Fluorescence detectors for HPLC

When compared to fluorescence detectors for HPLC, the design of a fluorescence detector for CE presents some technical problems. In order to obtain acceptable sensitivity, it is necessary to focus sufficient excitation light on the capillary lumen. This is difficult to achieve with a conventional light source but is easily accomplished using a laser. The most popular source for laser-induced fluorescence (LIF) detection is the argon ion laser, which is stable and relatively inexpensive. The 488-nm argon ion laser line is close to the desired excitation wavelength for several common fluorophores. The CLOD for a laser-based fluorescence detector can be as low as 10 12 M. 

Classical LC detectors (refractive index, fixed wavelength UV absorbance at 254 or 280 nm) have lacked the sensitivity to allow direct analysis of cannabi-noids in biological fluids. However, recent development of variable wavelength absorbance detectors extending into the 195-220nm UV region and of fluorescence detectors for HPLC led the authors to initiate... 

Fluorescence detectors for HPLC use come in many designs from the manufacturer. Differences in detector... 

Fluorescence detectors for HPLC are similar in design to the fluorometers and spectrofluorometers described in Section 15B-2. In most, fluorescence is observed by a photoelectric transducer located at 90° to the excitation beam. The simplest detectors use a mercury excitation source and one or more filters to isolate a band of emitted radiation. More sophisticated instruments are based on a xenon source and use a grating monochromator to isolate the fluorescence radiation. Laser-induced fluorescence is also used because of its sensitivity and selectivity. 

It has already been pointed out that earlier attempts to use HPLC for Be vitamer quantification were hampered by either laborious (microbiological) or insensitive (UV) detection procedures. Modern HPLC analysis of the different Be vitamers now exploits the fluorescence characteristics of these vitamers, which allow quantification in the nanomolar range. Modern fluorescence detectors for HPLC are easy to use and are unrivaled as far as both sensitivity and selectivity for vitamin Bg analysis are concerned. A thorough understanding of the fluorescence characteristics of the different B vitamers is therefore essential before different published HPLC methods can be evaluated. 

Quantitative accuracy and precision (see Section 2.5 below) often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials. The most widely used detector for HPLC, the UV detector, does not have such selectivity as it normally gives rise to relatively broad signals, and if more than one component is present, these overlap and deconvolution is difficult. The related technique of fluorescence has more selectivity, since both absorption and emission wavelengths are utilized, but is only applicable to a limited number of analytes, even when derivatization procedures are used. 

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

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. 

The beauty of absorbance and fluorescence modes as complimentary detectors for HPLC lies in their nondestructive nature. Fractions eluting from these detectors are readily available for confirmation by mass spectrometry. 

HPLC column technology has produced highly effective and efficient analytical columns and, as has been previously stated (7), this development has led to a demand for more sensitive and versatile detectors for HPLC systems. HPLC detector development within the past several years has been aimed at increasing sensitivity, as evidenced by the development of fluorescence detectors capable of quantitating subnanogram levels of PAH s. Similarly, UV/VIS detectors have been developed which can detect nanogram levels of PAH s. 

The main attraction of fluorescence detection for HPLC is that for strongly fluorescent molecules, it can offer limits of detection two or three orders of magnitude lower than UV absorbance methods. The reason for this lies in the difference in the nature of the measurements. In a UV absorbance detector the photodetector is constantly illuminated at a high... 

HPLC has been one ofthe most widely used analytical methods for determining PAHs in complex environmental samples. The development of a chemically nonpolar stationary phase for HPLC has provided a unique selectivity for separation of PAH isomers that are often difficult to separate by GC columns. For example, chrysene, benz[a]anthracene, and triphenylene are baseline resolved with a C-18 reverse phase column packing. A detection limit of subpicogram to picogram levels of PAHs per sample has been achieved by HPLC with fluorescence detector (For and Staley 1976 Furuta and Otsuki 1983 Futoma et al. 1981 Golden and Sawicki 1978 Lawrence and Weber 1984 Marcomini et al. 1987 Miguel and De Andrade 1989 Nielsen 1979 Risner 1988 Tomkins et al. 

Other widely used detectors for HPLC include refractive index (RI), fluorescence and evaporative light-scattering (ELS). The use of Rl and ELS detectors for pantothenic acid analysis in multivitamin dietary supplements has not been reported. The main reason is that the two detectors are not selective and thus cannot resolve pantothenic acid from other components existing in a multivitamin dietary supplement. Although fluorescence detection can be highly selective depending on the application, pantothenic acid does not have fluorescence excitation and emission and so fluorescence detection cannot be used for pantothenic acid analysis unless derivatization methods are applied (Pakin et al. 2004 Takahashi et al. 2009). Derivatization adds more complexity to analytical method and should not be used unless neeessary. For deteetion and quantitation of pantothenic add in multivitamin dietary supplements with HPLC/UHPLC, a highly selective detector such as MS should be the instrument of choice. 

Table 28-1 lists the most common detectors for HPLC and some of their most important properties. The most widely used detectors for LC are based on absorption of ultraviolet or visible radiation (see Figure 28-8). Fluorescence, refractive-index, and electrochemical detectors are also widely used.. Mass spectrometry (MS) detectors are currently quite popular. Such LC/MS systems can greatly aid in identifying the analytes exiting from the HPLC column as discussed later in this section. 

The available detectors for HPLC involve either bulk properties of the mobile phase (such as refractive index, conductance, dielectric constant) or specific properties of the solute, e.g. ultraviolet, visible or infra-red absorbance, fluorescence, or electrochemical characteristics. The latter class are generally more selective and have a wider dynamic range. 

Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

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