Introduction to Raman SpectroscopyNew Hire Sales Training

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What is Raman Spectroscopy?A Versatile Vibrational Spectroscopy TechniqueApplicable toOrganics and inorganicsSolids, liquids, and occasionally gasesMicro and macro samplingQualitative and quantitative analysis

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Raman Effect

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Raman SpectraSample excitation yields an entire Raman vibrational spectrum

Excitation Frequency/ Rayleigh ScatteringStokes Raman ScatteringAnti-Stokes Raman Scatteringcm-13500Frequency of Vibrational Transitions03500

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Measurement Wavelengths400nm80010001400VisibleNear Infrared

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Shifted Raman Spectra400nm80010001400VisibleNear InfraredAnti-Stokes Raman Spectrum03500

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Conceptual Raman Spectrometer

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How Raman Spectroscopy is PracticedReflection TechniqueSample geometry not importantSample surface exposed to sampleSize of sample can be as small as 1 mApplicable SamplesSolidsLiquidsOccasionally gasesusually need to be pressurized

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Advantages Offered by RamanComplementary to IRBoth techniques provide information-rich spectraWeak IR absorbers often strong Raman emittersRaman provides easy access to FIR vibrationsTypical spectrum extends to 100 cm-1Great for analysis of organics and inorganicsRaman bands typically sharper than IROften more suited to interpretationUseful for conformational analysesDrug polymorphsMaterial stress

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Advantages Offered by RamanSample preparation usually trivialSamples analyzed neatAnalysis possible through glass and plastic packagingUseful for analysis of aqueous solutionsWater bands weakerRemote sampling with fiber-opticsCommon quartz fibers can be usedFibers can be stretched 100s of metersUseful for microscopyHigh spatial resolution possibleless than 1 m with visible excitationDepth profiling possible with confocal optics

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Vibrational Techniques of ChoiceWhy doesnt everyone use Raman all the time?At one point they didHistorical Raman use1928 Raman discovered by C.V. RamanRaman developed considerable popularity during the 1930sBy 1939 Raman had become a principle analysis technique1945 more sensitive IR detectors had been developedIR becomes relatively inexpensive and uncomplicatedIR gradually eclipses Raman as the vibrational technique of choice

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Current State of Raman SpectroscopySignificant developments made a dramatic impact on Raman1965 Laser recognized as ideal light source1986 CCD arrays available for Raman useRecent developmentsRapid development in solid state and diode lasersRapid development in optical filtersRaman currently reemerging as the technique of choice in some areas New generation of highly automated Raman instruments appearing todayServing as very productive investigative tools

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Drawbacks of RamanRaman spectroscopy still has some drawbacksMore expensive than IRIR most cost effective for routine samplingFluorescenceSerious obstacle to collecting Raman with some samplesPotential of laser damageSome samples very sensitive to laser energy

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FluorescenceFluorescenceBroad band emissionsStokes emissions (lower energy than excitation energy)Results from excitation to higher electronic states followed very quickly by decay to lower electronic states producing emissionConsiderably more intense than Raman emissionsCan completely obscure Raman emissions in some cases

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Fluorescence AvoidanceSpectral correction after collectionSeveral specialized baseline correction algorithms existNot viable in cases where fluorescence saturates detectorConfocal opticsCan work well when the source of fluorescence is the substrate rather than the sampleExcitation laser changeMost reliable means of avoiding fluorescenceSwitch to an excitation frequency that does not stimulate fluorescence in the sampleTypically this means switching to longer (NIR) wavelengthsFT-Raman operating with 1064 excitation rarely exhibits fluorescence

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Fluorescence AvoidanceDispersive Raman, 532 nm laserDispersive Raman, 785 nm laserFT-Raman, 1064 nm laser 500 1000 1500 2000 2500 3000 3500 Example of fluorescing pharmaceutical

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Accuracy of Raman IntensityRaman is an emission techniqueEmission measurements are absolute measurements IR is an absorbance techniqueAbsorbance is relative measurement insensitive to instrumentAlways ratioed to background reference

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Accuracy of Raman IntensityEmission measurements start with raw emissions from sampleIntensity is further attenuated by wavelength dependence ofDetector responseOptical throughputIntensity differs between excitation lasers and optical designs

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Accuracy of Raman IntensityRaman spectra of the same sample collected under different conditions can be quite different in appearance

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White Light CorrectionIntensity differences can be corrected forWhite light correction is an intensity normalization procedureUtilize a white light black body standard to develop a wavelength dependent scaling factor that can be applied to correct intensityNIST also has several standards available with emission characterized at specific excitation frequencies.Important when comparing to common reference spectra

500 1000 1500 2000 2500 3000 Raman shift (cm-1) 500 1000 1500 2000 2500 3000 Raman shift (cm-1)785 nm laser785 nm laser white light corrected

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White Light CorrectionComparison of white light corrected spectra

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Selecting Between IR and RamanFactors to considerSensitivity differences with some compoundsDifferences in sample preparationInterferences from waterAre low frequency vibrations significant?Is microscopy important?

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Sensitivity DifferencesCompounds for which Raman offers increased sensitivityWeak IR Absorbers often strong Raman emittersSymmetric bonds represented more (S-S, C-C, etc.)Molecular backbone emphasized moreEnd groups de-emphasizedSpectral range offers more information on inorganics

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Lack of Sample PreparationSample prep advantages obtained becauseRaman emission is weakSamples can be analyzed neatMeasurement typically performed with visible or NIR wavelengthsSample through glass and plastic packagingUtilize remote fiber-optic sampling easilySaves time and can be important to the analysisExample: pharmaceutical polymorph analysis

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Interference from WaterLess interference from liquid waterMore applicable to solution studiesWater still emits and can still be a significant interferenceWater vapor usually insignificantAllows humidity cells for environmental studies

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Access to Low Frequency VibrationsAccess to low frequencies is routine with RamanGenerally limited only by Rayleigh rejection mechanismFilters allow access to 100 cm-1 or lowerOften can be tuned to 50 cm-1Atmospheric water vapor not a concern48.7581.56150.67217.78244.61 50 100 150 200 250 300 Raman shift (cm-1) Sulfur spectrum collected on Almega XR with 532 nm laser

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Raman MicroscopyTypically performed with visible wavelengthsDiffraction limit is much smaller than IRTypical limit for IR instrumentation is 10 mRaman can typically get to 1 m or smallerProvides higher spatial resolutionDepth profiling possible with confocal opticsUsually little absorbance of Raman frequenciesto within 2 mDetectorObjectiveSampleAdjust z-axiswith scope focus knob

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Raman Microscopy - MappingEffervescent cold medicine tabletLarge area captured with video mosaic - spliced video imagesCollected on Almega XR with 785 nm LaserInvestigate the distribution of active and inactive ingredientsPain relieverAntacidDye350 Microns350 Microns

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Raman Microscopy - MappingMultiple compounds identified by library search and mapped using spectral correlation

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Emission TechniqueEmission technique means no reference necessary for RamanIR requires a referenceNo reference needed for RamanGood Raman spectra can be collected on nearly any substratemetalglasspaperpolymerssilicon

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Reflectance TechniqueReflectance requires minimal sample preparationIR reflectance capabilitiesDirect Reflectance limited to thin samples on metal substratesATR more versatile but still limitedCan not access very small samples in crevicesRequires good sample contactRequires physical contact with the sampleRaman collected as direct reflectanceNo sample contactCan sample deep in crevicesMicrowell plate sampling is routine

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Performance of Raman Systems - SNRHighly dependent on how you are samplingSignal-to-Noise Ratios (SNR) a function ofEnergy on sampleLaser power density at sample depends onlaser powerhow tightly focused it isDetector sensitivity range (CCD, Ge, InGaAs)Raman emission strengthConfocal aperturesTrade off between SNR and resolutionLarger apertures let in more energy but also more stray lightThroughput of optics

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Raman DetectorsDetector Sensitivity RangeSi CCD most sensitiveOnly sensitive in visible rangeBest response typically with 532 nm and 633 nm lasersGe and InGaAs sensitive in NIRGe more sensitive but Liquid N2 cooled

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Intensity of Raman EmissionsRaman emission is excitation wavelength dependentStronger emissions with shorter excitation wavelengthsRaman emission proportional to (1/)4Intensity of Raman Emissions relative to 1064 nm excitationat 780 nm - 3.4X strongerat 633 nm - 8.0X strongerat 532 nm - 16.0X strongerat 473 nm - 25.6X strongerActual measurements further attenuated by wavelength dependent collection efficiencyTheoretical emission gains often not fully realized

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Intensity of Raman Emissions 0 to +3300 cm-1 ranges for selected Raman lasers 200 400 600 800 1000 1200 1400 1600 1800 Wavelength (nm) 244 266 325 458 473 488 514 532 633 780/785 830 1064 Relative Emissions strength and Stokes spectral range with some common Raman Lasers

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Selection of Excitation LasersFactors to consider in excitation laser selectionFluorescenceOrganic fluorescence commonly between 400 and 800 nmGlass fluorescence can be significant between 850 and 1000 nmVisible CCD performance falls off below 500 nmGlass optics transmission falls off below 350 nmHigher spatial resolution obtainable with shorter wavelengthsDetector technology creates a division at border of visible and NIRSi CCDs and dispersive technology used in visibleOffer higher sensitivity but more susceptible to fluorescenceGe and InGaAs detectors and FT technology used in NIRSensitivity less, but nearly always fluorescence free

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Selection of Excitation Lasers

Special Regions of Interest

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Wavelength (nm)

Arbitrary Scale

Resonance Raman

Fluorescent Organics

Glass Fluorescence

Water Absorbance

Glass Optics

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Dispersive vs. FT TechnologySelection dictated by detector behaviorInGaAs and Ge detectors limited by 1/f noiseBenefit from having more frequencies/energy on the detectorFT measures all frequencies simultaneouslySi CCD detectors limited by combination of dark current and quantum efficiencyNo benefit provided by measuring frequencies simultaneouslyDispersive technology discriminates frequencies before detection

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Dispersive TechnologyDispersive systems utilize a grating to break light into component frequencies before it arrives at the detector

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FT TechnologyFourier Transform (FT) technology obtains frequency discrimination by introducing an interference pattern with an interferometer which allows individual frequencies to be differentiated after detection with the Fourier Transform. All frequencies are measured by the detector simultaneously.

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Dispersive vs. FT TechnologyFT-RamanInterferometerNear Infrared laserDetector is InGaAs (room temperature) or Germanium (LN2 Cooled)Dispersive Raman Grating spectrograph Visible lasersSilicon CCD array detector

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Spectral RangeHigh cm-1 spectral range typically limited by detector cutoffSi CCDs limited to 1050 nmAt 830 nm excitation this is 2500 cm-1 StokesAt 780 nm excitation this is 3300 cm-1 StokesAt 633 nm excitation this is 6275 cm-1 StokesBands above 4000 cm-1 are almost exclusively overtone and combination modes with very weak Raman emissionsAntiStokes high end limited by distribution of excited state moleculesGenerally this only goes a few hundred cm-1Low cm-1 spectral range limited by Rayleigh blocking mechanismUsually between 100 cm-1 and 50 cm-1 for filtersPremonochrometers can extend this limit

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ResolutionSpectral resolution limited byWavelength discrimination mechanismDispersiveGrating resolutionCCD pixel spacingFocal length of spectrographFTOptical retardation introduced by interferometerExcitation laser line widthMost Raman lasers have line width approaching 1 cm-1Defines the effective limit for Raman spectroscopy at 1 cm-1 for all but the most specialized applications

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Quantitative RamanRaman emission proportional to concentrationRequires a calibration set specific to the analysis and experimental conditionsApplicable for use with a number of quantitative algorithms Beers LawCLSMLRPLS

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Example of Quantitative RamanFT-Raman spectra collected directly through gelcap wallsCalibration using known concentrations of active ingredientAdditional known samples collected for method validation

Gel-Caps with Ketoprofen active ingredientPure Drug spectrum

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Example of Quantitative Raman024680246810CalibrationPredictionR = 0.9993RMSEC = 0.0310SMLR at 999 cm-1Actual % KetoprofenPredicted % Ketoprofen

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SummaryRaman spectroscopy is a powerful tool for a wide range of chemical analyses.Raman offers a number of benefits over other vibrational techniques with specific samplesMost labs with an investigative focus can benefit from Raman technology

So how do you select the laser that is right for you?We will go through this in a lot more detail in the Almega XR talk later, but for now, just decide vs.. visible and NIRA more thorough explanation of the two technologies will follow. This description is being presented at this time merely to support the discussion of how to select an excitation frequency.This is quantitative analysis study of an active ingredient in real formulations of an anti-inflammatory medication. Spectra where acquired directly through the gel-cap dosage form. We were given a set of gel-caps that contained known concentration of the active for calibration, and another set of gel-caps with known concentration for verifying the method.

This is the results of the actual concentration in the validation samples vs the concentration predicted by the calibration model. Agreement is excellent. Also note the low end concentrations, single percent levels shows the sensitivity of the technique.