New Developments in Raman Spectroscopic Analysis
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1 New Developments in Raman Spectroscopic Analysis Mike Kayat B&W Tek, Inc 19 Shea Way Newark, DE United States of America
2 Overview Raman spectroscopy is now an established and evolving scientific method for analyzing molecular compounds and crystalline solids There have been some recent new developments where the Raman technique has been enhanced to increase the inherently low emissions These new approaches include: Coherent anti-stokes Raman spectroscopy (CARS) Surface enhanced Raman spectroscopy (SERS) Surface enhanced resonance Raman spectroscopy (SERRS) Spatially offset Raman spectroscopy (SORS) A review of these new Raman spectroscopy enhancements will be presented, together with several practical application areas, including: Biomedical Forensics Security o Counterfeit goods o Explosives o Narcotics identification. 2
3 Raman Scattering The Raman effect comprises a very small fraction, about 1 in 10 7 of the incident photons. 3
4 Raman Scattering Raman Shift o o Independent of laser output Excitation wavelength is determined by analytical and sample needs (avoid Fluorescence interference) Raman Intensity o o Depends on laser wavelength The larger the excitation wavelengths the lower Raman peak counts (at same power output) 4
5 New Raman Methods Resonance Raman Scattering (RRS) Raman shift is constant UV: resonance enhanced, R < F, photo damage, low penetration Visible: Raman -4, fluorescence overlaps with Raman signal NIR: low fluorescence, deep penetration, Raman -4 Raman scattering No overtones More modes in spectrum No electronic information Weak scattering Resonance Raman scattering Overtones Same modes are selectively enhanced Electronic information Strong scattering 5 5
6 New Raman Methods UV RRS Example RRS response of various carotenoid solutions under 488-nm excitation, shown as a function of increasing concentrations: β-carotene zeaxanthin Lutein lycopene The response is linear up to ~5 μg/ml, which is a carotenoid concentration exceeding that found in human skin Slight variations in the slopes for each carotenoid solution are in very good agreement with their respective excitation efficiencies. RRS responses in the spectral vicinity of the C==C stretching frequency, measured for various antioxidants as a function of concentration in ethanol under identical excitation and detection conditions 6
7 New Raman Methods UV RRS Example System measures carotenoid levels in the skin (β-carotene) Skin carotenoids correlate significantly to serum carotenoid levels The significant correlation between skin biopsy-levels of carotenoids and serum carotenoid levels eliminates the need for routine skin removal Highly significant correlation between serum total carotenoids and skin carotenoids as assessed by Raman Spectroscopy Use UV RRS as a method to assess skin carotenoid status as an indication of broad-spectrum antioxidant status, without the inconvenience of skin and blood samples 7
8 New Raman Methods SERS SERS absorbs molecules onto a rough gold or silver surface and provides 10 3 to 10 7 amplification of the Raman signal In addition, the use of gold or silver in this technique removes any interference from fluorescence for Vis-NIR wavelengths Effects depend on size, morphology, and composition of gold and silver nanoparticles Source: Renishaw P Raman = N Raman.σ Raman.I Laser P SERS = N SERS.σ SERS.I Laser. F Laser 2. F Stokes 2 Occurs due to the presence of a metal surface near the scattering molecule and contains two components; (1) electromagnetic and (2) chemical - electromagnetic enhancements due to the excitation of a surface plasmon which generates a strong, localized, secondary field, that Raman scatters from the molecules together with the incident field 8
9 New Raman Methods SERS Fabrication Colloidal SERS 10 ppm benzenethiol in methanol Raman neat benzenethiol SERS-active plates ~1-mm thick coating of silver (Ag)-doped solgel chemistry on glass bottoms with ~ μl of each analyte in solution was added to individual SERS-active wells for analysis. Can be used for explosives, chemical warfare agents, pharmaceuticals, and biological compounds SERS 100 ppm and 5 wt% herring sperm fragments in water Raman 100 ppm and 5 wt% herring sperm fragments in water 9
10 New Raman Methods SERS Examples 10
11 New Raman Methods SERS Examples 11
12 New Raman Methods Portable SERS-based System Yeast bacteria Substrate is a Si wafer of 5 mm X 5 mm with nanostructures glued on a standard microscope glass slide (3 inch by 1 inch) ppb to ppm detection levels Melamine Trycyclazole 12
13 New Raman Methods Portable SERS-based System Substrate Example detection limits Source: Nanova 13
14 New Raman Methods Portable SERS-based System Portable Analyzer is based on SERS Capable of identifying hazardous materials at the ~ O(100 ppb) Can precisely measure the Raman spectra of materials The analysis algorithms in the embedded computer provide fast identification of samples. Can be used for rapid detection of: o o o o o explosives chemical warfare agents contraband narcotics toxic industrial chemicals food contaminants 14
15 New Raman Methods SERRS SERS and Resonance Raman Spectroscopy (RRS) have been combined to produce surface enhanced resonance Raman scattering (SERRS) This combines the signal enhancement of both RRS and SERS and the SERS's avoidance of fluorescence to produce ultrasharp spectrographs To date, SERRS has proven to be extremely useful in DNA detection 15
16 New Raman Methods SORS Source: Rutherford Appleton Labs, UK Use the diffuse component of scattered light Measure Raman spectra from sublayers 16
17 New Raman Methods SORS Application SORS used for screening of liquids, aerosols and gels (LAGs) Can screen substances inside non-metallic containers such as plastic and glass bottles SORS is typically successful through opaque plastic and colored glass 17
18 New Raman Methods CARS CARS spectroscopy eliminates the effects of fluorescence Combines two high intensity, pulsed lasers beams (pico-second or femto-second) to create a single high energy beam Difference in frequency is the same as the target Raman mode of the sample Raman spectra can be detected over background fluorescence This system also has the advantage of being able to determine a molecule's chirality CARS has a big disadvantage it cannot distinguish between small equally sized molecules Still at early research stage, very expensive Two laser beams with frequencies v1 and v2 (v1 > v2) interact coherently, and because of the wave mixing, produce strong scattered light of frequency 2v1 - v2 If the frequency difference between two lasers v1 - v2 is equal to the frequency vm of a Raman-active mode then a strong light of frequency v1 + vm is emitted. 18
19 New Raman Methods CARS Example CARS has been used for monitoring the temperature of hot gases and flames CARS has been utilized as a method for non-invasive imaging of lipids in biological samples, both in vivo and in vitro 19
20 New Raman Methods Advantages of SERS and SERRS Sensitivity similar or better than fluorescence Selectivity many peaks, minimal interference Simplicity only one excitation wavelength required Multiplexing different labels per well, easy to identify by PCA Stability highly stable reagents, not light sensitive Shelf life & repeatability advances in reusable substrates for specific targeted applications Enable use of handheld Raman instruments for trace identification 20
21 Contact Information Michael Kayat, Ph.D. Vice President B&W Tek, Inc 19 Shea Way Newark, DE USA
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