Single column methods

Figure 10.4. Single-column method configurations (a) sample load and cleanup (b) sample elution.

While single-column methods work quite well, these methods have two main drawbacks. First, when coupling turbulent-flow directly to a mass spectrometer, the mobile-phase effluent (4 or 5 mL/min) has to be split to make the effluent flow compatible for mass spectrometers (< 1 mL/min). Although narrow-bore TFLC columns (0.5 mm i.d.) can be operated at 1-1.5 mL/min, only some mass spectrometers are capable of operating at these flow rates. The splitting will result in lower detection limits. The lower sensitivity is not due to the detection limit of the mass spectrometer, which acts as a concentration detector, but to the fact that the analyte is more diluted when contained in the higher TFLC flows. There is also more mobile-phase waste to dispose. Of course, this may not be a major drawback, if one were to collect fractions... 

Samples are injected onto the turbulent-flow column similar to single-column methods. The analytes of interest are retained in the turbulent-flow column while the large macromolecules are eluted to waste. Once the analytes are separated from the matrix, the samples are then eluted into the analytical column. The characteristics of the analytical column determine the peak shape and separation seen at the MS detector. Flow rates which are compatible with the mass spectrometer can then be used and the chromatograms are based on conventional HPLC parameters. The key to dual-column methods is that the retentive properties of the analytical column must be sufficiently stronger than that of the turbulent-flow column the dual-column approach is performed in such a manner so that the mobile-phase composition needed to elute the analyte from the turbulent-flow column does not elute the analyte from the analytical column. The sample is then focused at the head of the analytical column until the mobile-phase conditions are changed to elute the analyte. The choice of columns is critical to the success of dual-column methods. Table 10.2 lists some of the applications of dual-column methods found in the literature. 

The most obvious way to use immobilized antibodies for analytical affinity chromatography is to simply use it in a traditional single-column method to determine an antigen s concentration and/or purity. However, there are a number of ways this technique can be advanced to more sophisticated analyses. For example, instead of immobilizing an antibody, the antigen may be immobilized to quantify the antibody as has been done with the Lewis Y antigen [3]. However, the analysis is still a single-column method. 

Amino acid analyzer single column method... 

Numerous methods are described in the literature for effecting the separation of amino acid components from complex mixtures. Most methods are derived from the two-column system of Spackman, et al (1958), wherein sodium citrate buffer solutions of both increasing pH and ionic strength are used. Benson (1972, 1975) described a single-column method in which amino acids are eluted by solutions with essentially constant pH but in which the dominant driving force is the increasing cation molarity. The chief benefit of this system is that the baselines are quieter and more stable. 

The cytostatic agent 5-[ F]fluorouracil (5-[ F]FU) has been prepared by electrophilic addition of [ F]F2 (Fowler et al. 1973) and [ F]AcOF (Fowler et al. 1982 Diksic et al. 1986 Visser et al. 1986) to uracil. Subsequent elimination of the 6-fluoro or 6-acetoxy groups results in high regioselectivity for the five positions (see Fig. 42.6). Laborious purification methods to separate 5-[ F]FU from unreacted uracil have been overcome by using a single column method reported by Brown et al. (1993). Radiochemical yields >20% are obtained using this method. 

P.N. Nesterenko and P. Jones. Single-column method of chelation ion chromatography for the analysis of trace metals in complex samples. Journal of Chromatography A 770 129-135, 1997. 

In spite of the substantial progress in the field of LC coliunn and instrumentation and the considerable efforts made by analysts to develop chromatographic techniques taking full advantage of these developments, the separation efficiency required in many cases overwhelms the capability of any single-column method. 

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