Fluorescent species, identification

Identification of the Fluorescent Species. Figure 2 compares the fluorescence excitation spectra of the polymers with the absorption spectrum of a simple ,/3-unsaturated carbonyl compound (pent-3-ene-2-one) (13). The three spectra are very similar. Figure 2 shows also that the fluorescence from the polymers in the region 300-400 nm cannot be caused by the presence of polynuclear aromatic hydrocarbons such as naphthalene as postulated earlier by Carlsson and Wiles (13). Furthermore, as shown below, the excitation spectrum also differs significantly from that of a fully saturated aldehyde or ketone. 

The need for high-throughput screening methods of human mutations has stimulated the development of CE-based methods for SSCP analysis. For detection of ssDNA, PCR is carried out with primers labeled at the 5 site with fluorescence dyes. Two different labels may be used for identification of the forward and reverse strands. Advantages of CE-SSCP are speed of electrophoresis (ca. 10 min), high sensitivity, reproducibility, and the possibility of automation (Andersen et al., 2003 Hestekin et al., 2006). In food analysis, CE-SSCP has been used to identify bacteria (see Section 5.4.4) but, to the knowledge of the author, not to species identification of meat, fish, or other food up to now. 

Ultraviolet Raman resonance (UVRR) spectroscopy provides for chemical species identification from both the characteristic vibrational structure and electronic spectra. The resonance enhancement also increases the absolute sensitivity of detection, making it easier to detect the structures. The advantages of UVRR spectroscopy are high sensitivity, lack of fluorescence and suitability for use in aqueous solutions. 

Methods for meat species identification based on DNA analysis benefit from the heat stability of the DNA molecule and its high specificity. Originally, DNA methods consisted of immobilization of partially purified and denatured DNA, extracted from the meat product sample, on a nylon membrane, followed by hybridization of a species-specific segment of labeled (colorimetric, fluorescent, or chemiluminescent) DNA with any complementary sequences of DNA present on the membrane. More recently, a DNA amplification method - the polymerase chain reaction - has been used, but this is a relatively expensive and technically demanding technique. 

Atomic and molecular species identification via motional resonance-based detection is also possible in more complex systems, namely in large multispecies ion crystals of various size, shape, and symmetry [47,52,70] (Figure 18.19). The basic principle of the method is as follows. The radial motion of the ions in the trap is excited using an oscillating electric field of variable frequency applied either to an external plate electrode or to the central trap electrodes. When the excitation field is resonant with the oscillation mode of one species in the crystal, energy is pumped into the motion of that species. Some of this energy is distributed through the crystal, via the Coulomb interaction. This, in turn, leads to an increased temperature of the atomic coolants and modifies their fluorescence intensity, which can be detected. 

In order to relate material properties with plasma properties, several plasma diagnostic techniques are used. The main techniques for the characterization of silane-hydrogen deposition plasmas are optical spectroscopy, electrostatic probes, mass spectrometry, and ellipsometry [117, 286]. Optical emission spectroscopy (OES) is a noninvasive technique and has been developed for identification of Si, SiH, Si+, and species in the plasma. Active spectroscopy, such as laser induced fluorescence (LIF), also allows for the detection of radicals in the plasma. Mass spectrometry enables the study of ion and radical chemistry in the discharge, either ex situ or in situ. The Langmuir probe technique is simple and very suitable for measuring plasma characteristics in nonreactive plasmas. In case of silane plasma it can be used, but it is difficult. Ellipsometry is used to follow the deposition process in situ. 

Widengren, J., Kudryavtsev, V., Antonik, M., Berger, S., Gerken, M. and Seidel, C. A. (2006). Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal. Chem. 78,2039-50. 

Miller P, Scholin C. Identification and enumeration of cultured and wild Pseudo-nitzschia (Bacillariophyceae) using species-specific LSU rRNA-targeted fluorescent probes and filter-based whole cell hybridization. J Phycol 1998 34 371-382. 

The fluorescence spectrum of a compound may be used in some cases for the identification of species, especially when the spectrum exhibits vibronic bands (e.g. in the case of aromatic hydrocarbons), but the spectra of most fluorescent probes (in the condensed phase) exhibit broad bands. 

The emitting species was found to be the singlet-excited state of 3-aminophthlate ion in both protic and aprotic solvents. This identification was made based on the equivalence of the chemiluminescence spectrum of luminol and the fluorescence spectrum of 3-AP ion . In different reaction media, slightly different maximum chemiluminescence wavelengths are observed (Table 2). The spectral shift observed when the system changes from aqueous media to DMSO or other aprotic solvents can be ascribed to a quinoidal form of 3-aminophthalate (26) formed in aprotic solvents (Scheme 15). ... 

Some observations of the solutions also help confirm the earlier identification of TMAE as the emitting species. TMAE fluorescence is green from liquid n-decane, but becomes blue in frozen n-decane. The same sudden shift occurs in chemiluminescence. 

As seen in Chapter 9.C.2, a very wide variety of organics are found in particles in ambient air and in laboratory model systems. The most common means of identification and measurement of these species is mass spectrometiy (MS), combined with either thermal separation or solvent extraction and gas chromatographic separation combined with mass spectrometry and/or flame ionization detection. For larger, low-volatility organics, high-performance liquid chromatography (HPLC) is used, combined with various detectors such as absorption, fluorescence, and mass spectrometry. For applications of HPLC to the separation, detection, and measurement of polycyclic aromatic hydrocarbons, see Wingen et al. (1998) and references therein. 

The present review summarizes contemporary views of the problems, achievements, and prospects involved in the deep desulfurization of gas oils, including identification and reactivity of sulfur species in the feed, the reaction pathways and mechanisms, activity and selectivity of the conventional catalysts, and concerns of fluorescence color production. Process schemes and guidelines for the development of the next-generation catalysts for improved deep desulfurization technology based on these discussions are also proposed. The structure and nature of the active sites of current catalysts will not be extensively covered in this review, because several excellent reviews have been published on these subjects within the past two years (1-3). 

HPLC units have been interfaced with a wide range of detection techniques (e.g. spectrophotometry, fluorimetry, refractive index measurement, voltammetry and conductance) but most of them only provide elution rate information. As with other forms of chromatography, for component identification, the retention parameters have to be compared with the behaviour of known chemical species. For organo-metallic species element-specific detectors (such as spectrometers which measure atomic absorption, atomic emission and atomic fluorescence) have proved quite useful. The state-of-the-art HPLC detection system is an inductively coupled plasma/MS unit. HPLC applications (in speciation studies) include determination of metal alkyls and aryls in oils, separation of soluble species of higher molecular weight, and separation of As111, Asv, mono-, di- and trimethyl arsonic acids. There are also procedures for separating mixtures of oxyanions of N, S or P. 

A particular type of within-array analysis is the so called self-self hybridization [9], in which two dyes are used to label the same RNA species, so that the fluorescence values acquired by the scanner for each gene is supposed to be the same for the two channels. This approach allows the identification of the variability which depends only on systematic biases or on stochastic processes. Some authors suggest the performance of some self-self hybridization for each experiment, to establish an error model used to correct data derived from experimental measurements. 

The transition from amorphous carbon-containing deposits to graphite-like species and finally to graphitic carbon typically proceeds via polyaromatic heterocycles (Guisnet and Magnoux, 2001), which are not easily detected by conventional Raman spectroscopy because of fluorescence problems (Chua and Stair, 2003 Li and Stair, 1996). The use of UV excitation provides a powerful means to circumvent fluorescence problems and tackle the identification of the carbonaceous deposits (Chua and Stair, 2003). This subject was discussed in detail by Stair (2007). Polyaromatic deposits were burned off very quickly upon restoration of oxidizing conditions (Boulova et al., 2001 Mul et al., 2003 Puurunen and Weckhuysen, 2002 Puurunen et al., 2001). 

This pale-yellow quaternary alkaloid, picrate mp 189°, [a]D of chloride — 930° (in water), was first isolated from a calabash curare (12) it was subsequently isolated from other calabash curare preparations (34, 35) and has been identified chromatographically in extracts from the bark of S. mitscherlichii (33) and other Strychnos species (7). The identification of C-fluorocurarine is greatly helped by its deep-blue fluorescence in UV-light. Analyses of the crystalline iodide and anthraquinone sulfonate showed its molecular formula to be C2oH23N20+ (12), which is in agreement with all subsequent work. The properties of C-fluorocurarine, particularly its low toxicity and high Rc values, suggest that it is a C20 alkaloid rather than a double molecule. This was confirmed (102) by application of the partial quaternization method to one of its derivatives (see subsequent discussion) the method cannot be applied to C-fluorocurarine itself, since on pyrolysis it is not smoothly demethylated to the... 

While luminescence in vapor-deposited matrices accordingly should be a powerful technique for detection and quantitation of subnanogram quantities of PAH in complex samples, it suffers from two major limitations. First, it is obviously limited to the detection of molecules which fluoresce or phosphoresce, and a number of important constituents of liquid fuels (especially nitrogen heterocyclics) luminesce weakly, if at all. Second, the identification of a specific sample constituent by fluorescence (or phosphorescence) spectrometry is strictly an exercise in empirical peak matching of the unknown spectrum against standard fluorescence spectra of pure compounds in a hbrary. It is virtually impossible to assign a structure to an unknown species a priori from its fluorescence spectrum qualitative analysis by fluorometry depends upon the availabihty of a standard spectrum of every possible sample constituent of interest. Inasmuch as this latter condition cannot be satisfied (particularly in view of the paucity of standard samples of many important PAH), it is apparent that fluorescence spectrometry can seldom, if ever, provide a complete characterization of the polycyclic aromatic content of a complex sample. 

---