Column Coupling Technique

2 Method Development and Optimization of Enantiomeric Separations Using. .. 

Each glycopeptide CSP has unique selectivity as well as complementary characteristics, and a considerable number of racemates have been resolved on all three of them. Interestingly, most of the resolved enantiomers have the same retention order on these macrocyclic CSPs. When they are mixed or coupled with each other, the selectivity on one CSP will not be canceled by another. Even if some compounds may not have the same retention order, the complementary effects will result in an identifiable selectivity. Therefore, the coupled chiral columns can be used as a screening tool and save chromatographers substantial time in method development. 

One potential problem associated with column coupling in reversed phase is relatively high back-pressure ( 2600 psi at 1 mL miir ). This will place a limit on the flow rate, which in turn limits the further reduction of analysis time. Also, compared to the new polar organic mode, the retention in reversed phase on coupled columns is deviated more from the average retention on the individual stationary phases. 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

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Zelinski et al. [30] applied the technique to the determination of 0.02-0.lmg L 1 quantities of chloride, nitrate, sulphate, nitrite, fluoride and phosphate in river water. Approximately 25min was required for a full analysis. These workers used a column coupling technique which, by dividing the analysis into two stages, enables a high load capacity and a low detection limit to be achieved simultaneously without an appreciable increase in the analysis time. 

This section deals with the working techniques that extend the possibilities of applying isotachophoresis to the samples which, analyzed by simple procedures, provide mixed zones (counter-flow technique, cascade technique). It also describes the techniques which facilitate the analysis of very dilute samples (continuous sampling technique) or of small amounts of substances in the presence of a large excess of other components (column coupling technique). Furthermore, the spacer technique which provides a more efficient fractionation of complicated samples, e.g. in protein separations, is dealt with. 

Another development arising from FAB has been its transformation from a static to a dynamic technique, with a continuous flow of a solution traveling from a reservoir through a capillary to the probe tip. Samples are injected either directly or through a liquid chromatography (LC) column. The technique is known as dynamic or continuous flow FAB/LSIMS and provides a convenient direct LC/MS coupling for the on-line analysis of mixtures (Figure 40.2). 

An on-line supercritical fluid chromatography-capillary gas chromatography (SFC-GC) technique has been demonstrated for the direct transfer of SFC fractions from a packed column SFC system to a GC system. This technique has been applied in the analysis of industrial samples such as aviation fuel (24). This type of coupled technique is sometimes more advantageous than the traditional LC-GC coupled technique since SFC is compatible with GC, because most supercritical fluids decompress into gases at GC conditions and are not detected by flame-ionization detection. The use of solvent evaporation techniques are not necessary. SFC, in the same way as LC, can be used to preseparate a sample into classes of compounds where the individual components can then be analyzed and quantified by GC. The supercritical fluid sample effluent is decompressed through a restrictor directly into a capillary GC injection port. In addition, this technique allows selective or multi-step heart-cutting of various sample peaks as they elute from the supercritical fluid... 

Chlorophenoxy acids are relatively polar pesticides which are usually determined by LC because volatile derivatives have to be prepared for GC analysis. This group of herbicides can be detected by multiresidue methods combined with automated procedures for sample clean-up, although selectivity and sensitivity can be enhanced by coupled-column chromatographic techniques (52). The experimental conditions for Such analyses are shown in Table 13.1. 

While most preliminary SFC-plasma coupled techniques employed microwave-induced plasmas (MIPs), the use of ICP-MS is now increasing [469]. An advantage of microcolumn SFC-ICP hyphenation is the significantly reduced flow-rates of microcolumns compared with those of conventional columns. Both pSFC-ICP-AES [470,471] and cSFC-ICP-AES [472] were described. In the case of elemental detector selectivity (e.g. AES) complete chromatographic resolution is not required. The detector possesses linearity over several orders of concentrative magnitude. Minimum detectable quantities for nonmetals range from sub to low ng mL"1. 

To point out the relevance of the new coupling technique, Fig. 17.13 shows a precolumn (Fig. 17.13a) and a main column (Fig. 17.13b) chromatogram of a raspberry extract (variety Rucami) measured by MDGC-P-IRMS. The concentrations of ( )-a-ionone and ( )-/l-ionone were adjusted to the linearity range of the isotope ratio mass spectrometer (peak amplitude 4-7 V). 

High-performance LC coupled to capillary GC is a technique in which fractions from one or more LC columns are transferred into the GC column for further separation. This coupled technique is used more to separate a particular compound and/or class of compounds from an unknown matrix. Another field in HPLC serves for the preseparation of closely related classes or subclasses of compounds (64). 

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