Radio-flow detection

Planar radiochromatography was the first widely used technique for separation and detection of radiolabeled metabolites (Veltkamp, 1990). The technique is very sensitive, fast, and has relatively low equipment and operating costs, however, due to low separation resolution and poor precision in quantification, it has largely been replaced by HPLC-based radiochromatographic techniques in the pharmaceutical industry. Online radio flow detection (RED) and off-line liquid scintillation counting (ESC) have been routinely used for analysis of radiolabeled metabolites for the last 15 years (Veltkamp, 1990). 

Liquid chromatography is routinely used for the quantitative analysis of a drug and its metabolites in radiolabeled in vitro metabolism experiments and in humans and animal ADME studies. Online radio-flow detection (RFD) and offline microplate scintillation counting (MSC) are the most commonly used radiochromatographic techniques in radiolabeled metabolite profiling and quantification (Boernsen et al., 2000 Nassar et ah, 2003 Bruin et al., 2006). LC/RFD is compatible with ESI mass spectrometry and provides high analytical speed and excellent separation resolution (Athersuch et al., 2008). 

A radiometric detector, also called a radio-flow detector, is used to measure radioactivity of radioactive analytes in the HPLC eluent passing through a flow cell. Most are based on liquid scintillation technology to detect phosphors caused by the radioactive nuclides. A liquid scintillator can be added post-column with a pump or a permanent solid-state scintillator can be used around the flow cell. This detector is specific only to radioactive compounds and can be extremely sensitive. This detector is used for experiments using tritium or C-14 radiolabeled compounds in toxicological, metabolism, or degradation studies. 

The basic set-up and compounds of an ICP-AES and ICP-MS are shown in Fig. 2. The ICP part is almost identical for AES and MS as detection principle. The ICP torch consists of three concentric quartz tubes, from which the outer channel is flushed with the plasma argon at a typical flow rate of 14 1 min-1. This gas flow is both the plasma and the cool gas. The middle channel transports the auxiliary argon gas flow, which is used for the shape and the axial position of the plasma. The inner channel encloses the nebulizer gas stream coming form the nebulizer / spray chamber combination. This gas stream transports the analytes into the plasma. Both the auxiliary and the nebulizer gas flow are typically around 1 1 min-1. The plasma energy is coupled inductively into the argon gas flow via two or three loops of a water-cooled copper coil. A radio frequency of 27.12 or 40.68 MHz at 1-1.5 kW is used as power source. The plasma is... 

Occasionally, a laboratory will need an in-line detector of radio-labeled molecules. These detectors take the flow from the column or from an initial detector, mix it with fluorescing compound, and measure the fluorescence due to radioactive breakdown. A different system uses beads in the flow cell with an immobilized fluorescing compound, but these systems suffer from ghosting and cannot be used with very hot labeled compounds because of secondary radiation problems. These systems are very useful with tritiated samples and less so with carbon14 labeled compounds. Some success has been reported with sulfur32 label detection. 

All titrimetric methods of analysis require some means of detecting the equivalence point. This could be an abrupt change of color (colorimetric titrations), a sudden change in the potential difference between two electrodes (potentiometric titrations), a change in current flow through two electrodes (amperometric titrations), and so on. Similarly, the radioactivity of either the titrant or the substance titrated can be employed for detecting the equivalence point. This type of analysis is called radio-metric titration. It should be noted that the sole purpose of the radioactivity is to signal attainment of the equivalence point and that it takes no part in the titration process. The technique can be employed in all classes of titrations, provided that a phase separation can be effected. 

An alternative strategy for detection, where cellular systems or enzymatic fluxes are being investigated, is the use of radioactively labelled arachidonic acid or glutathione, which can function as precursor molecules and facilitate detection of the synthesised radio-labelled leukotrienes. The labelled molecules can be detected either directly by a radioactive flow monitor, or indirectly by fraction collection and scintillation counting. 

Nakagama T, Maeda T, Uchiyama K, Hobo T (2003) Monitoring nano-flow rate of water by atomic emission detection using helium radio-frequency plasma. Analyst 128(6) 543-546... 

The release of nine different peptides from 40 of the same pipettes was also examined by radio immunoassays, after the electrode was withdrawn from the slice and the tip immersed in 0.2 ml of 4 mM sodium acetate. Assayable amounts of peptide could be detected after about 1 hr in the absence of pressure and shown to be between 0.2 and 2.0 fmol per second. The application on average of 400-1000 kPa of pressure led to an eightfold increase in release. This would represent a flow of fluid of between 1-12 pl/sec. However, during repeated trials, the release from the same pipette was extremely variable and quite unlike the consistent results of Sakai et al. (1979). However, since in part their method depended on the direct measurement of the ejection of droplets into oil, it is possible that their electrodes were not only selected more carefully, but had bigger tips. It is also not clear how the use of siliconized glass and ethanolic solutions improved the performance of their pipettes. 

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