Detection fluorescence

The fluorescence detector is used for naturally fluorescent compounds or compounds that have been derivatized to compounds that emit fluorescent light. 

There are different constructions of fluorescence detectors, filter fluorimeters, and spectrofluorimeters with different hght sources, the most common one is the deuterium lamp. 

Quantum yields vary between 0 and 1, for strongly fluorescent compounds above 0.5. Dissolved oxygen (in the mobile phase) may reduce the quantum yield. 

Detection limits (depending on the molecular structure, the light source, and the column size) are in the picogram-femtogram range. 

Fluorescent compounds often contain condensed aromatic rings with electro-donating substituents, such as polycyclic aromatic hydrocarbons (PAHs). Rigid structures give better signals than flexible structures, since the latter lose more energy as vibration energy. 

Fluorescence detection is often used where no other property of the solute (e.g. UV of RI detection) is convenient and can be either an intrinsic property of the solute itself or a derivatised form of the solute. Solution studies have indicated that the sensitivity of detection can be increased by up to three orders of magnitude over UV. This has increased the popularity of post-column fluorescence detection methods for may compounds, including physiological fluids, catecholamines, and other polyamines. A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chro-mophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. 

The conventional fluorescence detector consists of the following components  

The increased sensitivity of fluorescence detectors means that the presence of contaminating material in the mobile phase can become a major problem. One method of circumventing this is to distill the solvents over a fluorescent reagent prior to use (Stein and Brink, 

In ion chromatography, fluorescence detection is mainly utilized in combination with postcolumn derivatization, because inorganic anions and cations, with the exception of the uranyl cation do not exhibit any intrinsic fluorescence. 

Fluorescence results from the excitation of molecules via absorption of electromagnetic radiation it is the emission of fluorescence radiation when the excited system returns to the energetic ground level. The emitted wavelength is characteristic to the kind of molecule while the intensity is proportional to concentration. 

The best known and most widely used fluorescence method was developed by Roth and Hampai [85] for the detection of primary amino acids and was 

The only drawback of the fluorescence method developed by Roth and Ham-pai is the relatively small stability of the Af-substituted l-alk)dthioisoindoles that are formed from a-amino acids after reaction with OPA. Thus, the reaction product has to be injected into the chromatography system at a defined time when applying OPA for the precolumn derivatization of primary amines. 

Much higher stabilities and partly higher fluorescence yields were obtained by Stobough et al. [86]. Naphthaline-2,3-dialdehyde (NDA), which reacts with primary amines in the presence of cyanide ions to Af-substituted l-cyanobenz[f isoindoles (CBI), was used as a reagent 

The best known and most widely used fluorescence method was developed by Roth and Hampai [71] for the detection of primary amino acids, and was described in Section 5.9.2. It is based mainly on the reaction of a-amino acids with o-phthaldialdehyde (OPA) and 2-mercaptoethanol to yield an intensively blue-fluorescing complex. Even at room temperature, the reaction occurs within seconds. At an excitation wavelength of 340 nm and an emission wavelength of 455 nm, this method has a detection limit in the low pmol range. The derivatization with o-phthaldialdehyde is applicable to all compounds carrying a primary amino group. This includes the ammonium ion, primary amines, polyamines, and peptides. 

The excitation spectrum of these derivatives shows maxima at 246 run and 420 nm the emission is measured at a wavelength of 490 nm. At an excitation wavelength of 246 nm, detection limits are obtained in the medium to low fmol range. However, secondary amines can be subjected to the two derivatization methods described above only after oxidation. 

One of the problems facing spin chemists performing these measurements is that the observed field effects can be rather small. Thus the method of detection should be as sensitive as possible. In some systems, it is possible to use the inherent fluorescence of one of the species involved in the reaction as a probe of RP activity. The most common of these approaches is the situation with the formation of RlPs that can often lead to spin-selective exciplex formation via the singlet RIP. Systems involving conjugated aromatic molecules, for example, anthracene and pyrene as electron donors/acceptors, amines as electron donors, and substituted benzenes (e.g., dicyanobenzenes) as electron acceptors, have been commonly employed and are now extremely well 

MAGNETIC FIELD EFFECTS ON RADICAL PAIRS IN HOMOGENEOUS SOLUTION 

Precolumn derivatization methods include 1,2-diphenylethylenediamine treatment, dansylation of E, NE and DA, derivatization of NE and DA by o-phthalaldehyde and mercaptoethanol and derivatization of catecholamines with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl). Derivatization with o-phthalaldehyde increases the sensitivity of NE and DA, but E is not measured because only primary amines are derivatized. Co-analysis of catecholamines, metanephrines and other related compounds by combined electrochemical oxidation and fluorescence derivatization had also been reported. ° This approach involves sequential chromatographic separation, coulometric oxidation and final chemical derivatization with 1,2-diphenylethylenediamine to fluorescent products. 

Chan et developed an assay for the simultaneous determination of catecholamines and metanephrines using FMOC derivatization. The assay is convenient for the simultaneous analysis of NM, MN, E and DA in human urine sample without prior extraction procedures. In this study, urine was directly derivatized and subjected to a simple extraction step with 

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Scherer N F, Carlson R J, Matro A, Du M, Ruggiero A J, Romero-Rochin V, Cina J A, Fleming G R and Rice S A 1991 Fluorescence-detected wave packet interferometry time resolved molecular spectroscopy with sequences of femtosecond phase-locked pulses J. Chem. Rhys. 95 1487... 

Scherer N F, Matro A, Ziegler L D, Du M, Cina J A and Fleming G R 1992 Fluorescence-detected wave packet interferometry. 2. Role of rotations and determination of the susceptibility J. Chem. Rhys. 96 4180... 

Stdhr J, Kollin E B, Fischer D A, Flastings J B, Zaera F and Sette F 1985 Surface extended x-ray-absorption fine structure of low-Z adsorbates studied with fluorescence detection Rhys. Rev. Lett. 55 1468-71... 

Figure B2.3.9. Schematic diagram of an apparatus for laser fluorescence detection of reaction products. The dye laser is syncln-onized to fire a short delay after the excimer laser pulse, which is used to generate one of the reagents photolytically.

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Brand L, Eggeling C, Zander C, Drexhage K FI and Seidel CAM 1997 Single-molecule identification of coumarin-120 by time-resolved fluorescence detection comparison of one- and two-photon excitation in solution J. Chem. Phys. A 101 4313-21... 

Blum L, Abruna FI D, White J, Gordon J G, Borges G L, Samant M G and Melroy 1986 Study of underpotentially deposited copper on gold by fluorescence detected surface EXAFS J. Chem. Phys. 85 6732-8... 

Better detection limits are obtained using fluorescence, particularly when using a laser as an excitation source. When using fluorescence detection, a small portion of the capillary s protective coating is removed and the laser beam is focused on the inner portion of the capillary tubing. Emission is measured at an angle of 90° to the laser. Because the laser provides an intense source of radiation that can be focused to a narrow spot, detection limits are as low as 10 M. 

The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

Extremely low level detection work is being performed ia analytical chemistry laboratories. Detection of rhodamine 6G at 50 yoctomole (50 x lO " mol) has been reported usiag a sheath flow cuvette for fluorescence detection foUowiag capiUary electrophoresis (9). This represeats 30 molecules of rhodamine, a highly fluoresceat molecule (see Electhoseparations, electrophoresis Spectroscopy, optical). 

Post-column in-line photochemical derivatization permits fluorescence detection of the common aflatoxins Bl, B2, Gl, and G2 (60). Chromatographic evidence indicates that photolysis causes the hydration of the nonfluorescent Bl and Gl components to B2a and G2a components, respectively. Analysis of naturally contaminated com samples show no interfering peaks and permits the deterrnination of 1 and 0.25 ppb for Bl and B2, respectively. 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

The performance of microwave-assisted decomposition of most difficult samples of organic and inorganic natures in combination with the microwave-assisted solution preconcentration is illustrated by sample preparation of carbon-containing matrices followed by atomic spectroscopy determination of noble metals. Microwave-assisted extraction of most dangerous contaminants, in particular, pesticides and polycyclic aromatic hydrocarbons, from soils have been developed and successfully used in combination with polarization fluoroimmunoassay (FPIA) and fluorescence detection. 

There have been compared the methods of mycotoxin control in food products with aflatoxin as an example, using both HPLC method with fluorescent detecting on the apparatus Thermo FL 3000 with a column BDS Hypersil C 2.1x150, as well as a chromatodensitometry method on the apparatus CAM AG TLS Scanner 3. 

After the dipped or sprayed chromatogram has been dried in a stream of cold air long-wave UV light (2 = 365 nm) reveals fluorescent yellow zones (flavonoids). Sterigmatocystine, which can be detected without derivatization on account of its red intrinsic fluorescence (detection limit 0.5 pg), also fluoresces pale yellow after being heated to 80°C [9] or 100°C [13] for 10 min on the other hand, citrinine, zearalenone and vomitoxin fluoresce blue. 

A stereoselective determination of enantiomers of 5, its A -oxide and N-desmethyl metabolites in human urine was developed by capillary electrophoresis using laser-induced fluorescence detection and sulfonated /1-cyclodextrin in the running buffer (01JC(B)169). 

Electrodriven separation techniques are destined to be included in many future multidimensional systems, as CE is increasingly accepted in the analytical laboratory. The combination of LC and CE should become easier as vendors work towards providing enhanced microscale pumps, injectors, and detectors (18). Detection is often a problem in capillary techniques due to the short path length that is inherent in the capillary. The work by Jorgenson s group mainly involved fluorescence detection to overcome this limit in the sensitivity of detection, although UV-VIS would be less restrictive in the types of analytes detected. Increasingly sensitive detectors of many types will make the use of all kinds of capillary electrophoretic techniques more popular. 

EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

E. R. Brouwer, A. N. J. Elermans, El. Lingeman and U. A. Th Briknman, Determination of polycyclic aromatic hydrocarbons in surface water by column liquid cliromatogr a-phy with fluorescence detection, using on-line micelle-mediated sample preparation , J. Chromatogr. 669 45-57 (1994). 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

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