Fluorescence detectors HPLC

Consequently, analysis of PAHs is mainly accomplished by GC-MS in simple or tandem mode (GC-MS/MS) and HPLC coupled with fluorescence detector (HPLC-FLD) since most of the PAHs are strong fluorophores. Multiple reaction monitoring (MRM) in a triple quadrupole GC-MS/MS is inherently more selective and sensitive than either scan or selected ion monitoring (SIM) as many matrix interferences are minimized or even removed. For this reason various groups have used tandem MS (Matozzo et al. 2010 Xia et al. 2012), however sufficient detection was accomplished also with GC-MS-SIM (Webster et al. 2006). HPLC-UV has also been used with less sensitivity as expressed by LOD values, although in one work LOD values with UV detection are reported quite low (Cravo et al. 2012). PAHs as in almost all UV or FLD methods were identified on the basis of retention time and quantification on an external standard method. 

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. 

In hplc, detection and quantitation have been limited by availabiHty of detectors. Using a uv detector set at 254 nm, the lower limit of detection is 3.5 X 10 g/mL for a compound such as phenanthrene. A fluorescence detector can increase the detectabiHty to 8 x 10 g/mL. The same order of detectabiHty can be achieved using amperometric, electron-capture, or photoioni2ation detectors. 

In the chemiluminescence-based HPLC detection system, illustrated schematically in Figure 6, the oxalate ester and hydrogen peroxide are introduced to the eluent stream at postcolumn mixer Mj, which then flows through a conventional fluorescence detector with the exciting lamp turned off or a specially built chemiluminescence detector. The two reagents are combined at mixer Mj, rather than being premixed, to prevent the slow hydrolytic reactions of the oxalate ester. 

Similarly to the methods used to characterize natural chlorophylls, RP-HPLC has been chosen by several authors to identify the individual components in Cn chlorophyllin preparations and in foods. The same ODS columns, mobile phase and ion pairing or ion suppressing techniques coupled to online photodiode UV-Vis and/or fluorescence detectors have been used. ° ... 

Polar or thermally labile compounds - many of the more modern pesticides fall into one or other of these categories - are not amenable to GC and therefore LC becomes the separation technique of choice. HPLC columns may be linked to a diode-array detector (DAD) or fluorescence detector if the target analyte(s) contain chromophores or fluorophores. When using a DAD, identification of the analyte(s) is based on the relative retention time and absorption wavelengths. Similarly, with fluorescence detection, retention time and emission and absorption wavelengths are used for identification purposes. Both can be subject to interference caused by co-extractives present in the sample extract(s) and therefore unequivocal confirmation of identity is seldom possible. 

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. 

In order to achieve detection limits below the ng mL-1 range only amperometric, chemiluminescence, radiometric, or conventional fluorescence (CF) can be applied (Table 4.41). Fluorescence detectors are generally about 100 times more sensitive and more selective than UV detectors. The selectivity of fluorescence detection is due to the fact that only aromatic and conjugated molecules can be analysed, and by applying specific excitation and emission wavelengths the selectivity can even be increased. Pre- or postcolumn derivatisation in HPLC is a technique that is most commonly performed prior to UV absorption or fluorescence detection... 

High Performance Liquid Chromatographic (HPLC) Analysis. A Waters HPLC system (two Waters 501 pumps, automated gradient controller, 712 WISP, and 745 Data module) with a Shimadzu RF-535 fluorescence detector or a Waters 484 UV detector, and a 0.5 pm filter and a Rainin 30 x 4.6 mm Spheri-5 RP-18 guard column followed by a Waters 30 x 3.9 cm (10 pm particle size) p-Bondapak C18 column was used. The mobile phase consisted of a 45% aqueous solution (composed of 0.25% triethylamine, 0.9% phosphoric acid, and 0.01% sodium octyl sulfate) and 55% methanol for prazosin analysis or 40% aqueous solution and 60% methanol for naltrexone. The flow rate was 1.0 mL/min. Prazosin was measured by a fluorescence detector at 384 nm after excitation at 340 nm (8) and in vitro release samples of naltrexone were analyzed by UV detection at 254 nm. 

The basic theory, principles, sensitivity, and application of fluorescence spectrometry (fluorometry) were discussed in Chapter 8. Like the UV absorption detector described above, the HPLC fluorescence detector is based on the design and application of its parent instrument, in this case the fluorometer. You should review Section 8.5 for more information about the fundamentals of the fluorescence technique. 

FIGURE 13.10 The HPLC fluorescence detector. When a mixture component that exhibits fluorescence elutes from the column, the light is detected and a peak appears on the chromatogram. 

As mentioned above, the most commonly used method for the analysis of cationic surfactants has been HPLC coupled with conductometric, UV, or fluorescence detectors, the latter typically utilizing post-column ion-pair formation for enhanced sensitivity. Analysis by GC is only possible for cationic compounds after a derivatisation step [33] because of the ionic character of this compound. However, structural information might be lost. 

Fig. 4.3.7. Reversed-phase HPLC (A + B) and normal-phase HPLC (C + D) chromatograms of wastewater (A + C) and sludge (B + D) taken from an industrial wastewater treatment plant. Detector fluorescence detector. Chromatograms used for calculation of the results shown in Table 4.3.1. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

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. 

Apparatus. The HPLC instrument used was a Water s Associates model 6000A pump for the solvent supply, a U6K septumless injector and a radial compression module with standard Radial Pak columns. Immediately after the column a low dead volume tee was inserted and another 6000A pump was used to deliver a solution of OPT for the post-column derivatization of histamine. Twenty feet of 9 thousandths (id) coiled stainless steel tubing was used as a mixing chamber and held at 60 C in a water bath. The reaction mixture then passed through a Water s 420 fluorescence detector which was connected to a recorder. The detector was equipped with a 340-nm excitation filter and a 440-nm emmission filter. 

Measurements were performed on a Waters 470 HPLC fluorescence detector equipped with a JASCO cuvette accessory and connected to a Perkin Elmer 561 strip chart recorder. Excitation and emission band-widths were 18 nm. Emission spectra were measured for the three excitation wavelengths mentioned above and emission starting from 10 nm higher than excitation up to 700 nm. Fluorescence at fixed wavelengths was measured four minutes after cuvette insertion and expressed as per-millage of the 275/303 fluorescence of 3.0 pM tyrosine in 50 mM HEPES, pH 7.4. Corrections were made for buffer- and blank collagenase fluorescence, and for signal attenuation. 

The HPLC gradient system consisted of two pumps for solvent delivery, an injector, a 120 x 4.6 mm Polyspher AA-NA column (Merck, 75°C), a pump for reagenf delivery, a mixing tee with reaction coil, and a fluorescence detector lined to an integrator (Waters). 

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