Fluorescence spectra chromatography

Figure 2. Fluorescence spectrum in a nitrogen matrix at 15 K (excited by a 2.5-kW mercury-xenon lamp) of an adsorption chromatography fraction from a coking plant water sample. Compounds BbF, benzo[b]fluorene C, chrysene BeP, ben-zo[e]pyrene P, pyrene BkF, benzo[k]fluoranthene BaP, benzo[a]pyrene U, unknown ( ).

The product of MMOL and DMOL decomposition was isolated by us using thin-layer chromatography. Its spectral properties (Table 1) are close to those of the product of decomposition of oxyluciferin with broken C-S-bond. The fluorescence spectrum of this product lies in the shorter wavelength region and this indicates the... 

Perhaps the most important feature of development chromatography is that the sample is separated by distance rather than time. This freedom from time constraints permits the utilisation of any of a variety of techniques to enhance the sensitivity of detection, such as reactions which increase light absorbance or fluorescence emission and wavelength selection for optimum response of each compound measured. The separation can be scanned as many times as desired, at a variety of wavelengths, and a complete UV visible or fluorescence spectrum can be easily plotted out for each component. Thus, the detection process in HPTLC is more flexible and variable than that for HPLC. Detection limits under optimum conditions are approximately the same for the two techniques. 

Tsukida K, Saiki K, Takii T and Koyama Y (1982) Separation and determination of cis/lrans-P-caTOtenes by high-performance liquid chromatography. J Chromatogr 245 359-364 Wasielewski MR, Johnson DG, Bradford EG and Kispert LD (1989) Temperature dependence ofthe lowest excited singlet-state lifetime ofall-fi o/w-/8-carotene and fully deuterated all-trans-P-carotene. J Chem Phys 91 6691-6697 Watanabe J, Takahashi H, Nakahara J and Kushida T (1993) Subpicosecond dynamic Stokes shift in /3-carotene solution probed by excitation energy dependence of fluorescence spectrum. Chem Phys Lett 213 351-355 Zechmeister L (1962) Cis-trans isomeric carotenoids vitamins A and arylpolyenes. Academic Press, New York... 

Other compounds that fluoresce may need to be removed from the system if the spectra overlap. This can be done, for example, by column chromatography. Peaks may appear in the fluorescence spectrum that are due to other emission and scattering processes Rayleigh, Tyndall, and Raman scattering may be seen because of the high intensity of the light source used. Peaks due to fluorescent impurities may occur. 

In these systems, a high-energy intermediate excites a suitable fluorophore, which then emits its characteristic fluorescence spectrum consequently, they are termed indirect or sensitized chemiluminescence. The most common analytical application has been as a postcolumn reaction detector for liquid chromatography. Various fluorescent analytes (polycyclic aromatic hydrocarbons and polycyclic aromatic amines) and compounds derivatized using dansyl chloride, fluorescamine, or o-phthalaldehyde have been determined with sub-femtomole detection limits. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

The separation of trichothecene mycotoxins from biological materials by UV absorption or fluorescence absorption is difficult, but the most suitable analytical methods are gas chromatography mass spectrum analysis. Recently, radioimmunoassay and enzyme linked immunosorbant assay have been developed for T-2 toxin and diacetoxyscirpenol (DAS) and deoxyverrucarol, which are highly sensitive as compared to other biological and chemical methods. 

Chemical identification infrared spectrum (Fer-slew etal, 1986 Shreenivasan and Boese, 1970) UV spectrophotometry (Ferslew et al, 1986) fluorescence spectrophotometry (Ferslew et al, 1986) gas chromatography (Zerba and Ruveda, 1972) isothermal gas chromatography (Jane and Wheals, 1972) ion mobility spectrometry (Allinson et al, 1998) NMR spectra (Ferslew et al., 1986 Mesilaakso, 1996) GC-MS (Smith et al., 2002). 

Fluorescence detection is often used in liquid chromatography. It has a low detection limit, but sometimes it does not cover the requirements of some compounds. To improve the detection limit, visible diode laser-induced fluorescence has been used as a detection system in liquid chromatography.208 Spectra are automatically corrected for differences in excitation efficiency in the red region of the spectrum. The automation of all components of analytical instruments improves the reliability of the analytical information. 

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15 -cA-spheroidene, were studied by absorption, fluorescence, CD, fast transient absorption and EPR spectroscopies in solution and after incorporation into the RC of Rb. sphaeroides R-26.1. High performance liquid chromatography (HPLC) purification of the synthetic molecule reveal the presence of several Ai-cis geometric isomers in addition to the mono-c/x isomer of locked-15,15 -c/x-spheroidene. In solution, the absorption spectrum of the purified mono-cA sample was red-shifted and showed a large c/x-peak at 351 nm compared to unlocked all-spheroidene. Spectroscopic studies of the purified locked-15,15 -mono-c/x molecule in solution revealed a more stable manifold of excited states compared to the unlocked spheroidene. Molecular modeling and semi-empirical calculations revealed that geometric isomerization and structural factors affect the room temperature spectra. RCs of Rb. sphaeroides R-26.1 in which the locked-15,15 -c/x-spheroidene was incorporated showed no difference in either the spectroscopic properties or photochemistry compared to RCs in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 RCs which naturally contain spheroidene. The data indicate that the natural selection of a c/x-isomer of spheroidene for incorporation into native RCs of Rb. sphaeroides wild type strain 2.4.1 was probably more determined by the structure or assembly of the RC protein than by any special quality of the c/x-isomer of the carotenoid that would affect its ability to accept triplet energy from the primary donor or to carry out photoprotection. 

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