Coupling separations with

Figure 12.8 RNA Chromatogram of the entire pool of tRNAs purified from coli. By coupling separation with RTPCR it is possible to identify and quantify the individual tRNAs. A typical population of tRNAs will contain around 50 species.

Although GC/MS is the most widely used analytical method that combines a chromatographic separation with the identification power of mass spectrometry, it is not the only one. Chemists have coupled mass spectrometers to most of the instruments that are used to separate mixtures. Perhaps the ultimate is mass spectrometry/mass spectrometry (MS/MS), in which one mass spectrometer generates and separates the molecular ions of the components of a mixture and a second mass spectrometer examines their fragmentation patterns ... 

Figure 8.17 Schematic diagram of a cross-section (a) tlvough the clamped plates, and views from above (b-d) of coupled plates serially connected to achieve multidimensional separation with stationary phases with different characteristics (hatched lines, glass plate light shading, stationary phase A dark shading, stationary phase B wavy lines, stationary phase C).

In 1995, Moore and Jorgenson used the optically gated CZE system to obtain extremely rapid separations with HPLC coupled to CZE. The rapid CZE analysis made possible more frequent sampling of the HPLC column, thus increasing the comprehensive resolving power. Complete two-dimensional analyses were performed in less than 10 min, with the CZE analyses requiring only 2.5s. A peak... 

Supercritical fluid extraction (SFE) has been extensively used for the extraction of volatile components such as essential oils, flavours and aromas from plant materials on an industrial as well as an analytical scale (61). The extract thus obtained is usually analysed by GC. Off-line SFE-GC is frequently employed, but on-line SEE-GC has also been used. The direct coupling of SEE with supercritical fluid chromatography (SEC) has also been successfully caried out. Coupling SEE with SEC provides several advantages for the separation and detection of organic substances low temperatures can be used for both SEE and SEC, so they are well suited for the analysis of natural materials that contain compounds which are temperature-sensitive, such as flavours and fragrances. 

The first bioanalytical application of LC-GC was presented by Grob et al. (119). These authors proposed this coupled system for the determination of diethylstilbe-strol in urine as a replacement for GC-MS. After hydrolysis, clean-up by solid-phase extraction and derivatization by pentafluorobenzyl bromide, the extract was separated with normal-phase LC by using cyclohexane/1 % tetrahydrofuran (THE) at a flow-rate of 260 p.l/min as the mobile phase. The result of LC-UV analysis of a urine sample and GC with electron-capture detection (ECD) of the LC fraction are shown in Ligures 11.8(a) and (b), respectively. The practical detection limits varied between about 0.1 and 0.3 ppb, depending on the urine being analysed. By use of... 

Analogous to two-dimensional LC, the on-line coupling of LC and CE has been carried out both in the heart-cut and the comprehensive mode. In a heart-cut LC-CE study, a protein G immunoaffinity LC column was used to selectively preconcentrate insulin from serum (171). A 1 p.1 elution plug comprising the insulin was switched on-line to the CE system where a part was injected into the capillary for final separation. With CE, efficient separations can be obtained in a... 

Since the first separation of enantiomers by SMB chromatography, described in 1992 [95], the technique has been shown to be a perfect alternative for preparative chiral resolutions [10, 21, 96, 97]. Although the initial investment in the instrumentation is quite high - and often prohibitive for small companies - the savings in solvent consumption and human power, as well as the increase in productivity, result in reduced production costs [21, 94, 98]. Therefore, the technique would be specially suitable when large-scale productions (>100 g) of pure enantiomers are needed. Despite the fact that SMB can produce enantiomers at very high enantiomeric excesses, it is sometimes convenient to couple it with another separation... 

In a very recent study, it has been demonstrated116 that zinc 5,15-bis(3,5-di-tert-butylphenyl)-porphyrin (13) without any activating halogen atoms at the chromophore can be directly linked in a very simple oxidative coupling reaction with silver(I) hexafluorophosphate to a mixture of porphyrin dimers, trimers and tetramers. The separation of the product mixture was achieved by gel-permeation chromatography based on the molecular weights of the oligomers. The dimer when re-exposed to the same reaction conditions yielded 25% of the tetramer.116... 

The basic aim of PEC applications in clinical chemistry, apart from the recovery of standards of endogenous substances, consists of structural identification of isolated (without further separation) substances of relatively high purity. Therefore, the majority of works devoted to this topic pertain to semipreparative separation. Obtaining low amounts of analytes, achieved by coupling TEC with modem... 

An analysis using perturbation theory shows that the influence of the coupling terms with Ck (Q) and A x is small when the electronic energy levels k(Q) and i(Q) are not closely spaced. Since the ground-state electronic energy of a molecule is usually widely separated from the first-excited... 

Applications If an extract needs further cleanup, it is possible to couple it with multidimensional chromatographic techniques such as LC-LC or LC-GC. The first chromatographic step can then be used for the on-line cleanup and concentration of the extract, and the second one for the final separation. Large-volume, on-column injection (LVI-COC) is particularly useful for coupled LC-GC in which 100-350 xL fractions of eluent from the NPLC cleanup separation step are transferred on-line to the GC column. For example, on-line removal of high-MW interfering material, such as polymers from a polymer/additive dissolution, can be achieved easily by using SEC before the fraction containing additives is transferred to the GC. 

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