Capillary Separation Techniques

Institut fur Organische Chemie, Universitat Tubingen, Tubingen, Germany 

Moreover, an easy exchange of the detection capillary is possible. This feature is of enormous importance, because in the current status of prototype development cracking of the capillaries is a routine occurrence. 

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Fig. 2.22. Coupling of capillary separation techniques with NMR (taken from Ref. [72] with permission of the publisher).

Schefer AB, Albert K. Capillary Separation Techniques. In Albert K, ed. On-line LC-NMR and Related Techniques. New York Wiley, 2002 237-246. 

Sample collection and preparation are crucial issues for any bioanalytical application in order to address the complexity of samples originating from biological tissues and fluids. It is necessary to cope with the lack in concentration sensitivity typical for capillary separation techniques, to avoid interference from matrix components as well as to ensure analyte stability. In peptide analysis, a strong focus exists on handling small-volume samples and on selective concentration of the analyte in order to overcome limitations with respect to loadability. In addition, loss of analyte frequently occurs due to degradation by proteases and due to adsorption to surfaces, which accordingly needs to be minimized. 

As advantages, capillary separation techniques demonstrate high separation efficiency. On occasion, the number of theoretical plates available from these approaches has exceeded 1 million [29]. Also, very small sample volumes, on the order of 100 to 0.5 nL, are needed for these techniques. This can be an advantage for sample-limited situations, which are often encountered in bioanalysis. High mass sensitivity (the absolute weight of analyte injected) can be achieved, as the narrow capillary concentrates the sample plug and allows less opportunity for band broadening. 

Phosphorus is not a TE but a major nutrient element. Nevertheless, fractionation of this element is essential for environmental studies, and hence it seems reasonable to highlight here some relevant SEPs. Four different procedures for the fractionation of P in lake sediment samples have been tested in an interlaboratory study in the framework of the SM T program (Ruban et al., 1999). As a result, a novel scheme based on the Williams protocol (Williams et al., 1976) has been developed aimed at the restoration of lake sediments. The scheme comprises three separate assays (1) sequential extraction of NaOH-extractable (Fe- and Al-bound) and HCl-extractable (Ca-bound) fractions, (2) sequential extraction of inorganic and organic phosphorus and (3) single extraction, after calcination, of concentrated HCl-extractable (total P) fraction (see Table 12.3 for further details). Further discrimination of specific compounds is made feasible by the use of chromatographic and capillary separation techniques as reviewed by Spivakov et al. (1999). 

The two capillary separation techniques mentioned above are not considered routine, however, partially because they require homemade parts. 

P Gfrorer, J Schewitz, K Pusecker, E Bayer. On-line coupling of capillary separation techniques with 1H NMR. Anal Chem 73 315A-321A, 1999. 

Online coupling of HPLC with nuclear magnetic resonance spectroscopy (NMR) has proved useful for a wide range of applications. The shortcoming of suppression of eluent signals can be circumvented by use of capillary separation technique. In this mode detection cells with internal volumes in the nanoliter scale and miniaturized probe heads have been developed by Albert et al. in Tuebingen. The system can be used in either HPLC, CE, or CEC, and consists of a capillary inserted into a 2.5 or 2.0 mm NMR microprobe equipped with a Helmholtz coil. In experiments, a capillary mbe of 315 /rm can create a detection volume of 900 nl. The flow rate of the capillary... 

Due to the rapidly growing importance of capillary columns in bioanalytical applications, special attention will now be devoted to sampling techniques associated with capillary GC. Small samples are typical for this type of chromatography and, consequently, a direct introduction of such samples is an apparent technological problem. In most biochemically interesting applications (typically, trace analysis problems), there is no general discrepancy between the demands of such analysis and the performance and sensitivity of capillary separation techniques. However, the manipulation of samples presents difficulties, as reliable methods for measurement, disposal, and introduction of nanoliter volumes are not readily available. Ironically, in many capillary GC applications, the solvent serves only as a sample vehicle we... 

Kang Choon Lee, Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, Jangan-ku, Suwon, Korea, Capillary Separation Techniques... 

Summarizing this chapter, it can be stated that the linking of NMR spectrometers (from 400 to 750 MHz) to various separation techniques has been developed into rather a powerful tool. Stereochemical problems in metabolism studies can be easily solved if the concentration of the present compounds is above the detection threshold. The coupling of capillary separation techniques together with NMR detection will stimulate high-throughput screening applications in modem dmg development. 

The problem of cross-talk between several microcoils has been solved by Professor Andrew Webb and his coworkers, but application in parallel HP LC-NMR detection has stiU yet to be achieved. For this, adequate NMR instrumentation will be necessary. Parallel HPLC-NMR detection with four microcoils in a hyphenated system will surely be realized within the next few years, and in the longer term a configuration of 16 microcoils in four parallel horizontal detection planes, as introduced by Professor Raftery, will be realized. Besides parallel detection, the capillary technique in principle opens the door for the hyphenation of NMR spectroscopy with other capillary separation techniques such as gas chromatography and supercritical fluid chromatography (SFC). The long spin-lattice relaxation times in the supercritical and gaseous states can be reduced by relaxation agents present in the separation capillary prior to the actual NMR detection. 

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