Introduction and Fundamentals of Raman Spectroscopy

Introduction and Fundamentals of Raman Spectroscopy
Dr. Katherine A. Bakeev B&W Tek, Inc September 4, 2013 Copyright 2013 B&W Tek, Inc.

Outline Raman Spectroscopy
Instrumentation for B&W Tek Portable Raman Systems Raman Applications Copyright 2013 B&W Tek, Inc.

Over 15 years We are Your Spectroscopy Partner
Global leader with > 10,000 Portable and handheld Raman systems B&W Tek, China Ming Shen Commercial Plaza 400 CaoBao Rd., Suite 2206, Shanghai , China Phone: B&W Tek, Japan F, Kamiochiai, Chuo-ku, Saitama-city, Saitama , Japan Phone: +81(0) B&W Tek, Corporate Headquarters 19 Shea Way Newark, DE 19713, USA Phone: Web: B&W Tek, Europe Seelandstraße 14-16, Lübeck, Germany Phone: +49(0) Over 15 years Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

Unchanged Elastic Scattering
Raman Scattering Raleigh Unchanged Elastic Scattering Laser 785 nm Excitation Raman Inelastic Scattering 1,000,000 photon vs.1 Copyright 2013 B&W Tek, Inc.

Energy Diagram for Raman Scattering
Stokes Raman Rayleigh Anti-Stokes Raman Fluorescence Electronic States Raman Scattering: Independent of laserλ Vibrations → shifts Intensity ∝ laser power Raman efficiency10-8 Virtual State Vibrational States Ground State Copyright 2013 B&W Tek, Inc.

Quantum Energy Scheme for Photon Scattering
The Raman effect comprises a very small fraction (about 1 in 107) of the incident photons. Copyright 2013 B&W Tek, Inc.

Information From Raman Spectroscopy

Raman Spectrum 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. (CC) Aliphatic Ring (CC) Aromatic Ring C=C Copyright 2013 B&W Tek, Inc.

Raman Spectral Information
500 1000 1500 2000 2500 3000 Raman Shift (cm-1) (CH3)2C=O CH3CH2OH (CH3)2S=O CH3CH2O2CCH3 C6H5CH3 C=O CH3 Aromatic-H S=O CH2 C-O Aromatic Copyright 2013 B&W Tek, Inc.

Raman Strengths Compatible with fiber optics and glass cells
High information content, including non-composition info such as crystallinity, orientation, particle size,… Can be used to analyze aqueous solutions without interference of water (as can be case in FTIR or NIR) Generally robust to temperature Does not necessarily require chemometrics, therefore valuable in early process research and development work Little or no sample prep Applicable to very tiny samples or locations on samples (micro- work) Copyright 2013 B&W Tek, Inc.

Raman Limitations Weak signal (efficiency ~ 10-8): typical LOD ~0.1%
Fluorescence interference Sample heating or photobleaching can interfere; can’t examine black or deeply colored materials High information content (interferences) Laser source may have fluctuations Need an internal standard or standardization for quantitative work Copyright 2013 B&W Tek, Inc.

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. Copyright 2013 B&W Tek, Inc.

Components of a Raman Spectrometer
Laser Narrow Linewidth Small Form Factor Low Power Consumption Extremely Stable Power Output Spectrometer High Resolution Low Noise Small Form Factor Low Power Consumption Glacier T BWN-OEM Copyright 2013 B&W Tek, Inc.

Excitation Wavelengths
532nm 785nm 1064nm Sesame Seed Oil Second diagram showing why flour … Copyright 2013 B&W Tek, Inc.

What can Raman do for you?
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 Copyright 2013 B&W Tek, Inc.

Raman can measure through a broad class of packaging
Bottles Thickness Amber Glass < 2 mm Clear Glass < 3 mm High Density Polyethylene (HDPE) < 1 mm Teflon FEP Polystyrene Vials Amber and Clear Glass Bags Polypropylene (PP) < 0.1mm Polyethylene (PE), Low-Density Polyethylene (LDPE) Copyright 2013 B&W Tek, Inc.

Portable Raman Instrumentation

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 Copyright 2013 B&W Tek, Inc.

NanoRam Handheld Innovations
Only handheld with high resolution touch screen interface Only handheld offering easy data management and reporting Best methodologies for robust analysis Fastest in adding Methods and Libraries Only handheld allowing editing Method Full method development and validation support Copyright 2013 B&W Tek, Inc.

i-Raman Plus Portable Raman Platform
Research applications Quantitative analysis Final product inspection Counterfeit detection PAT process applications – proof of concept and at line applications Field testing for various applications require portability Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

i-Raman® EX Comparative spectra for an Alka Seltzer tablet

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 Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

Spectral Analysis Examples
PLS Regression Modeling for the Development of concentration curve for quantitative prediction. Correlation Based Library Searching for Unknown Material Identification using HQI Measure 20 representative spectra Build method based on PCA and establish threshold p-value (typically 0.05) Test Method  PC1 PC2 Method Development for verification of “known” materials using multivariate classification modeling to determine p-value. Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

Applications in the Pharmaceutical Industry
Identification Verification of the identity of incoming raw materials. Identification and analysis of counterfeit drug products. Process Control Real time quantitative analysis for process analytical control (PAT) such as blending, titration, and polymorphic transition monitoring. Copyright 2013 B&W Tek, Inc.

Verification of Incoming Raw Materials
Pharmaceutical manufacturing facilities are moving toward 100% inspection of incoming raw materials, to support cGMP and PIC/S guidelines. Raman is ideal because % 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. Copyright 2013 B&W Tek, Inc.

Process Analytical Technology
Near Infrared Spectroscopy for PAT NIR2011 Process Analytical Technology 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. Copyright 2013 B&W Tek, Inc. NIR Katherine Bakeev

Counterfeit Drug Identification
According to the World Health Organization’s estimates, ~10-15% of the world’s drug supply (and about 1% in the US) is counterfeit - at a value of about $200B in 2010 Raman Spectroscopy is currently being used for not only identification of counterfeit drug products but also to analyze the quality and purity For example, the FDA is currently using portable Raman spectrometers for the identification of glycerin contaminated with DEG. From Forbes 7/25/2012 Roger Bate, a scholar at the American Enterprise Institute, has spent six years researching fake and otherwise inferior medicines, and his field work has been pulled together in a worthy new book, Phake: The Deadly World of Falsified and Substandard Medicines. He estimates, probably conservatively, that more than 100,000 people are killed worldwide by dangerous drugs every year. And that statistic does not take into consideration the incalculable morbidity and misery caused by such products. Bate describes the widespread distribution of fakes, noting that they are more likely in the poorest locations, whereas substandard drugs — those legally but poorly made — are almost never found in countries with average annual per capita income above $20,000. Bate and his colleagues sampled thousands of drugs from 22 cities and found that roughly 12 percent failed quality tests and were substandard, degraded or counterfeit. Bate’s work is more than a detailed analysis; it is also a revelatory first-hand account of the counterfeit drug trade. His adventures in Nigeria and India are fascinating and his exposure of a Middle Eastern counterfeit ring is alarming, not least because the most recent examples of fake drugs in America probably originated in the Middle East. An interesting tidbit in Bate’s book is that criminals in the Middle East sold fake drugs in Iraq that were purchased with our tax dollars. In his book, Bate proposes solutions to this pernicious, worldwide problem. Among them is the introduction of an international treaty to penalize counterfeits. He acknowledges that this will take a while to implement, but he calls it a necessary condition to combat the fake drug trade. Bate also suggests track-and-trace technologies and the deployment of handheld spectrometers to spot fakes. Although these are expensive, he explains how they are used even in impoverished Nigeria and argues that if the Nigerians can do this, then so can other richer nations. (There are other, easy-to-use high-tech devices for this kind of “molecular tracking” as well, which via a unique “barcode” determined by ratios of radioisotopes found naturally in the drug, can ascertain whether any given batch of drug is from one or another legitimate manufacturing site.) Bate argues persuasively that ”[u]ntil international bodies clean up their act and stop blurring the boundaries between safe medicines and sub-standard copies and until there are harsh local and international penalties for manufacturing and carrying counterfeits, the pirates will continue to get away with murder.” Literally. Copyright 2013 B&W Tek, Inc. Best 785

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 Bio-warfare Agents Trace Forensic Evidence Analysis: Including Fibers, Fabrics, Pigments, Inks, etc. Copyright 2013 B&W Tek, Inc.

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, a 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 Talk about SERS in more detail. SEM image of SERS substrate used in the measurement. S. Botti et al., J. Raman Spectrosc. 2013, 44, 463–468 Copyright 2013 B&W Tek, Inc.

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. Methanol, a potent toxicant in humans, occurs naturally at a low level in most alcoholic beverages without causing harm. However, illicit drinks made from “industrial methylated spirits” [5% (v/v) methanol:95% (v/v) ethanol] can cause severe and even fatal illness. Since documentation of a no-adverse-effect level for methanol is nonexistent in the literature a key question, from the public health perspective, is what is the maximum concentration of methanol in an alcoholic drink that an adult human could consume without risking toxicity due to its methanol content? Published information about methanol-intoxicated patients is reviewed and combined with findings in studies in volunteers given small doses of methanol, as well as occupational exposure limits (OELs), to indicate a tolerable (“safe”) daily dose of methanol in an adult as 2 g and a toxic dose as 8 g. The simultaneous ingestion of ethanol has no appreciable effect on the proposed “safe” and “toxic” doses when considering exposure over several hours. Thus, assuming that an adult consumes 425-ml standard measures of a drink containing 40% alcohol by volume over a period of 2 h, the maximum tolerable concentration (MTC) of methanol in such a drink would be 2% (v/v) by volume. However, this value only allows a safety factor of 4 to cover variation in the volume consumed and for the effects of malnutrition (i.e., folate deficiency), ill health and other personal factors (i.e., ethnicity). In contrast, the current EU general limit for naturally occurring methanol of 10 g methanol/l ethanol [which equates to 0.4% (v/v) methanol at 40% alcohol] provides a greater margin of safety. Source: doi: / A.J. Paine and A. D Dayan , Hum Exp Toxicol November (11) Copyright 2013 B&W Tek, Inc.

Example using Methanol-Laced Coconut Rum
CH3 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 Copyright 2013 B&W Tek, Inc.

Applications in Art & Archeology
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. Painting on cathedral ceiling, pigments on a budha, cave paintings, old artwork, coatings on sculptures Copyright 2013 B&W Tek, Inc.

Portable Raman Microscopy of Ancient Pigments
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. Courtesy of M.J. Ayora Cañada y A. Dominguez Universidad de Jaén Lead oxide (Pb3O4) Cinnabar (HgS) Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

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. Images Courtesy of Prof. Rull University of Valladolid Copyright 2013 B&W Tek, Inc.

Analysis of Garnet Gemstones
Garnets are a class of silicate minerals which include a number of varieties with the general form X3Y2(SiO4)3. Raman spectroscopy’s 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). Copyright 2013 B&W Tek, Inc.

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. Copyright 2013 B&W Tek, Inc.

Process Analytical Technology
Near Infrared Spectroscopy for PAT NIR2011 Process Analytical Technology 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 Copyright 2013 B&W Tek, Inc. NIR Katherine Bakeev

Polymer Reaction Monitoring
C=C Breathing mode (for normalisation) styrene -> polystyrene Follow the conversion of the vinyl band Henryk Herman, Ph.D. – Gnosys Global Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

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 G D MWCNT SWCNT Copyright 2013 B&W Tek, Inc.

Applications in the Biomedical Diagnostics
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. There are a number of important functional groups related to biomedical testing, which have characteristic Raman frequencies. Tissue samples include components such as lipids, fatty acids and protein all of which have vibrations in the Raman spectrum. The most significant spectral regions include: X-H Bonds (e.g. C-H stretches): cm-1 region Multiple Bonds (e.g. N≡C): cm-1 region Double Bonds (e.g. C=C, N=C): cm-1 region Complex Patterns (e.g. C-O; C-N and bands in the fingerprint region): cm-1 region Copyright 2013 B&W Tek, Inc.

Applications in Polymer Science
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 Copyright 2013 B&W Tek, Inc.

Polymer Crystallinity
These bands are related to crystalline and amorphous content, (as well as the interphase) Dr. Henryk Herman - GnoSys Global Copyright 2013 B&W Tek, Inc.

Polymer Identification

Summary Increase analysis
Raman Increase analysis Cost reduction Improve operations Competitiveness Summary Raman has proven to be a promising tool to increase operational capabilities and reduce cost while providing rapid material identification. Copyright 2013 B&W Tek, Inc. 54

Thank You Lab Science Solution 27, Anggerik Aranda C31/C Kota Kemuning 40460 Shah Alam Selangor DE, Malaysia Dr. Katherine A. Bakeev B&W Tek Inc. 19 Shea Way Newark, DE 19713, USA Copyright 2013 B&W Tek, Inc.

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-1or 65cm-1) The anti-Stokes scattering does not provide any additional information (except in thermal studies) so it is also filtered out. Copyright 2013 B&W Tek, Inc.

Mid-IR NIR reflectance Raman
Mechanism: absorption by fundamental molecular vibrational modes Molecular Specificity: high Signal Strength: strong Energy: 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: ,500 cm-1 Sampling: non-contact, but close proximity to material required (<5 mm) Hardware: longer fiber optics possible (meters); quartz optics; dispersive, interferometry common Mechanism: inelastic scattering by vibrational, rotational, low frequency molecular modes. Energy: cm-1 shift Sampling: container interference is mitigated by accessories Hardware: CCD detection; quartz optics; long fiber optics common (kilometers in some cases) Copyright 2013 B&W Tek, Inc.

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) Copyright 2013 B&W Tek, Inc.

Permanent Wavelength Calibration
NIST traceable wavelength calibration ensuring Raman shift accuracy with standards- Tylenol and cyclohexane Follows ASTM E (2007) Copyright 2013 B&W Tek, Inc.

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 Copyright 2013 B&W Tek, Inc.

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