Background fluorescence

Fluorescence Interference. The historical drawback to widespread use of Raman spectroscopy has been the strong fluorescence background exhibited by many materials, even those which are nominally nonfluorescent. This fluorescence often arises from an impurity in the sample, but may be intrinsic to the material being studied. Several methods have proved useflil in reducing this background. One of the simplest is sample purification. 

Another method, called photobleaching, works on robust soHds but may cause photodecomposition in many materials. The simplest solution to the fluorescence problem is excitation in the near infrared (750 nm—1.06 pm), where the energy of the incident photons is lower than the electronic transitions of most organic materials, so fluorescence caimot occur. The Raman signal can then be observed more easily. The elimination of fluorescence background more than compensates for the reduction in scattering efficiency in the near infrared. Only in the case of transition-metal compounds, which can fluoresce in the near infrared, is excitation in the midvisible likely to produce superior results in practical samples (17). 

This property can be applied to the detection of substances that absorb in the UV region For on layers containing a fluorescent indicator or impregnated with a fluorescent substance the emission is reduced in regions where UV-active compounds partially absorb the UV light with which they are irradiated. Such substances, therefore, appear as dark zones on a fluorescent background (Fig. 4A). 

Detection and result The chromatogram was freed from mobile phase in a stream of warm air, immersed in the reagent solution for 1 s and heated to 120°C for 20 min. Intense yellow to brown zones of various hues were produced these appeared as dark zones on a fluorescent background under long-wavelength UV light (X = 365 nm). 

Detection and result The developed chromatogram was freed from mobile phase by drying for 10 min at 110°C, allowed to cool and immersed for 1 s in the reagent solution. The plate was evaluated as rapidly as possible while it was moist since the fluorescent background increased in intensity as the plate dried out. Cholesterol appeared as a yellow-green fluorescent zone hR 20—25). 

Under UV light (A = 254 nm or 365 nm) the chromatogram zones appear as pale yellow fluorescent zones on a weakly yellow fluorescent background. 

In long-wavelength UV light (2 = 365 nm) carbohydrates, e.g. glucose, fructose and lactose, yield pale blue fluorescent derivatives on a weakly fluorescent background. In situ quantitation can be performed at = 365 nm and 2fi = 546 nm (monochromatic filter M 546) [19]. Further differentiation can be achieved by spraying afterwards with p-anisidine-phosphoric acid reagent [8]. 

Fluorescent chromatogram zones are produced on a dark or fluorescent background under long-wavelength (2 = 365 nm) and occasionally short-wavelength UV light (2 = 254 nm). 

Fig. 17. Changes in fluorescent background on changing the excitation wavelength. Raman spectra of o-xylene using different exciting lines (a) Ar+ 488 nm (b) Kr+ 647.1 nm (c) Ar+ 514.5 nm (d) Kr+ 568.2 nm. Fluorescent background was substantially reduced in spectrum (b). (Courtesy Spex Industries, Inc.)...

Fiq. 20b. The pulsed Raman spectrum of Mn-doped ZnSe with a 1 /xsec detection interval. The fluorescent background was significantly reduced from that observed with a 200 nsec detection interval in Fig. 20a (37). 

Funk et al. have used a low-pressure mercury lamp without filter to liberate inorganic tin ions from thin-layer chromatographically separated organotin compounds these were then reacted with 3-hydroxyflavone to yield blue fluorescent chromatogram zones on a yellow fluorescent background [22]. Quantitative analysis was also possible here (XoK = 405 nm, Xji = 436 nm, monochromatic filter). After treatment of the chromatogram with Triton X-100 (fluorescence amplification by a factor of 5) the detection limits for various organotin compoimds were between 200 and 500 pg (calculated as tin). 

Observation under short- and long-wavelength UV light (X, = 254 nm or 365 nm) reveals yellow to orange fluorescent chromatogram zones on a yellow-green fluorescent background. 

Under long-wavelength UV light (X = 365 nm) yellow-orange fluorescent chromatogram zones are observed on a pale light-blue fluorescent background. 

Phenylethylamine (h/ f 60-65), tyramine (h/ f 45-50), serotonin (h/ f 35-40) and histamine (hRf 20-25) yielded yellow-orange fluorescent zones on a pale light-blue fluorescent background under long-wavelength UV light (X, = 365 nm). 

It is not recommended that the chromatogram then be treated with liquid paraffin - n-hexane (1+4) since the intensity of the pale light blue fluorescent background is also increased, so that the difference in emission of the chromatogram zones is reduced. 

On excitation with long-wavelength UV light (X = 365 nm) ketoprofen (hcf 35-40) and flurbiprofen (h/ f 50-55) appeared as yellow or blue fluorescent chromatogram zones on a pale blue fluorescent background. The detection limits of, for instance, flurbiprofen were 10 ng subtance per chromatogram zone. 

Exposure of rhodamine 6G-impregnated silica gel layers to iodine vapor for two to five minutes followed by irradiation with UV light leads to the sensitive blue coloration of the chromatogram zones on a greenish fluorescent background [8, 10]. 

It can be advantageous to heat the chromatogram to 160 °C for 15 min before treating with nitrous fumes and to place it in the reagent chamber while still hot [1]. Heating to 260 °C has even been recommended for the purpose of reducing the fluorescent background [14], whereby the layer is previously immersed in 1 percent Ludox solution (silidc acid sol) to increase its stability [2]. The fluorescence of the substances detected usually remains stable for at least 2 weeks [2]. 

Brief exposure to nitrous fumes (up to 3 min) leaves the fluorescent power of the acid-instable fluorescence indicator 254. incorporated into most TLC layers, largely unaffected, so that the nitroaromatics so formed can be detected as dark zones on a green fluorescent background [1]. For purposes of in situ quantitation it is recommended that the fluorescence indicator be destroyed by 10 min exposure to nitrous fumes in order to avoid difficulties in the subsequent evaluation [1]. 

In situ quantitation The fluorimetric analysis was carried out at Xexc = 313 nm and at Xfl >560 nm (cut off filter FI 56). The chromatogram zones gave a negative signal (the fluorescent background was set at 100% emission). 

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