the scopolamine from other alkaloid and matrix components while the enantiomeric separation (also in the reverse-phase mode) was carried out on two coupled [3-cyclodextrin columns or a single acetylated (3-cyclodextrin column. The single... 

In chromatography, the separation efficiency of any single separation method is limited by the efficiency and selectivity of the separation mode, that is, the plate count of the column and the phase of the selected system. Adding more columns will not overcome the need to identify more components in a complex sample, due to the limitation of peak capacities. The peak capacity in an isocratic separation can be described, following Grushka (1970), as given in Equation (17.1) ... 

We will show several examples of the use of 2DLC for nonionic surfactants. The resulting resolution can be dramatically different depending on the two separation modes in the 2DLC system. As discussed previously, the separation of the hydrophobic groups can be accomplished with a reversed-phase column, and the separation of the... 

From quantification performed in the FIA mode, applying mixture analysis by FIA-MS-MS, reliable results can be achieved if product-, parent- or neutral loss scans (MRM mode), each of them of quite outstanding specificity, are recorded. Compared with FIA—MS, the time for recording has to be expanded marginally compared with column separation techniques. 

Fig. 9. Conjoint Liquid Chromatography (CLC). Separation of proteins from mouse ascites and isolation of monoclonal antibody IgG in one step obtained by a combination of CIM QA and CIM Protein A Disks. Conditions Separation mode CLC (first disk CIM QA, 12 x 3 mm ID, 0.34 ml second disk - CIM Protein A, 12 x 3 mm ID, 0.34 ml, inserted in monolithic column housing) Instrumentation Gradient HPLC system with extra low dead volume mixing chamber Sample Mouse ascites Injection volume 20 pL Mobile Phase Buffer A 20 mM Tris-HCl, pH 7.4 Buffer B Buffer A + 1 M NaCl Buffer C 0.1 M Acetic acid Conditions Gradient 0 - 50 % B in 50 s, 100% A for 40 s, 100% C for 30 s Flow Rate 4 ml/min Detection UV at 280 nm... 

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