Radioisotope detection

Other detection modes employed in capillary electromigration techniques include chemiluminescence [69-71], Raman spectroscopy [72,73], refractive index [74,75], photothermal absorbance [76,77], and radioisotope detection [78]. Some of these detection modes have found limited use due to their high specificity, which restricts the area of application and the analytes that can be detected, such as radioisotope and Raman-based detection that are specific for radionuclides and polarizable molecules, respectively. On the other hand, the limited use of more universal detection modes, such as refractive index, is either due to the complexity of coupling them to capillary electromigration techniques or to the possibility of detecting the analytes of interest with comparable sensitivity by one of the less problematic detection modes described above. 

What may prove to be the ultimate choice for an internal standard when using an MS (37) is the addition of a PGS standard as a deuterated compound to the initial sample preparation. The deuterated compound is quantified directly on the MS rather than having to subsequently subject the sample to conventional radioisotope detection methods. This procedure has been applied to ABA (29) and IAA (38, 39) analyses. A high deuterium content (labeled at five or more positions) should be sought to avoid confusion with naturally "heavy" isotopic compounds (39). 

Radioisotope detection is used widely in HPLC but has received little attention in capillary electrophoresis applications (3-61. 

The availability of an on-line radioisotope detector for CE is especially appealing for several reasons. First, state-of-the-art radiation detection technology offers extremely high sensitivity. Second, radioisotope detection affords unrivaled selectivity because only radiolabeled sample components yield a response at the detector. Third, the radiolabeled molecule possesses the same chemical properties as the un-labeled molecule, thereby permitting tracer studies. Fourth, radioisotope detection can be directly calibrated to provide a measurement of absolute concentration of the labeled species. Finally, a capillary electrophoresis system in which radioactivity detection is coupled with more conventional detectors adds extra versatility to this new separation technique. 

Radioisotope detection of P, 14C, and Tc was reported by Kaniansky et al. (7,8) for isotachophoresis. In their work, isotachophoretic separations were performed using fluorinated ethylene-propylene copolymer capillary tubing (300 pm internal diameter) and either a Geiger-Mueller tube or a plastic scintillator/photomultiplier tube combination to detect emitted fi particles. One of their reported detection schemes involved passing the radiolabeled sample components directly through a plastic scintillator. Detector efficiency for 14C-labeled molecules was reported to be 13-15%, and a minimum detection limit of 0.44 nCi was reported for a 212 nL cell volume. 

Figure 3. Exploded diagram showing the design of the coincidence radioisotope detection scheme. The fused-silica capillary is exposed to a 2-mm length of plastic scintillator material located between two photomultiplier tubes operated in the coincidence counting mode.

The large gain in sensitivity afforded by on-line radioisotope detection in comparison with the more commonly used UV-absorbance detector is illustrated in Figure 7. In this example, a UV-absorbance detector, monitoring at 254 nm, was positioned 8.5 cm downstream from a CdTe radioisotope detector, and 2P-labeled ATP was injected at a concentration of approximately 5 x 10 M. Under these conditions, ATP is detected with an excellent signal-to-noise ratio by the radioisotope detector but is completely undetectable by UV absorbance. 

Application 2 Capillary Gel Electrophoresis. Recently, Karger and co-workers demonstrated the use of polyacrylamide gel-filled capillaries to separate peptide/protein (SDS PAGE) Qfi) and oligonucleotide mixtures (12,22) by capillary electrophoresis. This mode of CE operation may prove to couple well with on-line radioisotope detection. The results of several preliminary capillary electrophoresis separations using gel-filled capillaries and on-line radioisotope detection using the coincidence unit described here are presented below. 

Figure 10. Capillary gel electrophoresis separation of a simple three-component nucleotide mixture with on-line radioisotope detection using the coincidence unit.

On-line radioisotope detection has been demonstrated to be a practical alternative to UV absorbance detection when gel-filled capillaries are used for CE separations. Significant Improvement in detection limits is realized with radioisotope detection. The greatest improvement is realized for small molecules and is roughly one to two orders of magnitude (for runs in which the residence time is not enhanced). 

The inherent time factor in radioisotope detection can also be partially compensated by the electronic controls. While counting times of 10-20 minutes are commonly used for liquid... 

Radioactive/Non-radioactive Detection Systems. Radioisotopic detection methods frequently employ molecules containing radionuclides of hydrogen, sulfur and phosphorus, and less frequently iodine and chromium. Oftentimes, the probe molecules themselves are directly radiolabeled for immediate detection. 

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