Fluorescence detection, alternative

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

Alternatively, LC is used for the separation and quantification of PAHs using both UV and fluorescence detection. The analytes are identified based on their relative retention times and UV and/or fluorescence emission spectra. For UV detection an efficient cleanup is a prerequisite since this detection method is not very selective (almost universal for PAHs), and hence it also responds to many coeluting compounds. Due to the high specificity of fluorescence detection for most PAHs, this LC detection method is less susceptible to potential interferences. As in the case of GC the apphcation of internal standard(s) is mandatory since solvents have to be evaporated during the cleanup, which may result in partial losses of some of the more volatile analytes. 

The current trends toward miniaturization and the need of massively parallel measurements led to the development of biochips. In this area, biocatalyzed and electrogenerated chemiluminescence reactions appear attractive and represent an alternative to fluorescence detection which is still widespread used despite the numerous problems of quantitative measurements and interference fluorescence emission. 

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

A simple alternative to direct detection of intrinsic protein fluorescence detection is the technique of indirect fluorescence detection proposed by Kuhr and Yeung.20 In this approach, the analysis buffer contains a fluorescent anion that produces a high background fluorescence signal. Nonfluorescent analyte anions displace the fluorescent species, producing a zone of reduced signal. Sensitivity in indirect fluorescence detection is determined by the dynamic reserve (ratio of signal... 

Fluorescence detection, because of the limited number of molecules that fluoresce under specific excitation and emission wavelengths, is a reasonable alternative if the analyte fluoresces. Likewise, amperometric detection can provide greater selectivity and very good sensitivity if the analyte is readily electrochemically oxidized or reduced. Brunt (37) recently reviewed a wide variety of electrochemical detectors for HPLC. Bulk-property detectors (i.e., conductometric and capacitance detectors) and solute-property detectors (i.e., amperometric, coulo-metric, polarographic, and potentiometric detectors) were discussed. Many flow-cell designs were diagrammed, and commercial systems were discussed. 

Finally, the use of CCD cameras as detectors in combination with short-pulsed light sources enables not only spatially resolved imaging, but also a time-resolved fluorescence detection. Time resolution offers two different new alternatives ... 

As an alternative, immunoaffinity procedures have been developed to selectively extract PAHs from environmental samples. Thomas and Li62 demonstrated the greater efficiency of immunoaffinity methods in comparison with conventional extraction procedures. Bouzige et al.48 prepared an immunosorbent for use in an on-line analytical procedure, followed by liquid chromatography coupled to fluorescence detection, to monitor surface water samples. The sensitivity of the fluorescence detection in combination with the selectivity of the immunosorbent (IS) extraction enabled PAH compounds to be detected at levels between 2 and 10 ng I. 

An alternative to derivatization of nonfluorescent compounds is to perform indirect fluorescence detection. The procedure is performed on-column, by incorporating a fluorescent ion into the electrolyte. When ionic analytes interact with the fluorophore, the result is either displacement of the fluorophore or ion pairing with it.47 Kuhr and Yeung51 explored indirect laser-induced fluorescence detection using 1.0 mM salicylate as the fluorophore in the electrolyte they analyzed 10 amino acids and obtained detection limits on the order of 10 5 M. 

If the sensitivity is still not sufficient, then a different detection techniques or analytical technique may need to be employed (for example, LC-MS and fluorescence detection, capillary electrophoresis, ion mobility spectrometry, etc.). These alternate methods and/or detection techniques may have higher sensitivity than HPLC with UV detection. Since the new method will be a simple method (peaks from APl(s) and/or functional excipients), development of these methods should be simple and fast. MS is generally more... 

In addition to its contribution described above, nanoscience can provide an alternative step forward to promoting biomolecular-fluorescence detection. Recent advances in nanoscience permits innovative assembly and fiibrication of nanomaterials for use as advanced biosensor substrates in fluorescence detection. Therefore, nanoscience may offer a much simpler and convenient route in promoting biomolecular fluorescence detection, even when using as-grown nanomaterials without any downstream modifications following their synthesis. In order to design such substrates comprised of nanonaaterials, four key characteristics of the candidate nanoscale materials should be carefiilly considered ... 

The analysis of cocaine and its metabolites has been performed by means of GC-MS, but this demands extensive sample pretreatment and derivatization. LC-MS has been evaluated as an alternative. Simultaneous LC-ESI-MS and fluorescence detection of cocaine, benzoylecgonine, and cocaethylene in human hair was described by Clauwaert et al. [84]. The effect of mobile-phase composition and pH on the ESI LC-MS performance of cocaine, ecgoitine methyl ester, and benzoylecgoitine was investigated by Jeanville et al. [85]. The best sensitivity was achieved with a 1 1 mixture of 60% acetonitrile-40% acetone in 100 mmol/1 ammonium acetate. The pH has little influence on the response, suggesting that gas-phase rather than liquid-phase ionization is important. 

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