UV and fluorescence detection

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. 

Ni Y. Liu Y. Kokot S. Two-dimensional fingerprinting approach for comparison of complex substances analysed by HPLC-UV and fluorescence detection. Analyst, 2011,136 (3), 550-559. 

Isocratic reversed-phase HPLC determinations of twelve natural corticosteroids in serum with online UV and fluorescence detection. (163)... 

Fig. 29.6.1 Chromatograms obtained with UV and fluorescence detection from analysis of catfish tissue containing incurred quinolones. (Reprinted from Ref. 196. Copyright, (1995), by AOAC INTERNATIONAL.)...

Fig. 3 Separation of standard and liver phospholipids by HPLC on a 5-yttm Nucleosil 5 NH2 stationary phase with an isocratic mobile phase consisting of acetonitrile, methanol, water, and methyl phosphonic acid and subsequent UV and fluorescence detection. The second column was cut off from the eluent stream by valve switching after about 30 min. (Reprinted from Ref. 43 with the kind permission of Analytical Biochemistry.)...

Residues of FLU and 7-OH FLU in edible sheep tissues (muscle, liver, kidney, and fat) were determined by HPLC with UV and fluorescence detection (191). Liquid-liquid extraction with ethyl acetate was described, with extraction recoveries of 90,82,89, and 82% for FLU in muscle, liver, kidney, and fat, respectively. Recoveries for 7-OH FLU were 91, 90, 86, and 84%. The method was validated for specificity, linearity, limits of detection and quantitation, and precision. 

Simultaneous determination of FLU, NALA, OXO, and PIRA in catfish muscle was developed for HPLC with fluorescence detection (192). Sample workup involves homogenizing tissue with acetone, defatting with hexane, and extracting QUINs into chloroform. The sample was purified by partitioning into a base and back-extracted into chloroform after acidifying the aqueous phase. After evaporating and dissolving the residues in the mobile phase, the supernatant was injected into an HPLC system with UV and fluorescence detection (for PIRA). Extraction recoveries were FLU 79.7%, RSD 5.7%, OXO 80.8%, RSD 6.3%, PIRA 75%, RSD 5.9%, NALA 87.1%, and RSD 10.0%. The limits of quantifications were about 5 /mg/kg for all analytes. 

Quantification. High Pressure Liquid Chromatography. In plasma sulphapyridine, acetylsulphapyridine, 5-aminosalicylic acid, and 5-acetamidosalicylic acid, sensitivity 500 ng/ml, UV and fluorescence detection—P. N. Shaw et al., J. Chromat., 1983, 274 Biomed. AppL, 25, 393-397. In plasma or urine 5-aminosalicylic acid and 5-acetamidosalicylic acid, sensitivity 20 ng/ml, fluorescence detection—C. Fischer et al., J. Chromat.,... 

As regards the sensitivity of the MS detection in HPLC analysis of flavonoids, this technique has proved to be the most sensitive as compared to UV and fluorescence detection. A very comprehensive comparison of the four detection systems—UV, fluorescence, and two MS systems [atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)]—for the determination of the previously identified 3, 4, 5 -trimethoxyflavone is presented in Table 1. Fluorescence detection is 10 times more sensitive than UV detection, whereas MS detection is 50 times more sensitive than UV detection and 5 times more sensitive than fluorescence detection. 

The final determination of the PAHs was performed by capillary gas chromatography using flame ionisation or mass spectrometric detection, or by high-performance liquid chromatography using UV- and fluorescence detection. Each participant had validated its method by performing experiments on recovery, extraction efficiency, procedure blanks and detector linearity. 

Polynuclear aromatic hydrocarbons (PAH) are formed by pyrosynthesis during the combustion of organic matter and have widespread occurrence in the environment. PAHs are found as trace pollutants in soil, air particulate matter, water, tobacco tar, coal tar, used engine oil, and foodstuffs such as barbequed meat. Many PAHs are carcinogenic in experimental animals (e.g., benzo(a)pyrene) and are implicated as causative agents in human cancers. Analytical techniques include HPLC and GC. Advantages of HPLC are the ability to resolve isomeric PAHs, and the selective and sensitive quantitation by UV and fluorescence detection. U.S. EPA methods for PAH are 550.1,610, and 8310. 

Detailed analyses by HPLC revealed that there were more than 60 minor contaminants in the implicated L-tryptophan from Showa Denko.10 Of these contaminants, six were considered to be associated with EMS. The structures of the three contaminants are known and have been described previously, but the full identities of the other three contaminants are unknown. Simat et al.17 used single reversed-phase high-performance liquid chromatography run using UV and fluorescence detection on implicated L-tryptophan and observed 17 contaminants. Most of these contaminants were classified as tryptophan metabolites, nonphysiological oxidation, or carbonyl condensation compounds of tryptophan. 

Figure 11.12 Comparison, following a chromatographic separation, of UV and fluorescence detection. Aflatoxins, which are carcinogenic contaminants present in certain hatches of grain cereals, are the subject of analysis by HPLC. Detection by fluorescence is much more sensitive to Gj and B2 than with UV detection (reproduced courtesy of SUPELCO). Below left, schematic of the different components of a LC-detector based upon fluorescence. This detector is able to find rapidely, for each compound eluted, the best coupling of excitation/emission without interrupting the chromatography underway (reproduced courtesy of a document from Agilent Technologies).

Until the 1990s, UV and fluorescence detection were the main detection methods used for a... 

Selective extraction method of polycyclic aromatic compounds from tobacco smoke condensate and quantitative determination by HPLC-UV and fluorescence detection ... 

The most commonly used analytical technique for sugars is HPLC with a refractive index detector (RID). Although the HPLC-RID method is simple, the RID lacks sensitivity and selectivity. Therefore, UV and fluorescence detection is frequently used, coupled with pre- or postcolumn derivatization, for analysis with higher sensitivity. Liquid chromatography-mass spectrometry (LC-MS) using electrospray ionization also requires pre- or postcolumn derivatization. LC-MS using atmospheric pressure chemical ionization does... 

UV absorbance detection has been most widely used for vitamin A analysis. However, because retinol and retinyl esters are highly fluorescent, detection limits of one order of magnitude better than in assays with UV detection can be obtained using fluorescence detection. Also, electrochemical detection is a valuable alternative to UV and fluorescence detection provided the eluent contains water to incorporate essential electrolytes. Another detector for LC is the mass spectrometer. The LC-MS approach has also been applied to the analysis of vitamin A and its metabolites. 

Solid-phase extraction is also often used to remove interfering coextracted compounds. Solid-phase extraction columns contain either non-polar reversed-phase Cig sorbents or polar sorbents (such as alumina, aminopropyl acid, and propylsulfonic acid). Matrix solid-phase dispersion cleanup using reversed-phase Cig material has been also employed for the determination of oxohnic acid in catfish muscle.In-tube solid-phase microextraction (SPME) based on poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith coupled to high-preformance liquid chromatography (HPLC) with ultraviolet (UV) and fluorescence detection (FED) was... 

This review gives an overview of current advances in nucleic acid separation by CE and microchip electrophoresis. The focus is on the separation mechanisms during CE, conventional separation matrices and thermoresponsive polymers solutions, UV and fluorescence detection, microchip-based CE, and entropic trapping networks. 

Detectors. In ion chromatography, suppressed conductivity detection is still the most frequently used technique. Indirect UV detection (Section 12.2.6.3) can also he a powerful tool for the detection of ions, although with respect to sensitivity. conductivity detection (detection ptower 10-100 ng/g) cannot be surpassed. In amino acid analysis, UV and fluorescence detection (after post- or precolumn derivatization) is often applied. 

HPLC with UV and fluorescence detection are the most common techniques used today. HPLC is the preferred technique for vitamin separation because of its high selectivity. Reverse-phase chromatography on a Cl8 column support with an isocratic mobile phase works efficiently for the resolution of thiamine. 

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