Fluorescent detection, instrument detector

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... 

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. 

A CEC instrument basically consists of a system for injection (pressure driven or electrokinetic), a column in which the separation takes place, a detector and a high voltage supply (Fig. 16.1). The most commonly used detector so far has been UV with transmission through the capillary outside of the packed bed. Laser induced fluorescence detection has been employed in several studies. Also, mass-spectrometry has been used. Normally, isocratic CEC is performed, but approaches to gradient CEC have been reported [29]. However, special equipment must be employed in most cases. 

The so-called micro-total analytical systems (/tTAS) can integrate sample handling, separation, and detection on a single chip [9]. Postcapillary reaction detectors can be incorporated as well [10]. Fluorescence detection is the most common method employed for these chip-based systems. A commercial instrument (Agilent 2100 Bioanalyzer) is available for DNA and RNA separations on disposable chips using a diode laser for LIF detection. In research laboratories, polymerase chain reaction (PCR) has been integrated into a chip that provides size separation and LIF detection [11]. 

Laser excitation for fluorescence detection has received much research interest, but as of yet there is no commercially available instrument. Fluorescence intensity increases with excitation intensity, and it is generally assumed that laser excitation would then offer improved limits of detection. However, as Yeung and Synovec have shown, various types of light scattering, luminescence from the flow cell walls, and emission from impurities in the solvent all increase with source intensity as well, yielding no net improvement in signal-to-noise ratio (53). Where laser excited fluorescence may prove useful is for the design of fluorescence detectors for microbore packed and open tubular LC columns, where the laser source can be focused to a small illuminated volume for on-column detection. 

A detector, for example, a UV absorbance detector, through which the solution flows, is placed near or at one end of the capillary. A focused beam is passed through the capillary and may be collected by an optical fiber coupled to a photomultiplier tube. The short pathlengths (10 to 100 /rm) involved make sensitive detection a challenge. But the small peak volumes, often less than 1 nL, lead to very low detection limits, even with moderately sensitive detectors (i.e., the solute is concentrated in a very small volume). The use of laser sources, especially for fluorescence detection, has pushed detection limits to zeptomoles (10"- mol) A capillary electrophoresis instrument is shown in Figure 21.19. 

EDCs in the environment are often analyzed using GC or LC based instrumental techniques. GC coupled with an electron capture detector (BCD), a nitrogen-phosphorus detector (NPD), or mass spectrometry (MS) has been the preferred method due to its excellent sensitivity and separation capability on a capillary column. High performance liquid chromatography (HPLC) with various detectors such as ultraviolet detection (UV), fluorescence detection (FLD), MS, and more recently tandem MS (MS/MS) has also been used for analysis of some EDCs, especially for the polar compounds. Analytical techniques for each class of EDCs will be discussed in the following section. 

Earlier methods used to determine mercury in biological tissue and fluids were mainly colorimetric, using dithizone as the com-plexing agent. However, during the past two to three decades, AAS methods - predominantly the cold vapor principle with atomic absorption or atomic fluorescence detection - have become widely used due to their simplicity, sensitivity, and relatively low price. Neutron activation analysis (NAA), either in the instrumental or radiochemical mode, is still frequently used where nuclear reactors are available. Inductively coupled plasma mass spectrometry (ICP-MS) has become a valuable tool in mercury speciation. Gas and liquid chromatography, coupled with various detectors have also gained much importance for separa-tion/detection of mercury compounds (Table 17.1). 

In addition to adsorption, issues to be addressed for the routine use of microchips for clinical analysis include the development of lower cost, compact instrumentation for detection on microchips. Shrinivasan et al. detailed the development of a miniaturized LIF detector applicable for fluorescent detection of DNA on microchips, which could also be applied to proteins with the necessary fluorescent tags. By replacing the argon-ion laser with a diode laser, the cost and power requirements were significantly decreased, with only a small loss in sensitivity. Integration of fluid flow and mixing... 

Table 3.4 compares detection limits with secondary fluorescers to the results with the RMF method and 15-kV broadband excitation [16,17]. Four different fluorescence analyzers were tested (units A, B, C, and D), and the results were corrected for differences in performance for the energy-dispersive spectrometers employed on each unit. Unit A used a chromium anode tube, and unit B used a tungsten anode tube. Unit A was a commercial, general-purpose instrument. Unit B was specifically designed for atmospheric aerosol analysis, where closer coupling between the tube, fluorescer, sample, and detector could be employed with some sacrifice of insensitivity to specimen-positioning errors. Table 3.5 lists the x-ray tube operating conditions required for Table 3.4. For medium- to high-atomic-number elements, the secondary fluorescer method provides detection limits equivalent to the RMF element, but requires much higher x-ray tube power. For light elements.

There s No Detector Which Is More Sensitive than a Mass Spec. This phrase touches the same misapprehension as the previous one. Sensitivity and the LoD and LoQ in mass spectrometry are not by default superior to any other detector. Under favorable conditions, like high ion formation yield and good ion transmission through the mass analyzer to the mass detector, mass spectrometers are indeed very powerful, allowing LoQs down to a femto- or even attomol level. However, in case of poorly ionizable analytes, an inappropriate ionization principle and/or perhaps not the most sensitive MS instrument design, there maybe other detection principles that are clearly in favor, for instance electrochemical or fluorescence detection. 

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