Fluorescence interference with absorbance measurement

M. Chergui Dimers absorb at -207 nm in rare-gas matrices and excitation of this band does not yield any fluorescence [Chergui et a]., Chem. Phys. Lett. 201,187 (1993)]. Furthermore, our detection is based on the fact that we record the depletion of the fluorescence of one of the A(0, tf") bands due to NO monomers. There is therefore no possibility that NO dimers could interfere with our measurements. 

Fluorescence is generally more sensitive to environmental factors than absorbance measurements. Signal intensity may be affected by pH, temperature, quenching, interfering substances, solvent, or interference from Rayleigh and Raman scattering. Many fluorescent species contain ionisable groups whose fluorescent properties are sensitive to pH. In some cases only one of the ionised species may be fluorescent. An example is the barbiturates which only fluoresce at elevated pH in the di-anionic form. The relationship of fluorescence intensity with pH should always be examined as part of the development of the method. 

Direct methods of analysis such as ultraviolet (UV) absorption, infrared spectroscopy (IR), fluorescence, phosphorescence [13], X-ray fluorescence [14-16] and thermal analysis [17] have been reported. However, these methods generally lack specificity [18]. In Fourier transform IR (FTIR), overlapping bands of other species may interfere with the absorbance bands of the analyte, and in UV analysis the absorbance bands of different antioxidants can be very similar. UV and FTIR analysis are especially useful techniques when an antioxidant system is already known. X-ray fluorescence and elemental analysis are fast and useful techniques for the determination of antioxidants containing phosphorus or sulfur. The measurement of oxygen consumption... 

Fluorescence will interfere with kinetic absorbance measurements for the duration of the fluorescence or of the pump pulse, whichever is longer. Scattered light and... 

Chlorophyll a fluorescence induaion is a widespread method to evaluate the photosynthetic activity. This method is noninvasive, highly sensitive, fast, and easily measured. When chlorophyll molecules in photosystem II absorb light, that light may be assimilated into the hght reactions of photosynthesis or may be released as fluorescence or heat energy. In vivo fluorescence increases when photosynthesis declines or is inhibited. Numerous environmental f ors can affect the rate of electron transport between photosystem II and photosystem I due to interference with electron carriers between the two photosystems. For example, when the diuton is added in the measured sample, electron transport from photosystem II to photosystem I is blocked resulting in maximum fluorescence. This method was often employed to detect the photosynthetic activity of immobilized photosynthetic material. ... 

Cell responses to physical or chemical cues are measured in microfluidic devices primarily via optical or electrochemical means. Huorescence is the most widely used optical detection technique, because absorbance detection (commonly used for macroscale assays) is of limited value in microchannels because of the short path lengths. Fluorescence detection, characterized by its unparalleled sensitivity, is easy to implement in microfluidic systems. Chemiluminescence and bioluminescence also offer low detection limits and have less background noise than fluorescence [8]. Electrochemical detectors are even more easily integrated with microfluidic devices and often are much less expensive than optical systems. However, fabrication of electrodes in microchannel devices is a technical challenge, and the electrical fields used in detection can interfere with on-chip processes such as electrophoresis. Electrochemical techniques include potentiometry, amperometry, and... 

The discussions of various flame analysis techniques in section 2.2 are equally applicable to the determination of rare earth elements in a complex mixture of these elements./Net intensity or absorbance measurements usually provide adequate precision, so that an added reference element is not needed. In addition, there is no evidence of inter-element effects, and line interferences, even in small monochromators, are rarely a serious problem. This contrasts sharply with the selective enhancement and absorption effects observed in X-ray fluorescent spectrometric measurements. Analyses of rare earth mixtures by AAS have been described by Jaworowsk et al. (1967), Kriege and Welcher (1968) and many others. 

One of the main advantages of Raman spectroscopy over IR is that water is a weak Raman scatterer. The spectrum of water causes little interference so that spectra of solutes can be measured in aqueous solutions. A good example of the reduced interference from water is shown for two pharmaceuticals in Fig. 7-28. The Raman spectra of damp and dry samples of acetaminophen and ibuprofen are shown in the figure. Bands due to water are not observed in the spectra. Near and mid-IR of these same samples exhibited relatively strong absorbances due to water. These Raman spectra were measured on a dispersive instrument and were excited with an Ar-ion laser emitting at 488 nm. The background for the acetaminophen sample is flat, whereas ibuprofen exhibits a background characteristic of fluorescence. 

FP assays are known to be susceptible to artifacts (Turek-Etienne and Small, 2003). In principle, the assays are ratiometric and should normalize for variations in total excitation energy applied as would occur with inner filter effects, and newer generations of red-shifted fluorophores should help to eliminate interference (Vedvik et al., 2004). However, introducing a test compound with fluorescent or absorbent properties at 5 or 10 pM with the typically sub-micromolar concentrations of fluorophores in an FP assay can significantly skew the measurements. For example, if the compounds are insoluble, they can scatter and depolarize light. A concentration-dependent effect on an FP assay could result from an increase in the amount of insoluble compound. 

At excitation wavelengths and concentration ranges where the simple absorbance fluorescence is linear with concentration, the fluorimetric detector is susceptible to the usual interferences that hinder fluorescence measurements, mainly background fluorescence and quenching. 

Fluorescence detection is also inherently more selective than absorbance detection, since both the excitation and emission wavelengths may be chosen to suit a particular reaction product. For example, assays employing dehydrogenase enzymes may monitor NAD+ or nicotinamide adenine dinucleotide phosphate (NADP+) absorbance at 340 nm with reasonable sensitivity and selectivity. However, if excited at 340 nm, the nicotinamide coenzymes fluoresce at 460 nm. Not only do the fluorescence measurements inherently have lower detection limits, but they also provide selectivity against potential interferents that may also absorb at 340 nm but do not emit at 460 nm. 

The method of Fan and Dasgupta (1994) relics on tlie reaction of formaldehyde with 1,3-cyclohexane-dione in acidified ammonium acetate to form the fluorescent dihydropyridine derivative in a flow injection analysis system. Formaldehyde trapped in water can be reacted with pararosaniline and sodium sulfite under mild conditions (neutral pH, room temperature equilibration) to produce a colored product that is measured at 570 nm (Petreas et al. 1986). The presence of bisulfite is an interference in this reaction so the method cannot be used to sample atmospheres that contain sulfur dioxide. In addition, the method is reported to suffer from interferences resulting from the presence of other aldehydes and phenol (Hoogenboom et al. 1987). The indirect method of Hoogenboom et al. (1987) relies on the reaction of excess bisulfite in an aqueous solution of formaldehyde with 5,5 -dithiobis(2-nitrobenzoic acid) to form a colored product, the absorbance of which is measured at 412 nm. The method reported by Naruse et al. (1995) relies on the formation of a colored product obtained by reacting the aqueous formaldehyde with acetylacetone and ammonium acetate in acetic acid. Absorbance is measured at 414 nm. 

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