Fluorescent detection, instrument

An absorption peak at an excitation wavelength available on the fluorescence detection instrument (large extinction coefficient at the wavelength of excitation)... 

Photomultipliers are appreciably more sensitive sensors than the eye in their response to line or continuum sources. Monochromators are fitted to the light beam in order to be able to operate as substance-speciflcally as possible [5]. Additional filter combinations (monochromatic and cut-off filters) are needed for the measurement of fluorescence. Appropriate instruments are not only suitable for the qualitative detection of separated substances (scanning absorption or fluorescence along the chromatogram) but also for characterization of the substance (recording of spectra in addition to hR and for quantitative determinations. 

The ultimate in fluorescence detection is a detector that uses a monochromator to select the excitation wavelength and a second monochromator to select the wavelength of the fluorescent light. This instrument is ideal, giving the maximum versatility and allowing the... 

The most common final separation techniques used for agrochemicals are GC and LC. A variety of detection methods are used for GC such as electron capture detection (BCD), nitrogen-phosphorus detection (NPD), flame photometric detection (FPD) and mass spectrometry (MS). For LC, typical detection methods are ultraviolet (UV) detection, fluorescence detection or, increasingly, different types of MS. The excellent selectivity and sensitivity of LC/MS/MS instruments results in simplified analytical methodology (e.g., less cleanup, smaller sample weight and smaller aliquots of the extract). As a result, this state-of-the-art technique is becoming the detection method of choice in many residue analytical laboratories. 

Instrumentation for fluorescence spectroscopy has been reviewed [8]. For standards in fluorescence spectroscopy, see Miller [138]. Fluorescence detection in HPLC has recently been reviewed [137], Phosphorescence detection of polymer/additive extracts is not being practised. 

The instrumentation for real-time PCR includes a thermal cycler with a computer, a spectrophotometer for fluorescence detection, and software for acquisition and analysis of data.105,109... 

They allow sensing at a distance from analyzed object. Because the fluorescence reporter and the detecting instrument are connected via emission of light, the sensing may occur in an essentially noninvasive manner and allow formation of... 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Fluorescent detection technology applicable to biochips is evolving rapidly, resulting in detection instruments that are more powerful, user-friendly, and less expensive. Most systems employ photomultiplier tube (PMT) technology in conjunction with multiple colors, lasers, and a variety of filters. It is essentially a fluorescent microscope that... 

LOD is defined as the lowest concentration of an analyte that produces a signal above the background signal. LOQ is defined as the minimum amount of analyte that can be reported through quantitation. For these evaluations, a 3 x signal-to-noise ratio (S/N) value was employed for the LOD and a 10 x S/N was used to evaluate LOQ. The %RSD for the LOD had to be less than 20% and for LOQ had to be less than 10%. Table 6.2 lists the parameters for the LOD and LOQ for methyl paraben and rhodamine 110 chloride under the conditions employed. It is important to note that the LOD and LOQ values were dependent upon the physicochemical properties of the analytes (molar absorptivity, quantum yield, etc.), methods employed (wavelengths employed for detection, mobile phases, etc.), and instrumental parameters. For example, the molar absorptivity of methyl paraben at 254 nm was determined to be approximately 9000 mol/L/cm and a similar result could be expected for analytes with similar molar absorptivity values when the exact methods and instrumental parameters were used. In the case of fluorescence detection, for most applications in which the analytes of interest have been tagged with tetramethylrhodamine (TAMRA), the LOD is usually about 1 nM. 

Progress in instrumentation has considerably improved the sensitivity of fluorescence detection. Advanced fluorescence microscopy techniques allow detection at single molecule level, which opens up new opportunities for the development of fluorescence-based methods or assays in material sciences, biotechnology and in the pharmaceutical industry. 

Finally, we note that future instrument for lifetime-based sensing and imaging can be based on laser diode light sources. At present it is desirable to develop specific probes which can be excited from 630 to 780 nm, the usual range of laser diodes. The use of such probes will allow us to avoid the use of complex laser sources, which should result in the expanded use of fluorescence detection in the chemical and biomedical sensors. 

This paper reports on research involved the design, construction, and evaluation of a portable instrument, a "luminoscope", for detecting skin contamination by coal tars via induced fluorescence. The instrument has been used in the laboratory to measure the fluorescence of various coal tars and recycle solvents from liquefaction processes spotted on filter paper on rat and on hamster skin. The practical use of the devices in field test measurements to monitor skin contamination of workers at coal gasifier is discussed. The paper also discusses the practicality and usefulness of the luminescence method for detecting skin contamination. 

Chemiluminescent labeling systems have been developed, based on the incorporation of fluorescein-11-dUTP into a DNA probe. An anti-fluorescein antibody covalently bound to the enzyme HRP is then bound to the incorporated fluorescein label. HRP catalyses the breakdown of luminol and the chemiluminescent signal is detected by autoradiography with X-ray film or by fluorescence scanning instrumentation. Chemiluminescence is more sensitive than enzyme-based color detection systems. Furthermore the labeled gene probes are stable and give results quickly (160). 

Routine laboratory equipment vortex mixer, laboratory centrifuge, analytical balance, water bath, pH meter, HPLC system with fluorescence detection at 350/440 nm (for neopterin see 6.1.3.1, subheading Instrumentation ). 

Methylcarbamate insecticides have been recently labeled with DNS-C1 [145]. The procedure involves the hydrolysis of the carbamates with 0.1 M sodium carbonate to form a phenol and methylamine [166]. The two hydrolysis products are labeled with DNS-C1 and subsequently detected and determined quantitatively by TLC on silica gel layers by scanning spectrofluorimetry in situ. The reaction conditions were examined, and optimum conditions for hydrolysis and labeling were established [167]. The overall reaction scheme is shown in Fig. 4.62. The phenol derivatives of a number of N-methylcarbamates have been separated by one- and two-dimensional TLC [168], and the fluorescence behaviour and stability of the derivatives have been examined [169]. Most of the DNS derivatives fluoresce at similar wavelengths (excitation, ca. 365 nm emission, ca. 520 nm). The fluorescence spectrum of a typical DNS derivative is shown in Fig. 4.63. The method has been applied successfully to the analysis of low concentrations of carbamates in water and in soil samples with little or no clean-up being required [170,171]. Amounts as low as 1 ng of insecticide can be detected instrumentally. Visual limits of detection are ca. 5-10 ng per spot. 

Radioactivity, chemiluminescence or fluorescence have traditionally been used to monitor signals with protein arrays. The use of fluorescent dyes allows for increased sensitivity of detection, the ability to multiplex detection of several targets, and easy interfacing with detection instrumentation and automation. Surface plasmon resonance spectroscopy is another alternative, but successful development and commercialization has not yet been achieved. 

Bio-Logic Instrument and Laboratories (Meylan, France) manufactures an SFM-3 stopped-flow instrument (Fig. 4.17) that consists of three independent drive syringes driven by stepping motors, two mixers and a delay line, three observation windows, replaceable cuvettes, no stop-syringes, and efficient temperature regulation. At maximum flow rate, the minimum dead times range from 1.0 to 4.9 ms for fluorescence detection and 1.3 ms for transmittance. Currently, the Bio-Logic MOS-IOOO optical system employs fluorescence or absorbance detection, which is not suitable... 

Pesticides Separated on a CN-Bonded Polar Phase. Figures 3 and 4 contain the chromatograms obtained for several of the pesticides listed in Table 2. Various mobile phases were used to facilitate separation on the CN-bonded polar phase. Detection is shown in both absorbance and fluorescence modes. It would appear that fluorescence detection is more sensitive for some pesticides, while absorbance detection is more sensitive for others. However, comparisons of one manufacturer s absorbance monitor with another manufacturer s fluorescence monitor could be misleading. (I will refer to this under Instrumental Parameters.) Furthermore, the ultimate useful comparison is obtained when practical samples are chromatographed since these... 

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