Quantitative and qualitative analysis of fluorescent substances and binary mixtures by use of shifted excitation Raman difference spectroscopy

Shifted Excitation Raman Difference Spectroscopy (SERDS) implemented with two wavelength-stabilized laser diodes with fixed wavelength separation is discussed as an effective method for dealing with the effects of fluorescence in Raman spectroscopic analysis. In this presentation we discuss the results of both qualitative and quantitative SERDS analysis of a variety of strongly fluorescing samples, including binary liquid mixtures. This application is enabled by the Volume Bragg Grating® (VBG®) technology, which allows manufacturing of compact low-cost high-power laser sources, suitable for extending the SERDS methodology to portable Raman spectrometers. INTRODUCTION Raman spectroscopy has been experiencing a period of growing interest for qualitative and quantitative analysis in a vast scope of applications pertinent to various industries, including pharmaceuticals [1-5], petrochemical [6-8] and law enforcement [9-13]. This growth is enabled by increase in the offering of affordable Raman instrumentation, some is portable and ruggedized for field use. Such increase in availability and portability is powered in turn by compact, highperformance wavelength-stabilized laser diodes. However, with widening of the scope of applications for Raman spectroscopy the challenge of sample fluorescence becomes more and more evident. Besides the natural fluorescence that many substances possess, there are issues of sample contamination with fluorescent compounds in the field that one must deal with in real life situations [9-10]. One example of that is fluorescent agents such as caffeine and flour regularly used cutting illicit street drugs [10]. To combat sample fluorescence laser excitation at longer wavelengths has long been used [14-16]. Indeed, if it were not for sample fluorescence nearly all Raman spectroscopy could be performed with the same short-wavelength laser, especially since blue and violet diode lasers are becoming more and more powerful and affordable. As it is right now, 785 nm and 830 nm laser excitation wavelengths have been an acceptable compromise for substances with weak short-IR fluorescence, and 1064 nm excitation has to be resorted to in order to deal with samples exhibiting stronger short-IR fluorescence. Although the long-wavelength, typically1064 nm, Raman instruments have been considered the benchmark in dealing with fluorescent substances, dealing with that excitation wavelength presents a number of issues. First of all, as is well known, the Raman signal diminishes as the 4 power of laser wavelength, which means that it is ~ 3.4 times weaker with 1064 nm excitation as compared with commonly used 785 nm excitation. Generally it means that significantly longer collection times are necessary to collect as much Raman signal when using 1064 nm laser. Furthermore, the use of 1064 nm excitation laser requires using InGaAs detectors instead of silicon CCDs. In case of dispersive Raman systems this means InGaAs arrays, which have more than an order of magnitude higher thermal noise than that of the silicon CCDs. As a result, InGaAs detector arrays for dispersive Raman instruments must be cooled to achieve satisfactory noise levels. Although cooling the detector is not a problem for laboratory instruments, it presents an obvious issue for portable, battery-operated Raman instrumentation, as it means more power consumption, larger battery and shorter battery life, in addition to increase in cost and complexity of the instrument. For these reasons the use of the Shifted Excitation Raman Difference Spectroscopy (SERDS) approach has long been considered attractive, effectively expanding the use of the conventional dispersive Raman instrumentation equipped with inexpensive and efficient CCD detectors to the classes of samples that exhibit fluorescence. Historically the SERDS method has been used in laboratories employing tunable-wavelength lasers. However, the use of this type of laser sources presents a significant challenge for portable Raman systems. Generally these lasers are much more costly than simple and efficient wavelength-stabilized laser diodes. But in addition to this there is an issue of the exact wavelength control over these lasers required to perform accurate qualitative and especially quantitative Raman analysis, as it is not fixed or stable, which requires constant active wavelength monitoring for accurate analysis. For these reasons we have studied a comparatively much more simple and practical approach to performing SERDS analysis using two affordable wavelength-stabilized laser diodes operating at slightly offset wavelengths. The lasers are stabilized by use of the Volume Bragg Grating (VBG) technology and are very compact, efficient and inexpensive, making them well suited for portable battery-operated Raman instrumentation. The use of such fixed-offset SERDS method is compared here with the conventional Raman analysis employing baseline fitting and also a longwavelength 1064 nm dispersive Raman system. EXPERIMENTAL The SERDS experiments were performed using a dual-laser SERDS laser source, LS-2, produced by PD-LD, Inc. The lasers operated at the 784.5 nm and 785.5 nm wavelengths. The wavelength separation was selected to correspond to the approximate line width of the Raman lines of the substances under study. Note that the exact wavelength separation is not significant for the accuracy and practicality of SERDS, however, wavelength separation that is much smaller than the width of the Raman bands would result in increase in noise in SERDS spectra. The LS-2 laser source delivers the output of either one of the lasers to the output port via a fiber-optic switch. A fiber-optic cable was attached to the output port of the LS-2 and then coupled into a bench-top Raman system. The system delivered the laser light to the sample and then collected the Raman signal in the back-reflection geometry, together with fluorescence and the Rayleigh scattering. The notch filters installed in the optical system suppressed the Rayleigh scattering and the collected signal was delivered to the input port of the spectrometer via the collection fiber in the probe. For the experiments with 785 nm excitation we used a laboratory F/3 bench top spectrometer with 30 cm focal length and 600 groves per millimeter diffraction grating. The spectrometer had and slit and resolution. The detector was a cooled CCD with 1024x256 pixel array from Princeton Instruments. For SERDS analysis the spectra were collected sequentially with laser #1 and then with laser #2. The lasers ran continuously during the course of the experiment to assure best stability of the output power. The wavelengths of the lasers were stable to < 5 pm over the course of the day. The samples under study were either in liquid or solid form. Majority of the samples (usually liquids) were placed in small glass vials and illuminated through the bottom of the vial. Figure 1. Raman spectra of a binary mixture of ethanol and methanol in the presence of large amount of fluorescence from rhodamine 6G dye. The spectra shown in this figure were collected at the two slightly offset laser wavelengths.