Raman spectroscopy fluorescence interference

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. 

Principles and Characteristics The prospects of Raman analysis for structural information depend upon many factors, including sample scattering strength, concentration, stability, fluorescence and background scattering/fluorescence from the TLC substrate. Conventional dispersive Raman spectroscopy has been considered as a tool for in situ analysis of TLC spots, since most adsorbents give weak Raman spectra and minimal interference with the spectra of the adsorbed species. Usually both silica and cellulose plates yield good-quality conventional Raman spectra, as opposed to polyamide plates. Detection limits for TLC fractions... 

Raman spectroscopy has enjoyed a dramatic improvement during the last few years the interference by fluorescence of impurities is virtually eliminated. Up-to-date near-infrared Raman spectrometers now meet most demands for a modern analytical instrument concerning applicability, analytical information and convenience. In spite of its potential abilities, Raman spectroscopy has until recently not been extensively used for real-life polymer/additive-related problem solving, but does hold promise. Resonance Raman spectroscopy exhibits very high selectivity. Further improvements in spectropho-tometric measurement detection limits are also closely related to advances in laser technology. Apart from Raman spectroscopy, areas in which the laser is proving indispensable include molecular and fluorescence spectroscopy. The major use of lasers in analytical atomic... 

Raman spectra (for both the solid state and aqueous solution) provide better fingerprints for heparins than their i.r. spectra.79 However, the application of Raman spectroscopy to glycosaminoglycans is less routine than with i.r., both for instrumental reasons and because of possible interference from traces of fluorescent impurities.77... 

Raman spectroscopy has been widely used to study the composition and molecular structure of polymers [100, 101, 102, 103, 104]. Assessment of conformation, tacticity, orientation, chain bonds and crystallinity bands are quite well established. However, some difficulties have been found when analysing Raman data since the band intensities depend upon several factors, such as laser power and sample and instrument alignment, which are not dependent on the sample chemical properties. Raman spectra may show a non-linear base line to fluorescence (or incandescence in near infrared excited Raman spectra). Fluorescence is a strong light emission, which interferes with or totally swaps the weak Raman signal. It is therefore necessary to remove the effects of these variables. Several methods and mathematical artefacts have been used in order to remove the effects of fluorescence on the spectra [105, 106, 107]. 

The limitations of Raman spectroscopy are its low sensitivity compared to IR absorption and fluorescence interference from impurities in the sample. Raman spectroscopy is a developing technology, and a good amount of research and planning is necessary before deciding whether or not to employ it. The cost of a Raman process analyzer exceeds that of other analyzers. To reduce cost, Raman analyzers often include multichannel capability. Up to four process streams can be analyzed with a single CCD camera by splitting the lasers. 

The use of picosecond pulses to minimize the Interference of fluorescence with the Raman spectrum was also demonstrated (5) at about that time. The use of vldicon detection in Raman spectroscopy was demonstrated (6) in 1976. The first resonance Raman spectrum taken for a photobiologlcal system (bacteriorhodopsin) in the nanosecond time scale was (7) in 1977. The resonance Raman spectra of bacteriorhodopsin have also been measured in the microsecond (8,9,10) and in the millisecond (11) time domain. Recently the time resolved resonance Raman spectra of photolyzed hemoglobin derivatives have been reported (12). 

The instrumentation for conventional Raman spectroscopy will be discussed in this chapter. Special techniques of Raman spectroscopy will be described in Chapter 3. Most Raman spectroscopic investigations have been performed on dispersive instruments. However, Fourier transform (FT) techniques have become increasing important as a means of reducing interference from fluorescence. Both dispersive and FT-Raman spectroscopy will be discussed in this chapter. 

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. 

Romero-Torres, S., Perez-Ramos, J. D., Morris, K. R., and Grant, E. R. (2006), Raman spectroscopy for tablet coating thickness quantification and coating characterization in the presence of strong fluorescent interference, J. Pharma. Biomed. Anal., 41, 811-819. 

The post-1986 developments that caused the Raman renaissance are mainly technological, but they largely overcome the fundamental problems of a weak Raman signal and interference from fluorescence. To be sure, there were major technical developments preceding 1986, such as photon counting detection and the introduction of the laser, but the more recent technical innovations have been responsible for the transition of Raman spectroscopy from the research lab to the real world. These advances are listed here and discussed in detail in subsequent chapters. 

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