Slide 1

Copyright @ B&W TEK Overview of Raman Spectroscopy and B&W Tek Portable Instruments and Applications August 2013 1

Slide 2

Copyright @ B&W TEK Outline Raman Spectroscopy Positioning of Portable Raman vs NanoRam Instrumentation for B&W Tek Portable Raman Systems Portable Raman Systems Sampling and Accessories Raman Software: BWID, BWSpec and BWIQ Raman Applications Hands-on use of iRaman Plus and iRaman EX Lunch 2

Slide 3

Copyright @ B&W TEK RAMAN SPECTROSCOPY 3

Slide 4

Copyright @ B&W TEK www.bwtek.com4

Slide 5

Copyright @ B&W TEK What is Raman Spectroscopy ? Raman spectroscopy is a form of molecular spectroscopy the scattering of electromagnetic radiation by atoms or molecules. The Raman signal is an invaluable tool for molecular fingerprinting. Advantages of Raman Spectroscopy Little to no sample preparation required Perform analysis directly through transparent containers (i.e. plastic bags, glass, etc.) Enables both qualitative and quantitative analysis Highly selective Fast analysis times Insensitive to aqueous absorption bands KEY: Sensitivity, S/N, performance/cost, reproducibility, qualitative/quantitative 5

Slide 6

Copyright @ B&W TEK Raleigh Unchanged Elastic Scattering Raman Inelastic Scattering Laser 785 nm Excitation 1,000,000 photon vs.1 Raman Scattering 6

Slide 7

Copyright @ B&W TEK Raman Spectrum www.bwtek.com7 (CC) Aliphatic Ring (CC) Aromatic Ring C=C A Raman spectrum is a plot of the intensity of Raman scattered radiation as a function of its frequency difference from the incident radiation (usually in units of wavenumbers, cm -1 ). This difference is called the Raman shift.

Slide 8

Copyright @ B&W TEK Raman Spectral Information 500 1000 1500 2000 2500 3000 Raman Shift (cm -1 ) (CH 3 ) 2 C=O CH 3 CH 2 OH (CH 3 ) 2 S=O CH 3 CH 2 O 2 CCH 3 C 6 H 5 CH 3 C=O CH 3 Aromatic-H S=O CH 3 CH 2 C-O CH 3 Aromatic www.bwtek.com8

Slide 9

Copyright @ B&W TEK Limitations of Raman Spectroscopy Weak signal (efficiency ~ 10 -8 ) Fluorescence interference 9

Slide 10

Copyright @ B&W TEK Raman Diagram A Raman Instrument consists of a laser, sampling optics (probe), and an optical spectrometer. Because of very weak Raman scattering signals lasers are used as intense excitation sources. 10

Slide 11

Copyright @ B&W TEK Sampling Fiber optic probes can also be easily adapted to a variety of different sampling chambers Liquid flow cells Gas flow cells Optical microscopes Direct sampling accessories allow for extreme portability and improve collection efficiency. Point & Shoot Vial Holder Large Bottle Sampler 11

Slide 12

Copyright @ B&W TEK 12 Excitation Wavelengths 532nm 785nm 1064nm Sesame Seed Oil

Slide 13

Copyright @ B&W TEK What can Raman do for you? www.bwtek.com 13 Strong Raman Signal Active Pharmaceutical Ingredients Alcohols Antibiotics Antioxidants Buffers Coatings Diluents Emulsifiers Excipients Flavors Fragrances Lubricants Monomers and polymers Polyatomic inorganics Preservatives Solvents Vitamins Weak Raman Signal Materials that are dark in color Highly fluorescing molecules Fillers/binding agents Glass Thin-walled plastics Water No Raman Signal Black materials Metals Mono-atomic ions

Slide 14

Copyright @ B&W TEK Raman can measure through a broad class of packaging BottlesThickness Amber Glass < 2 mm Clear Glass < 3 mm High Density Polyethylene (HDPE) < 1 mm Teflon FEP < 1 mm Polystyrene < 1 mm Vials Amber and Clear Glass < 1 mm Bags Polypropylene (PP) < 0.1mm Polyethylene (PE), Low-Density Polyethylene (LDPE) < 0.1mm 14

Slide 15

Copyright @ B&W TEK PRODUCT POSITIONING 15

Slide 16

Copyright @ B&W TEK Portable Raman Spectrometer Rugged design No moving parts for reliability Small size for on site analysis Light weight Fiber probe for easy sampling Battery option High performance to cost ratio 16

Slide 17

Copyright @ B&W TEK Raman Spectrometer Product Line www.bwtek.com17

Slide 18

Copyright @ B&W TEK www.bwtek.com Summary Product Positioning Compact, handheld, highly portable Portable computer required Rapid material identification in a regulated or non- regulated environment Laboratory analysis for identification, and general research applications including quantitative analysis Samples that fluoresce, such as highly colored samples, natural products, and samples with fluorescing impurities Easy operation and direct results - nonexpert users Flexible system that can be used by nonexpert users, in QA/QC labs and research applications Fully integrated all-in-one measurement and analysis tool in touchscreen GUI Flexibility in setting measurement parameters and in data analysis options 18

Slide 19

Copyright @ B&W TEK Target for Handheld and Portable Raman NanoRam is purpose-build for ID testing Portable Raman instruments are versatile for use in research and development applications The iRaman series is available with different laser excitation to suit particular applications and needs BWSpec and BWID software used for the data collection Quantitative analysis can be done with BWIQ Use of flexible fiber optic probe makes interfacing to different types of samples in lab, process, and field possible www.bwtek.com19

Slide 20

Copyright @ B&W TEK Similar features: Handheld and Portable Use class 3b laser source and carry warning labels; fully comply with laser safety requirements of 21CFR 1040.10 ANSI standard safe distance same as NanoRam ~ 30 cm Have flexibility of different sampling accessories Adjustable laser power settings 21 CFR11 compliant software available Data can be exported and further explored and analyzed 20

Slide 21

Copyright @ B&W TEK Some key differences: Software NanoRam Touchscreen user interface, with methods and libraries is for qualitative analysis Closed software system Full audit trail, login with different user levels comply with regulatory needs, and work well in manufacturing and QC environment Data management is with NID software; Portables Only BWID Pharma is compliant for regulatory and can be used for material release decision BWSpec software interface suited for laboratory use and analysts In depth data analysis requires software beyond BWSpec such as BWIQ Supports customer software development via SDKs www.bwtek.com21

Slide 22

Copyright @ B&W TEK Some key differences: Hardware NanoRam Integrated barcode reader Non fiber-based sampling Battery operated Dust and splash proof (IP64) Portables External bar code reader option Fiber-based sampling External rechargeable battery option Not IP rated- more suitable for lab environment Can be packaged in NEMA enclosure for process use www.bwtek.com22

Slide 23

Copyright @ B&W TEK Operational differences between portable and NanoRam NanoRam Little need to set measurement settings Intensity correction automatically applied Soft key for laser on/off Rapid analysis results displayed in GUI Purpose built for Identification and Verification i-Raman Series Set integration time, laser power, averaging Manually enable intensity correction Physical laser on/off key Data or results rapidly displayed depending on software used Quantitative analysis options with BWIQ 23

Slide 24

Copyright @ B&W TEK PORTABLE RAMAN INSTRUMENTATION 24

Slide 25

Copyright @ B&W TEK Key Instrumentation for Portable Raman Stabilized laser: 532nm, 785nm, 1064nm Multi-element CCD (Charge Coupled Device) array High Rayleigh rejection fiber optic probe Quiet detection system Software: BWID, BWSpec, BWIQ 25

Slide 26

Copyright @ B&W TEK Relative Intensity Correction In Raman spectroscopy, intensity correction is applied to correct relative intensities against a traceable standard for individual spectrometers For systems with 785 nm excitation use SRM2241 Enable the correction in the software using a factory- generated correction file (Ratio3 file) 26

Slide 27

Copyright @ B&W TEK Permanent Wavelength Calibration NIST traceable wavelength calibration ensuring Raman shift accuracy with standards- Tylenol and cyclohexane Follows ASTM E1840-96(2007) 27

Slide 28

Copyright @ B&W TEK i-RAMAN PLUS www.bwtek.com28

Slide 29

Copyright @ B&W TEK i-Raman Plus Positioning www.bwtek.com29 Research applications Quantitative analysis Final product inspection Counterfeit detection PAT process applications proof of concept and at line applications such as dispensing, mixing, granulation, reaction monitoring, etc.

Slide 30

Copyright @ B&W TEK i-Raman Plus Technical Specifications i-Raman Plus BWS465 DetectorBack-Thinned CCD Array Excitation532 nm785 nm mW< 50mW< 300mW Range / Resolution 532 H: 175 - 3300cm -1 ~ 3.0cm -1 @ 614nm 785 S: 175 - 3200cm -1 ~ 4.5cm-1 @ 912nm Detector Detail-2C TE Cooled Back-Thinned CCD Array Dynamic Range50,000:1 Power 5VDC at 5.5 amp Optional Battery w/ DC only 30

Slide 31

Copyright @ B&W TEK i-Raman Series System setup 31

Slide 32

Copyright @ B&W TEK i-Raman Plus Higher S/N ratio allows for greater specificity of measurement Greater range than any handheld allows you to see more C-H Stretching Comprehensive suite of accessories, software, allow versatility and portability BWIQ allows development of Quantitative Raman methods BWID allows user to develop libraries for Final Product Identification Counterfeit Detection www.bwtek.com32 CH stretching

Slide 33

Copyright @ B&W TEK Signal to noise compared to iRaman Plus 33

Slide 34

Copyright @ B&W TEK i-Raman EX Technical Specifications i-Raman EX BWS485 Detector TE Cooled InGaAs Excitation 1064 nm mW 450 mW max Laser Power Control 0 to 100% Range / Resolution 175 - 2500cm -1 ~ 9.5cm -1 @ 1296nm Dynamic Range 25,000:1 Digitization Resolution 16 bit or 65,535:1 Integration Time 200 s to >20 minutes DC Power 12V DC at 6.6 Amps Battery Optional 34

Slide 35

Copyright @ B&W TEK i-Raman EX Material fluorescence reduces Raman applicability on a small subset of materials especially those that are highly colored Utilize 1064 nm excitation Reduce fluorescence Increase productivity vs other techniques Research grade instrument provides enhanced material characterization opportunities www.bwtek.com35

Slide 36

Copyright @ B&W TEK i-Raman EX Comparative spectra for an Alka Seltzer tablet 36

Slide 37

Copyright @ B&W TEK RAMAN ACCESSORIES 37

Slide 38

Copyright @ B&W TEK Sampling Fiber optic probes can also be easily adapted to a variety of different sampling chambers Liquid flow cells Gas flow cells Optical microscopes Variety of sampling accessories allows for portability and improved collection efficiency. Industrial probe option for process applications 38

Slide 39

Copyright @ B&W TEK Fiber Optic Probe Standoff or direct contact with sample Excitation fiber is filtered to prevent interference from silicas Raman signal Collection fiber is filtered to remove wavelength of the excitation laser Fibers decrease the throughput of a system 39

Slide 40

Copyright @ B&W TEK B&W Tek Fiber Optic Probes Lab grade standard Industrial grade probe Standoff or direct contact with sample Sample form: solid, liquid, gas, powder Industrial immersive version: for process- type applications 40

Slide 41

Copyright @ B&W TEK Raman Probe Tip Black distance regulator is for direct measurement of solid samples Remove distance regulator if going through vial or quartz cuvette Some samples may burn, depositing soot on probe tip Clean probe tip with lint free cloth wetted with isopropyl alcohol 41

Slide 42

Copyright @ B&W TEK Cleaning & Care of Fibers End caps should be placed on fibers when not in use Leave the fiber probe connected to minimize the risk of contamination of the fiber ends Do not bend or coil fibers too tightly Clean fiber ends before connecting to ports for best results Cleaning is especially critical for Raman probes Any debris will be burned onto fiber tip Fiber must be re-polished to fix problem 42

Slide 43

Copyright @ B&W TEK Portable Video Microscope Sampling System (BAC151A) Compatible with all BWT Raman probes Precise target and focusing Good for precise measurement of small sample volume Digital camera and LED illuminator Coarse and fine XYZ adjustment Standard objectives from 10x to 100x Bright and dark field illumination for various sample surfaces Low cost and portability 43

Slide 44

Copyright @ B&W TEK B&W Tek Raman Probe Holder (BAC150) Compatible with BWT Raman probes Coarse and fine XYZ adjustment Z-axis adjustment allows for laser focusing on the desired plane 44

Slide 45

Copyright @ B&W TEK B&W Tek Enhanced Raman Cuvette Holder (BAC100A) Precise focusing Three-point locking mechanism Increase Raman signal up to 3 times in comparison to a standard cuvette holder Standard 10x10 mm (inside dimension) cuvette Liquid or powder sample 45

Slide 46

Copyright @ B&W TEK B&W Tek Liquid Sample Flow Cell Compatible with BWT Raman probes On-line process monitoring Kalrez O-ring, chemical resistant Offers high throughput and stability Quartz or sapphire window options 46

Slide 47

Copyright @ B&W TEK IQ/OQ available for i-Raman series OQ includes the performance qualification on installation BWID software has function for performance test Successfully executed at global pharmaceutical companies 47

Slide 48

Copyright @ B&W TEK RAMAN SOFTWARE 48

Slide 49

Copyright @ B&W TEK Chemometric (Spectral) Analysis 49 The use of mathematical and statistical methods for analysis of chemical or spectral data Correlation analysis for material identification and/or verification (Qualitative) Multivariate analysis and complex systems with large data sets (Quantitative / Qualitative) Uncover underlying trends in the data and detect outliers, Develop models based on data similarity for classification Develop quantitative models to predict response in unknown data sets Allows for real time monitoring and control

Slide 50

Copyright @ B&W TEK B&W Tek Software BWID Pharma and BWID Standard: Qualitative analysis by library searching BWID Pharma developed for use in regulated environment BWSpec: general purpose spectrometer software for data acquisition and basic data manipulation and viewing BWIQ: chemometric software for quantitative analysis classification and prediction www.bwtek.com50

Slide 51

Copyright @ B&W TEK www.bwtek.com51 Spectral Analysis Examples Correlation Based Library Searching for Unknown Material Identification using HQI PLS Regression Modeling for the Development of concentration curve for quantitative prediction. Method Development for verification of known materials using multivariate classification modeling to determine p-value. Measure 20 representative spectra Build method based on PCA and establish threshold p-value (typically 0.05) Test Method PC1 PC2 PC1 PC2

Slide 52

Copyright @ B&W TEK BWID Pharma Software 21 CFR Part 11 compliant software with electronic records and signatures Operator/Developer/Adminstrator users User-defined and commercial third-party libraries can be used One click Identification or Verification of sample spectra Reporting functions for analysis and instrument diagnostics www.bwtek.com52

Slide 53

Copyright @ B&W TEK BWID - PHARMA User Levels Operator: Run sample identification Perform diagnostic tests Print results for recently performed ID analysis and performance test Electronically sign an ID analysis record Change his/her own password Developer: Create/modify a data library Setup an operation preset method Configure performance test parameters Configure ID analysis report Select, view, and print a report for any ID analysis and performance test Electronically sign and approve an ID analysis record Configure graphical user interface (GUI) settings Configure licensed instruments Administrator: Manage user accounts: View user accounts Add/lock/unlock/reset/dis able a user account Modify user account properties Backup/restore user accounts file Configure access policy 53

Slide 54

Copyright @ B&W TEK Spectrum Search: Identification Results immediately when run sample or on stored data MATCH/NO MATCH results Ability to add new spectrum to library after search www.bwtek.com54

Slide 55

Copyright @ B&W TEK Spectrum Search: Verification Results immediately when run sample or on stored data To verify must choose product name PASS/FAIL results Ability to add new spectrum to library after search www.bwtek.com55

Slide 56

Copyright @ B&W TEK BWID Audit trail www.bwtek.com56

Slide 57

Copyright @ B&W TEK Instrument Diagnostics Performance test interface and tracking of results Performance tests include: Test of USB instrument connection and dark scan Noise test Peak position using standard www.bwtek.com57

Slide 58

Copyright @ B&W TEK e-signature Signature for review, approval, rejection available based on user level e-signature added to printed report 58

Slide 59

Copyright @ B&W TEK BWSpec General purpose spectrometer software www.bwtek.com59

Slide 60

Copyright @ B&W TEK BWSpec Software Interface 60

Slide 61

Copyright @ B&W TEK BWIQ TM : Chemometric Software Classification and Regression Range of methods and computational algorithms that provide accuracy and speed with reduced memory requirements State-of-the-art pretreatment methods for spectral smoothing and baseline correction Enhanced multivariable methods Vector processing for non-linear datasets 61

Slide 62

Copyright @ B&W TEK BWIQ: DEG in glycerin B&W Tek i-Raman Plus, 785nm Laser 16 samples with diethylene glycol (DEG) in glycerin DEG concentration from 0 48.3% Integration time 20 second, 300 mW laser power www.bwtek.com 62

Slide 63

Copyright @ B&W TEK RAMAN APPLICATIONS www.bwtek.com63

Slide 64

Copyright @ B&W TEK Portable Raman Applications Product contamination (i.e. alcohol, DEG in glycerin) Counterfeit drug identification Forensic analysis Food and agriculture industry applications Process Analytical Technology (PAT) QC: 100% incoming raw material identification Environmental analysis Geology and Gemology Minerals and rocks analysis Gemstone identification and examination Art and Archaeology Biomedical Analysis www.bwtek.com64

Slide 65

Copyright @ B&W TEK Applications in the Pharmaceutical Industry Identification Verification of the identity of incoming raw materials. Identification and analysis of counterfeit drug products. Research Polymorph screening; material identification; structural elucidation; Process Analysis Real time quantitative analysis for process analytical technology (PAT) such as blending, titration, reaction monitoring and polymorphic transition monitoring. 65

Slide 66

Copyright @ B&W TEK Pharmaceutical manufacturing facilities are moving toward 100% inspection of incoming raw materials, to support cGMP and PIC/S guidelines. Raman is ideal because 85 - 90% of common pharmaceutical ingredients are Raman active. Additionally Raman can measure through common packaging materials such as glass and plastic. Handheld Raman spectrometers are commonly used to provide on the spot "Pass/Fail" decision to verify the identity of incoming raw materials. Verification of Incoming Raw Materials 66

Slide 67

Copyright @ B&W TEK Polymorphic screening by Raman Mannitol has at least three polymorphic forms: alpha, beta, and delta. The beta form is considered to be the most stable The vapor-induced transition from the less stable form to form can be monitored by Raman spectroscopy and with dynamic vapor sorption 67 Raman spectra taken at 2-hour intervals for D-mannitol transition to form DVS Case Study 615: Majid Naderi, Jiyi Khoo, Manaswini Acharya and Dan Burnett, Surface Measurement Systems Ltd.

Slide 68

Copyright @ B&W TEK According to the World Health Organizations estimates, ~10-15% of the worlds drug supply (and about 1% in the US) is counterfeit - at a value of about $200B in 2010 Raman Spectroscopy is currently being use for not only identification of counterfeit drug products but also to analyze the quality and purity Counterfeit Drug Identification 68 Best 785 www.bwtek.com For example the FDA is currently using portable Raman spectrometers for the identification of glycerin contaminated with DEG.

Slide 69

Copyright @ B&W TEK Applications in Forensic Analysis Nondestructive Narcotic Drug identification Explosives Identification: Exact Chemical Compositions of Material (i.e. PETN, RDX) Binding Agents Within Explosive Materials Identification and Analysis of Toxic Solvents and Chemical and Bio-warfare Agents Trace Forensic Evidence Analysis: Including fibers, fabrics, pigments, inks, paint chips, etc. 69

Slide 70

Copyright @ B&W TEK Illicit Drug Analysis Portable Raman spectrometers are commonly used for the Identification of a frequently encountered illicit street drug. In the example to the right we are showing the spectrum of a confiscated white powder which was analyzed using correlation analysis against a spectral library of common illicit drugs and positively identified at cocaine with a HQI of 99.13. www.bwtek.com70

Slide 71

Copyright @ B&W TEK Identification of Explosives Portable Raman spectrometers are also well suited for the identification of explosives and hazardous materials. Typically surface enhanced Raman spectroscopy (SERS) is utilized for this application because it allows for the detection of trace levels of explosives. For example in a recent publication it was shown that PETN could be detected at concentrations as low as 5 pg. SERS Spectra of PETN 0.2 g 200 pg 5 pg SEM image of SERS substrate used in the measurement. S. Botti et al., J. Raman Spectrosc. 2013, 44, 463468 71

Slide 72

Copyright @ B&W TEK Product Contamination Methanol-Laced Spirits Over the past several years an alarming trend has become evident that there are serious issues with contaminated alcohol within the EU, and in particular Eastern Europe. Studies have shown that the maximum tolerable concentration of methanol in alcoholic beverages with about 40% alcohol is about 2% (v/v) by volume. In September of 2012 when the Czech Republic banned the sale of hard liquor after 20 people died from the consumption of methanol-laced spirits. After an exhaustive study of different screening tools the Czech Republic turned to the use of portable Raman spectroscopy as the screening tool of choice for the identification and quantification of methanol in contaminated spirits. www.bwtek.com72

Slide 73

Copyright @ B&W TEK Example using Methanol-Laced Coconut Rum CH 3 bending vibration at 1013 cm -1 increases with methanol concentration. A PLS regression method can be developed to readily measure concentration of MeOH in alcoholic beverage by portable Raman spectroscopy www.bwtek.com73

Slide 74

Copyright @ B&W TEK Applications in Art & Archeology 74 Portable Raman spectroscopy is widely used for the analysis of paintings, ceramics, statues (surface coatings), and other artifacts. The flexibility of fiber optics in conjunction with the non-destructive & non-contact nature of Raman allows measurements to be taken in-situ. This application has become so popular that in 2001 a biennial international conference was formed solely dedicated to the subject of the use of Raman spectroscopy in art and archeology.

Slide 75

Copyright @ B&W TEK 75 Portable Raman Microscopy of Ancient Pigments Courtesy of M.J. Ayora Caada y A. Dominguez Universidad de Jan Lead oxide (Pb 3 O 4 ) Cinnabar (HgS) Analysis of pigments on the ceiling of a cathedral in Spain using a portable Raman spectrometer connected to a tripod-mounted video microscope for precision alignment.

Slide 76

Copyright @ B&W TEK Analysis of metal corrosion Raman spectroscopy was used at the Guggenheim Museum in Bilbao Spain to analyze environmental effects on the corrosion of seven steel sculptures from Richard Serra. The figures below show the presence of lepidocrocite, magnetite, and iron sulfate from Richard Serras Inverse Blind Point sculpture, as well as a SEM image of the surface confirming the results of the Raman analysis K. Castro et al., COST Action D42 Extended Abstract. 2011, 113116 76

Slide 77

Copyright @ B&W TEK Applications in Geology and Mineralogy Portable Raman spectrometers are ideal for the identification of gemstones and minerals, including polymorphs and isomorphs. Non-contact, non-destructive sampling allows for analysis of precious or scarce samples, unlike other techniques such as LIBS. Anti-counterfeiting of precious, such as identification of diamond from zircon Images Courtesy of Prof. Rull University of Valladolid 77

Slide 78

Copyright @ B&W TEK Raman evaluated for Geological survey on Mars Before testing for life on other planets, feasibility studies are done on barren areas of the Earth. One such place is Rio Tinto in Spain, where conditions are analogous to Mars, where portable Raman spectrometers were evaluated for the joint NASA/EAS 2018 Mars rover mission. www.bwtek.com78 Images Courtesy of Prof. Rull University of Valladolid

Slide 79

Copyright @ B&W TEK Analysis of Garnet Gemstones Garnets are a class of silicate minerals which include a number of varieties with the general form X 3 Y 2 (SiO 4 ) 3. Raman spectroscopys high selectivity allows for the differentiation of the different garnet varieties. Andradite and grossular fall into the ugrandite group of garnets (calcium in X site), while spessartine falls into the pyralspite group (aluminum in Y site). 79

Slide 80

Copyright @ B&W TEK Diamond or Zircon? Raman spectra of diamond and zircon are distinctly different Diamond shows only one very strong and sharp Raman band at around 1328 cm -1, which corresponds to the C-C stretching mode. Zircon shows multiple Raman bands at around 349, 431, 967 and 1002 cm -1, which correspond to the Si-O bending mode and stretching mode. www.bwtek.com80

Slide 81

Copyright @ B&W TEK The use of rapid off-line, at-line, on-line or in-line analyzers to obtain analytical data giving the following benefits: Faster (real-time or near real-time) Data collected more often Higher precision data Provides the pulse of a process One example is Raman spectral change during HSWG (High Shear Wet Granulation) polymorphic transformation of a drug substance. Raman bands for Polymorph form B appear and for form A disappear during the process Process Analytical Technology www.bwtek.com81

Slide 82

Copyright @ B&W TEK 5 mW 375 nm 30 s Epoxy Cure Monitoring www.bwtek.com82

Slide 83

Copyright @ B&W TEK TiO 2 H 2 SO 4 H 2 O TiO 2 H 2 SO 4 TiO 2 (nanophase) TiO 2 (anatase) TiO 2 (rutile) Process Application: TiO 2 phase transitions www.bwtek.com83

Slide 84

Copyright @ B&W TEK Polymer Reaction Monitoring www.bwtek.com84 C=C Breathing mode (for normalisation) Follow the conversion of the vinyl band styrene -> polystyrene Henryk Herman, Ph.D. Gnosys Global

Slide 85

Copyright @ B&W TEK Food & Agriculture Industry Measuring chain length and extent of saturation of fatty acids in edible oils Meat product quality analysis Product contamination SERS analysis of food contaminants including bacteria, antibiotics, dyes, etc. Analysis of components in grain kernels Raw material identification/verification for the food and beverage industries 85

Slide 86

Copyright @ B&W TEK Contamination of extra virgin olive oils To improve profit margins there are many instances of the adulteration of extra virgin olive oil (EVOO) with light olive oil or other cheaper ingredients A feasibility study was performed with 8 samples spiked with 0.21- 19.15% and measured on the i-Raman EX BWIQ software used to develop a quantitative model for light oil in EVOO www.bwtek.com 86 2 factor PLS model Error ~ 1.6%

Slide 87

Copyright @ B&W TEK Structure and SEM image of a Klarite TM SERS active gold substrate Average SERS spectra (n = 4) acquired from extracts of wheat gluten containing different concentrations of melamine: 2.0% (a), 1.0% (b), 0.5% (c), 0.1% (d), 0% (e). Detection of Melamine in Gluten Using SERS www.bwtek.com87

Slide 88

Copyright @ B&W TEK Raman Analysis of Carbon Nanotubes RBM Radial breathing modes, which probe the lattice structure of the CNT allowing for the calculation of tube diameter. D Band Disorder band, measures the degree of amorphism of the CNT. G Band Tangential Mode, measures the degree of crystallinity (diamond like structure) of the CNT. RBM D G MWCNT SWCNT 88

Slide 89

Copyright @ B&W TEK Applications in the Biomedical Diagnostics www.bwtek.com89 Raman spectroscopy is becoming more pervasive in biomedical diagnostics because of the demand for near real time and minimally invasive analysis. Applications include: biopsies, cytology, drug efficacy studies, histopathology, surgical targets and treatment monitoring. Some of the most active research areas are the analysis of abnormalities in tissue samples such as brain, arteries, breast, bone, cervix, embryonic media; and the identification of biomarkers for early stage detection of various diseases. Raman has also been used to investigate blood disorders such as anemia, leukemia and thalassemias (inherited blood disorder), as well as understanding cell growth in bacteria, phytoplankton, viruses and other micro- organisms.

Slide 90

Copyright @ B&W TEK Assessment of Axillary Lymph Nodes in Breast Cancer with Raman Spectroscopy 90 Images Courtesy of Jonathan Horsnell, Gloucester Royal Hospital Department of Biophotonics Studies by researchers in the UK have shown that Raman spectroscopy can detect differences in tissue composition, and in particular a wide range of cancer pathologies. Using PCA-LDA data processing to evaluate the Raman spectra of lymph nodes they have confirmed that the technique can match the sensitivities and specificities of current histopathological techniques.

Slide 91

Copyright @ B&W TEK Applications in Polymer Science 91 Portable Raman spectroscopy helps to meet needs of the polymer, additive, compounding and masterbatch industries, by allowing for an audit trail to certify the link between the quality of raw materials and finished product. Raman has many applications in both QA and QC including incoming material inspections, measurement of polymer grade, blend ratios, additives, and ageing. It can aid in the optimisation of formulations for desired properties and performance by predicting: Processing properties MFI, liquid viscosity Thermal properties glass transition, melting point Physical properties density, mechanical modulus and strength, impact strength Fire properties UL scores, LOI, flame retardants

Slide 92

Copyright @ B&W TEK Polymer Crystallinity www.bwtek.com92 These bands are related to crystalline and amorphous content, (as well as the interphase) Dr. Henryk Herman - GnoSys Global

Slide 93

Copyright @ B&W TEK Polymer Identification www.bwtek.com93

Slide 94

Copyright @ B&W TEK Environmental Science Water pollution detection using SERS technology Identification of contaminants in water Petrochemical analysis Identification and analysis of sediments in water 94

Slide 95

Copyright @ B&W TEK Environmental Science Calcium Carbonate condensation in water system 95

Slide 96

Copyright @ B&W TEK In Situ Contaminant Study (quoted) Field-portable Raman spectrometer reported under DOE Waste site measurement conducted Spectral database of over 200 contaminants 96

Slide 97

Copyright @ B&W TEK Thank you for your attention www.bwtek.com97 B&W Tek, Inc. 302-368-7824 www.bwtek.com support@bwtek.com

Slide 98

Copyright @ B&W TEK BACK UP SLIDES www.bwtek.com98

Slide 99

Copyright @ B&W TEK Raman Scattering: Independent of laser Vibrations shifts Intensity laser power Raman efficiency10 -8 Ground State Virtual State Electronic States Vibrational States Stokes RamanRayleighFluorescence Anti-Stokes Raman Energy Diagram for Raman Scattering

Slide 100

Copyright @ B&W TEK Quantum Energy Scheme for Photon Scattering 7/28/2011100 The Raman effect comprises a very small fraction (about 1 in 10 7 ) of the incident photons.

Slide 101

Copyright @ B&W TEK Information From Raman Spectroscopy 101

Slide 102

Copyright @ B&W TEK Components of a Raman Spectrometer Laser Narrow Linewidth Small Form Factor Low Power Consumption Extremely Stable Power Output BWN-OEM Glacier T Spectrometer High Resolution Low Noise Small Form Factor Low Power Consumption 102

Slide 103

Copyright @ B&W TEK Importance of Rayleigh Rejection Since the Rayleigh scattering can be up to 10,000,000 times stronger than the Raman scattering, it is imperative that it be filtered out. The quality of the Long-pass filter will determine the wavenumber cut-on of the spectrometer (e.g. 175cm -1 or 65cm -1 ) The anti-Stokes scattering does not provide any additional information (except in thermal studies) so it is also filtered out. 103

Slide 104

Copyright @ B&W TEK CCD Detector Arrays Linear CCD Arrays Single Row of Pixels Mid Light Level Applications Low Cost Low Dynamic Range High Read Noise Mid Readout Speed Back Thinned CCD Multiple Rows of Pixels Low Light Level Applications Higher Cost High Dynamic Range Low Read Noise Slow Readout Speed 104

Slide 105

Copyright @ B&W TEK Stabilized Laser 105 Raman Spectrum Unstable Laser Diode VBG Stabilized Laser Diode Raman Spectrum VBG stabilization results in the correct center wavelength and avoids the phenomenon of mode hopping, this insures that the resultant shift is the same each measurement. It is also essential that the laser has a narrow bandwidth laser since resolution of the Raman peaks is directly correlated to the linewidth of the laser.

Slide 106

Copyright @ B&W TEK Optimized Fiber Optic Probe Design Optimized 6-8 orders Rayleigh rejection 65 cm -1 cut on version Excitation fiber filtered to prevent silicas Raman signal On probe trigger option 106

Slide 107

Copyright @ B&W TEK Raman Pros and Cons Applicable to aqueous solutions Less demanding to optics Easy sampling for solids Weak signal (efficiency 10 -6 ) Fluorescence interference www.bwtek.com107

Slide 108

Copyright @ B&W TEK Mid-IRNIR reflectanceRaman Mechanism: absorption by fundamental molecular vibrational modes Molecular Specificity: high Signal Strength: strong Energy: 600-4000 cm -1 Sampling: direct material contact required, glass and water appear opaque; can rarely see through container materials Hardware: probes expensive and fragile; typically short in length, and inflexible Mechanism: absorption by harmonics of fundamental vibrational modes of X-H bonds (e.g., N-H, O-H) Molecular Specificity: low Signal Strength: weak Energy: 4000-12,500 cm -1 Sampling: non-contact, but close proximity to material required ( 5 hours operation PC Software NanoRam ID Computer Interface USB to Ethernet, Wifi IP Protection Class IP64 - Protection from infiltration of dust and splashing water

Slide 121

Copyright @ B&W TEK Inspection of Raw Materials Pharmaceutical manufacturing facilities are moving toward 100% inspection of incoming raw materials. NanoRam allows rapid confirmation of the content of each container at the molecular level. Raman spectra can be acquired in warehouse through transparent packaging materials, bags and bottles. "Pass/Fail" decision Supports cGMP and 21 CFR Part 11 www.bwtek.com121

Slide 122

Copyright @ B&W TEK NanoRam Incoming non-destructive testing of Raw Materials Reducing Waste in the Dispensing Suite Plastic Packaging Identification At line identification of intermediates Final product identification Substandard, Adulterants, and Counterfeit Deterrent