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IlluminatIR™

Applications• Coating characterization • Compound validation • Consistency verification • Contaminant analysis• Counterfeit analysis • Crystal characterization • Drug precursor identification • Illicit drug identification • Explosive identification • Failure analysis • Fiber or film recognition • Forensic analysis • Laminate characterization • Process chemistry • QA/QC analysis • Reaction monitoring • Soil identification • Suspicious powder identification • WMD identification• Trace evidence analysis Features/Benefits• Unique accessory to enhance your light microscope • Minimal sample prep • Definitive spectral signatures in seconds • Combines infrared spectroscopy with advanced

microscopy techniques, polarized light microscopy, fluorescence microscopy and differential interference contrast microscopy

IlluminatIR™ adds FT-IR and advanced imaging software to your research-grade light microscope creating an easy to use and powerful platform for the identification, verification, and characterization of molecular compounds.

The IlluminatIR™ chemically identifies microscopic unknowns in seconds.

IR Technology Overview ....................................................... 14Product Overview ................................................................. 15Software ............................................................................... 18Technical Specifications ...................................................... 20Ordering Information ........................................................... 21Application Notes (IlluminatIR™) ......................................... 58

Table of Contents

Introduction


Product Overview

The IlluminatIR™ is the first and only Fourier-Transform infrared (FT-IR) spectrometer system to mount directly onto a research-grade light microscope frame. The optics and electronics of the spectrometer are contained in a compact enclosure that is mounted between the microscope frame and the observation tube mount.

Technology Overview IlluminatIR™

MicroscopeThe IlluminatIR™ optical interface takes advantage of the infinity corrected design of modern light microscopes. The output from the spectrometer is in the form of a parallel beam, the same geometry as the visible illumination. An optical element allows the simultaneous observation of the specimen as IR spectral data is collected. The sample can be viewed through the diamond during this process. All your traditional microscopy techniques and options such as polarized light microscopy (PLM) remain fully functional.

ATRThe attenuated total reflection (ATR™) infrared objective allows analyses with little or no sample preparation. The spectrum of the microscopic specimen is recorded by contacting the specimen with the diamond element at the end of the objective. This patented-design see-through objective enables visualization of the sample during ATR analysis.

AROThe all-reflecting infrared objective (ARO) allows samples to be measured in reflec-tion-absorption mode. The spectrum of the microscopic specimen is recorded by simply focusing on the specimen. This mode is useful for particles or thin samples that can be transferred to reflective microscope slides.

LibrariesAdding the IlluminatIR™ to a conventional light microscope, users are able to obtain an infrared (IR) spectrum, or “molecular fingerprint,” of a sample as small as 10 µm2. Once a spectrum is collected the SynchronizIR software compares the collected spectrum against libraries containing reference spectra of known materials for identification.


IlluminatIR™Application Brief AIL-P01

Characterization of Hydrates, Solvates, Salts & Polymorphs in Drug Development Using Infrared Microspectroscopy andThermomicroscopy

Introduction In drug development, characterizing the active pharma-ceutical ingredient’s (API) ability to form hydrates, sol-vates, salts and polymorphs is crucial to formulating a new drug product. To be successful, a drug must be effective and stable to provide consistent release. Analysis of solid drug substances is fundamental, providing a global under-standing of the material’s physical, chemical and pharma-ceutical properties.

Hydrates or solvates formations Hydrates or solvates are formed by the incorporation of water or small organic molecules into a crystal lattice. Detecting and characterizing hydrated or solvated crystals can be a significant challenge for the solid-state chemist. The solvating species can exist in a number of environ-ments within the crystal lattice and often the extent of hydrogen bonding indicates the stability of the solvate or hydrate. The formation of a solvate or hydrate effects properties such as solubility, hygroscopicity, chemical and physical stability and crystallinity.

Hydrates and solvates can create problems during either the production of drug substance or the formulation of a drug product. In the production stage, if excessive heat is used during drying, desolvation can occur. This can pro-duce either a hygroscopic form, an anhydrous crystalline form or alteration of the crystal structure and habit. In the formulation phase, any processes involving heat may produce changes to the degree of solvation. Hydrates can change as the relative humidity increases or decreases. Any of these changes can affect the performance of the API. Screening for the formation of hydrates or solvates takes place early in the development phase of a drug prod-uct.

Polymorphic; solid-state substances Many drugs are polymorphic; solid-state substances exist-ing in more than one crystalline structure. Polymorphic forms have the same chemical structure, but they have different crystalline structures. Each polymorphic form has its unique melting point, density, vapor pressure, X-ray

diffraction pattern, infrared spectrum and solubility. Since the solubilities vary between polymorphic forms, it can be understood that some polymorphic forms of a drug work better than others. The solubility can affect the biological availability of the drug.

Polymorphic forms can undergo changes from one form to another. When the change is reversible it is called enantiotropic. When the change of forms only goes one direction, it is called monotropic. The transition tem-perature in polymorphism is important because it helps to characterize the system and establish the room tem-perature stable form. While only one polymorphic form of a substance is thermodynamically stable at any specific temperature and pressure, unstable forms can exist that do not transform because of kinetic factors.

Salts of API’s Many drugs are polymorphic solid-state substances. Salts of API’s are often found to be the more effective than the free base and can improve the processing of a drug prod-uct. Because selection of an appropriate salt can signifi-cantly reduce time to market, rapid salt screening and salt selection is a critical part of the drug development process. Salts are used to alter the physical or chemical properties of a drug substance. If the correct salt is select-ed, subsequent development will be facilitated. Salts that exhibit advantageous properties may be patentable as new chemical compounds.

Changes in crystal structure that occur by salt formation can lead to improved properties. API’s containing free acid or base groups often have poor water solubility. Formation of salts often improves solubility, thus providing greater bioavailability. Sometimes, a salt provides increased chem-ical or physical stability compared to the parent drug sub-stance. Salts can also provide a means of purification and/

Figure 1. U.S. Patents issued between 1976 and 2003 that contain references to polymorphic forms of drugs. The data for 2003 is estimated based on patents issued through June, 2003.


IlluminatIR™Application Brief AIL-P01 (continued)

or a way to improve the processing of a drug substance. How important is solid-state characterization of APIs? While the general consensus is that solid-state charac-terization is very important, a review of the U.S. Patent literature provides a metric that indicates both the level and growth. U.S. Patents were issued that specifically site polymorphic drug products. More than half of the patents were issued after 2000 (see Figure 1). Clearly this is both important, and a growing area of pharmaceutical science.

Tools for solid-state characterizationTools for solid-state characterization of drug substances- X-ray crystallography, solid state NMR, PLM, IR, Raman, moisture sorption/desorption isotherms, thermomicroscopy, DSC, TGA, EGA, vapor pressure, inverse GC, dissolution rates, particle size, and crystal morphology, are tools that can be used to characterize drug substanc-es. X-ray crystallography, DSC/TGA, infrared spectroscopy and thermomicroscopy are the most common. X-ray dif-fraction is the primary method for determining the crystal lattice geometry. However, it can not determine chemical

composition. DSC/TGA and thermomicroscopy can record changes occurring as a thermal history is imposed on a sample. While detecting change is important, it is equally important to understand the nature of the change. Is the change a polymorphic transformation, desolvation, decom-position or melting? Infrared microspectroscopy combined with thermomicroscopy provides direct observation of the molecular chemistry before, during and after a thermally induced event is observed. Infrared thermomicroscopy is described in more detail.

What is infrared thermomicroscopy? Infrared thermomicroscopy is the combination of polarized light microscopy, mid-infrared spectroscopy and thermal microscopy.

What instrumentation is needed for infrared thermomicroscopy?A polarized light microscope is fitted with a hot stage and an infrared spectrometer to form a system for infrared thermomicroscopy. The system shown in Figure 2 consists of an Olympus™ BX-51 polarized light microscope, a Linkam™ THMS-600 heating/cooling stage and the IlluminatIR™ FT-IR spectrometer attachment.

What information do we get using infrared thermomicroscopy?Changes in state are observed directly using polarized light microscopy and the sample’s mid-infrared absorp-tion spectrum records changes in the molecular chemis-try before, during and after the change. In Figure 3, two polymorphic forms of 1-nitronaphthlene (MNN) are visible

Figure 2. The infrared thermomicroscopy system used for characterization of solid drug substances

Figure 3. Polymorphic transformation of 1-nitronaphthalene



Figure 4. Detailed spectra of 1-nitronaphthaline polymorphic forms showing minor differences expected for an enantiotropic phase transformation.

Dehydration of caffeine monohydrateCaffeine monohydrate was formed by recrystallization anhydrous caffeine from water solution at 27-deg C. On heating this substance at a rate of 2-deg C per minute, the monohydrate decomposed between 45 and 46 deg C. Dehydration was monitored by recording the infrared spectrum as the temperature increased. Spectra, shown in Figure 5, show a loss of water at 3300-cm-1 confirming that the caffeine was dehydrated.

Figure 5. Dehydration of caffeine upon heating at 2-degC. The loss of water is confirmed by the disappearance of the 3300-cm-1 absorption band.

ConclusionsInfrared microspectroscopy and thermomicroscopy provide vital information to the pharmaceutical chemist to confirm the type of change that occurs when a drug substance is heated or cooled. The ability to rapidly record the infrared absorption spectrum of a microscopic sample is a distinct advantage of this method. Molecular spectroscopy generates unique chemical information to compliment and support the results of X-ray diffraction and thermal analytical methods.

in the polarized light micrograph. The infrared spectra of these phases are similar, confirming that there is no chemical change. However, minor spectral differences, shown in Figure 4, are typical of an enantiotropic phase transformation.


IlluminatIR™Application Brief AIL-P02

Use of the IlluminatIR™ ATMS to Evaluate the Polymorphism and Solid-State Properties of Molecular CrystalsIntroduction The ability to identify polymorphic and other solid-state forms of molecular crystals has become significant to the pharmaceutical industry for a variety of reasons. Historically, the need to evaluate solid-state chemistry was of practical concern. Did the solid-state form of the drug developed meet the metabolic requirements for its indica-tion, and did it provide adequate stability to meet manu-facturing, shipping and shelf-life requirements? In 1984, however, the law governing the pharmaceutical industry changed.

With the enactment of The Drug Price Competition and Patent Term Restoration Act of 1984, the solid-state form of a drug product’s active pharmaceutical ingredient (API) became legally significant. Developers spend considerable amounts of time and money on research attempting to understand the solid-state properties of their patentable compounds at the earliest stages of the drug development process. Due to these changes as well as advances in instrumentation and technology, the role of infrared analysis within the drug development process has also changed. Developments in infrared microprobe technol-ogy married infrared spectroscopy with the use of the light microscope and its accessories (variable temperature and variable humidity stages). This integration makes it pos-sible to provide information and insight about the system being studied that would not otherwise be possible.

In this example, three polymorphic forms of a drug used to treat schizophrenia were prepared and subsequently identified using infrared microprobe analysis. Results of infrared experiments were confirmed using X-ray powder diffraction. The IlluminatIR™ ATMS system was also used to evaluate the sample visually and with the infrared microprobe. The IlluminatIR™ ATMS integrates infrared microprobe analysis with variable-temperature micros-copy. The ability to perform this type of analysis generated a more complete understanding of the behavior of the different solid-state forms of this drug including the identification of a fourth polymorphic form. Identification of Solid-State Forms To identify the three forms initially prepared, a Smiths

Detection IlluminatIR™ II with a 36-times attenuated total reflection objective was used. For each sample, ten spec-tra were collected from different areas of the sample and the average spectrum of these ten reported. An overlay of the average spectra for each sample is detailed within Figure 1. Differences in the high-wave number region due to hydrogen and non-hydrogen X-OH stretching are displayed in the graph at the top in this figure, while the fingerprint and carbonyl regions of the spectrum are dis-played in the graph on the bottom. Note the differences in the absorption bands in the fingerprint and carbonyl regions are much less dramatic than those observed in the high-wave number region. This is consistent with differ-ences due to polymorphism rather than molecular com-position. The “molecule-specific” information (fingerprint region) changes minimally, while the molecular bonding information (high-wave number region) changes more significantly. The identification of these three forms as polymorphs was confirmed using X-ray powder diffraction data shown within Figure 2.

Figure 2. X-ray Powder Diffraction of Polymorphic Forms of API

All three forms were distinguishable based upon their infrared spectra. A summary of band-specific information used to identify these three forms is displayed within Figure 3. When differentiating solid-state forms, the presence or absence of these bands was considered. In addition, differences throughout the entire spectrum were also considered. These differences included small shifts in wave number (<5cm-1) and differences in band intensity ratios.

Figure 1. Infrared Spectra of Polymorphic Forms I (Red), II (Black) and III (Blue) of API



Variable-Temperature Infrared Microprobe Analysis to Evaluate Relative StabilityPublic Health Laboratories (PHLs) face a myriad of analytical issues. Once the solid-state forms of an API are identified, the relative stability of these forms needs to be established in order to select the best candidate for development. Of critical concern is the affect of heat and/or humidity on the sample. Using the variable-temperature infrared microprobe, it has never been easier to generate meaningful chemical and structural information with microscopic samples.

In this example, the three polymorphic forms of the sam-ple identified were heated at a controlled rate of 10°C per minute while photomicrographs and infrared microprobe data were collected throughout each experiment. Although not necessary in this example, a variable humidity stage could also be used in a similar manner to develop a better understanding of the effect of humidity on the sample. All three forms exhibited differences when heated. These differences were observed in both the images and infrared spectral data, demonstrating how significant polymorphic forms can be when considering thermal stability.

As the temperature is increased from 25°C to 125°C, the visual appearance of Form I and Form II darken due to changes in the way light is scattered by the sample (see Figures 4 and 5). Form I then undergoes a second change in visual appearance that is clearly a change in solid-state form. This change is first seen in the image collected at 135°C. By the time the sample is heated to 145°C, the sol-id-solid conversion is complete. The final melt of this Form I sample occurs when the sample is heated to 155°C.While Form II darkens as the sample is heated to 125°C, it does not undergo a solid-solid conversion beginning at 135°C. In fact, the sample does not change significantly until melting, which is observed to occur between 175°C and 185°C. The difference in melting temperature between Form I and Form II is significant, especially when con-sidering differences in melt temperature of polymorphic forms are typically only a few degrees.

The visual changes observed in Form III are completely different from the changes observed in Form I and Form II (see Figure 6). By the time Form III is heated to 145°C, barely any darkening of the sample occurs. In fact, even at 185°C, minimal differences in appearance are observed. The melting temperature of the sample is observed to occur between 185°C and 195°C.

The visual images collected throughout the variable-tem-perature experiments confirm that each polymorphic form responds differently to heat. The infrared microprobe data collected during these experiments enables an understanding of what is chemically changing as these visual changes are occurring.

In Forms I and II, changes due to darkening of the sample do not have an impact on the infrared spectra. This indicates that these changes are not due to changes in polymorphic form. However, the spectrum of Form II changes significantly as the sample approaches the melt temperature showing that the sample decomposes with melting (see Figure 7). This same phenomenon is not observed for either Form I or Form III.

Figure 6. Photomicrographs of Form III Captured During Variable-Temperature Infrared Microprobe Analysis

Figure 4. Photomicrographs of Form I Captured During Variable-Temperature Infrared Microprobe Analysis

Figure 5. Photomicrographs of Form II Captured During Variable-Temperature Infrared Microprobe Analysis

Solid-State Form Characteristic Band (cm-1)I 1490, 2802II 1098, 2824III 868, 1480, 2814

Figure 3



Summary and ConclusionThe value of performing infrared analysis of pharmaceutical compounds is outlined in USP <197> Spectrophotometric Identification Tests which reads, ”The infrared absorption spectrum of a substance, compared with that obtained concomitantly for the corresponding USP Reference Standard, provides perhaps the most conclusive evidence of the identity of the substance that can be realized from any single test.” From this example, it is clear that infrared spectroscopy is just as useful for the identification of polymorphic form. When analyzing polymorphs, bands in the fingerprint region can be used to verify chemical composition while bands due to hydrogen- and non-hydrogen bonding can be used to verify structural changes. No other type of analysis provides such a comprehensive evaluation of the sample.Developments in infrared microprobe technology have married infrared spectroscopy with the use of the light microscope and its accessories (variable-temperature and variable-humidity stages). This integration makes it possible to provide information and insight about the system being studied that would not otherwise be possible.

Although Forms I and III do not decompose as they approach their melting temperature, Form I does undergo a polymorphic conversion prior to its melting. This data is evidenced in both the visual images in Figure 4, as well as in the infrared spectral data in Figure 8. Although the spectral features of Form I are more consistent with Form III as it approaches melt, there are some differences that differentiate these two forms (see Figure 9). This new form is a fourth polymorphic form of this API. The infrared spectral data establishes that these three polymorphs are anhydrates. During heat, there is no evidence of water evolving from any of the samples. Typically during heating of hydrates, water will evolve from the sample below the melt or decomposition temperature. This information is used to establish whether different solid-state forms of a compound are polymorphs or hydrates of each other.

Figure 7. Infrared Spectral Overlay of Form II Data (Red 25°C, Green 175°C)

Figure 9. Overlay of Form I at 145°C (Red) and Form III at 185°C (Blue)

Figure 8. Infrared Spectral Overlay of Form I Data (Red 25°C, Green 135°C, Black 145°C)


IlluminatIR™Application Brief AIL-F01

Screening for Biological Agents in Powder Mixtures with the IlluminatIR™ Introduction In this time of heightened national security, identifying the presence of biological agents in suspicious powder mixtures is a serious issue. The state National Guard Weapons of Mass Destruction Civil Support Teams (WMD-CSTs) and Public Health Laboratories that are charged with this mission need reliable and rapid analytical capabilities.

One approach is to combine polarized light microscopy (PLM) with infrared (IR) microspectroscopy to character-ize powdered mixtures on both microscopic and molecular levels. The synergism of these analytical techniques pro-vides significantly more information than can be derived from either technique alone. Whereas PLM can elucidate optical and morphological characteristics of solids, IR spectroscopy provides highly specific information on the covalent bonds within molecules. In fact, the IR spectrum of a substance is often called its “fingerprint,” and can be used to identify substances of unknown origin. The IlluminatIR™ accessory merges IR microspectroscopy with a conventional infinity-corrected light microscope, and its SynchronizIR™ software performs an automated analy-sis of the spectra to easily identify unknown samples. In this study, bacillus thuringiensis (BT), a bacterium that is harmless to humans but is a simulant of deadly bacillus anthracis (Anthrax), was analyzed within a matrix of non-biological solid materials to demonstrate the union of these techniques. Experimental The source of bacillus thuringiensis was a commercially available Mosquito Dunk™ pest control tablet (Summit Chemical Co., Baltimore, MD), obtained from a local agriculture supply store. According to the product packaging, each 12 g tablet contains 10% (w/w) bacillus thuringiensis and 90% undefined inert ingredients. Approximately 0.5 g of Dunk material was cut off of one tablet and ground into a fine powder using a mortar and pestle. An equal volume of CAB-O-SILTM M-4 amorphous fumed silica (Cabot Corp., Tuscola, IL) was added to the

Dunk material. Silica is a common material for aerosolizing Anthrax spores, and would likely be found in a weaponized biological agent mixture. The entire sample mixture was ground until finely powdered and homogeneous.

Approximately 1 mg of the final mixture, containing about 5% BT, was placed onto a ReflectIR infrared-reflective glass microscope slide and flattened with a roller knife. The unique ReflectIR slide allows the flattened sample to be simultaneously viewed with transmitted light (diascopic) illumination and analyzed by reflection-absorption infrared spectroscopy (RAS). Suspected pathogenic samples can be contained beneath a visible- and infrared-transparent window using a special sealing mechanism.

For these experiments, the sample was mounted on an unsealed slide, and analyzed with an Olympus™ BX-60 light microscope equipped with an IlluminatIR™. Visible light polarizing filters were used with the microscope to probe the birefringent nature of the various mixture components. The all-reflecting infrared objective (ARO), in concert with a 50mm IR source aperture mask, was used to collect the IR spectra, each of which represent 32 co-added scans at 8 cm-1 resolution.

Figure 1. Photomicrograph of solid sample mixture in oil (n = 1.630) viewed using cross-polarized light and a firstorder red plate


Product Overview

Application Brief AIL-F01 (continued) IlluminatIR™

Results and Discussion The photomicrograph of the mixture in Figure 1 shows some interesting features, but little information on the individual components can be derived from this image alone. Close inspection of different areas of the flattened sample, shown in the images in Figure 2, reveals the presence of different components.

The component in Figure 2A is translucent through its center where it is flattest. However, characteristic cracks approximately 10mm long appear at the edges where the particle broke apart when rolled. The dark spots are indicative of intact particles. The identity of this component was obtained by bringing it into the center of the field of view where the IR spot is located and collecting its spectrum.

As shown in Figure 3, the automated library search produced a match with calcium sulfate dihydrate, which was determined to be the main inert ingredient in the Dunk tablets. The IR spectra of all three components, shown in Figure 4, are strikingly different, allowing them to be easily identified and differentiated using the software.

Figure 4. Infrared RAS spectra of the three components shown in Figure 2. The spectra have been corrected for scatter and atmospheric CO2 interference.

In contrast to the crystalline calcium sulfate dihydrate, the amorphous silica in Figure 2B appears more irregular with interspersed dark and light areas and no obvious flat spots. The protein in Figure 2C (known to be BT from the product label) has its own characteristic appearance. The flattening across the particle area is smooth and uniform, with no apparent cracking as observed in the crystalline material. These observed morphological characteristics, combined with the IR spectral search results, help to discern the components in the mixture when the ensemble is analyzed.

All proteinaceous biological agents exhibit similar spectroscopic features, so they cannot necessarily be discriminated through inspection or even library searching of the IR data. Indeed, the IR spectra of BT and Anthrax appear similar to one another. However, their spectra are very different than non-biological materials, therefore, while other techniques such as the polymerase chain reaction (PCR) must be employed to characterize the exact nature of the biological substance, microscopy and IR microspectroscopy can play an important role in rapidly screening suspicious materials.

Summary Confirming the presence of a biological agent in suspicious powder mixtures is a major concern for National Guard WMD-CSTs and State Public Health Laboratories. The IlluminatIR™ microspectrometer accessory coupled to an optical microscope allows this problem to be approached quickly and accurately in a way not possible with either technique alone.

Figure 2. Photomicrographs ofA calcium sulfate dihydrate,B amorphous silica andC bacillus thuringiensis (BT)

flattened on ReflectIR slide.

Figure 3. Automatic library search result showing the identity of the component in Figure 2A to be calcium sulfate dihydrate.



Public Health Laboratory Applications of a Miniaturized FT-IR Light Microscope AccessoryIntroduction Public Health Laboratories (PHLs) face a myriad of analytically challenging samples, including unidentified particulates, food and drug contaminants, air and water borne materials, and architectural asbestos. Most recently, PHLs have been called upon to support HazMat response units to characterize the plethora of “suspicious white powders” threatening our national security. While often well-equipped with chromatography, mass spectrometry, and other instrumentation for environmental applications, as well as microscopy for particulate analysis, PHLs generally do not have capabilities for rapid identification of solids. The IlluminatIR™ Fourier Transform Infrared (FT-IR) light microscope accessory fills this gap.

With the IlluminatIR™, PHL technicians can quickly identify a wide range of particulates using their exist-ing light microscope capabilities, such as polarized light microscopy (PLM), differential interference contrast (DIC) and fluorescence microscopy. The IlluminatIR™ obtains the infrared (IR) spectrum, or “molecular fingerprint,” of a sample as small as 10 micrometers, then compares it to libraries containing reference spectra of known materials. This analysis provides complementary and non-subjective data which can be submissible in court. With specialized PHL libraries, including Common White Powders, Fibers, and Asbestos, the IlluminatIR™ is readily useful to any PHL analyst.

White Powder Screening In October 2001, a series of terrorist events involving the dissemination of Anthrax as white powders through the U.S. mail caused panic among American citizens. The white powder hoaxes that followed, while posing no real medical threat, were no less damaging to the American psyche. HazMat teams were inundated with calls, virtually all of which were false alarms, to identify the powders and bring calm to the public conscience. Even today, white powder identification remains a challenge for PHLs, and one more legitimate threat could set off another panic.

Commonly, a PHL will incubate a white powder culture for 24 hours to allow any bacteria to grow and be identified. However, for the majority of cases in which the samples are non-biological, the powders remain unidentified 24 hours later. As shown in Figure 1 (a), the IlluminatIR™ allows the user to identify a powder in seconds. In this example, a white powder of unknown origin received by a PHL was subjected to tests for almost 2 weeks. The presence of biological material was ruled out, but no tests could confirm the sample identity. In less than 60 seconds, the IR analysis showed the true identity of the powder: Boric Acid. When IR is combined with light microscope techniques like PLM, even powder mixtures can be characterized by isolating individual particles optically, as indicated in Figure 1 (b), then identifying each component spectroscopically.

Figure 1. (a, top) QualID Library search result, clearly indicating that Boric Acid is the most likely identity of the suspicious white powder. Cross-polarized light photomicrograph of a white powder mixture, indicating two different components that can be identified individually using the IlluminatIR™ (b, bottom).


IlluminatIR™Application Brief AIL-F02 (continued)

Particle and Fiber IDWhite powders are just one type of sample that PHLs face daily. Unidentified dust from office buildings, water borne particles from public and private water sources, and suspicious looking soil samples like that in Figure 2 (a), are submitted to PHLs for identification. The submitted samples typically contain many types of particulate and fibrous material, and identification of as many of the components as possible is essential. While particle and fiber ID has traditionally been the role of the skilled microscopist, the IlluminatIR™ makes it possible for a novice technician to fully characterize a complex particulate mixture.

In this example, a new property owner submitted a soil sample that appeared to be contaminated with a white granular substance, which he feared was toxic pesticide dust. After physically isolating individual particles on a microscope slide, the PHL technician analyzed them using the diamond ATR objective. The IlluminatIR™ software automatically compared the spectra to reference libraries and displayed the results. As shown in Figure 2 (b), the white granular particles were calcium carbonate, an agricultural product commonly known as “limestone” that is used to adjust the pH of acidic soil. The identification took only seconds, proving that PHLs can significantly increase sample analysis throughput and decrease sample backlog using the IlluminatIR™ system.

Figure 2. (a, top, left) Typical particle mixture sample submitted for identification and (right) photomicrograph of isolated particles. (b, bottom) QualID library search result identifying the white granules to be Calcium Carbonate

Asbestos CharacterizationPHLs also play a crucial role in Occupational Health, and verifying the presence of asbestos in building materials, and subsequently characterizing the type of asbestos present, is a major task. Methods based on PLM are well-established for this purpose. To complement these methods, Smiths Detection has developed a method for identifying bulk asbestos samples using the IlluminatIR™. Once individual asbestos fibers have been observed and characterized by PLM, as shown in Figure 3 (a), the bulk sample is analyzed using the ATR objective of the IlluminatIR™.

Figure 3a. Cross-polarized light photomicrograph of asbestos fibersThe frayed ends of the fibers are indicative of Chrysotile asbestos

A specialized library search method which excludes the Si-O stretch region form 1050 - 800 cm-1, and which performs a first derivative correlation algorithm, is executed to search a spectral library of reference asbestos materials. The discriminating characteristics of the asbestos spectra occur in the weaker Si-O sidebands and in the free O-H vibrations near 3650 cm-1, which are highlighted by the first derivative computation. As shown in Figure 3 (b), this method, combined with PLM analysis, provides a reliable and objective verification of the asbestos identity in a matter of seconds.

Figure 3b. Grams SpectralID library search results correctly identifying the fibers as Chrysotile asbestos



The IlluminatIR™: An FT-IR Microprobe for Forensic Science ApplicationsIntroduction The light microscope is a very familiar analytical instrument to the forensic scientist. The instrument is used in the examination of criminal evidence includ-ing drug crystals, hair, fibers, bullets, and documents. Microscopic images are powerful data in legal proceed-ings as people without a technical background have some understanding of the interpretation of images. All analyti-cal techniques have limitations, however, and stronger legal cases are made by collecting complementary data that support a common conclusion.

For chemical analysis, the interpretation of images from a light microscope is based on comparisons with known morphological forms, birefringence, refractive index matching, wet chemical methods, phase transitions, or combinations of these techniques. The forensic scientist may also use the microscope purely to detect physical changes, for example, the comparison of striations on bullets to identify weapons used in the commission of a crime.

Coupling spectroscopic analysis of materials under microscopic examination can significantly enhance the chemical analysis capabilities of a light microscope. In electron microscopy this has been amply demonstrated through the use of energy dispersive X-ray spectrometers coupled to the electron microscope. These spectrometers provide insight into the elemental composition of the specimen under examination.

Infrared (IR) spectroscopy has been proven invaluable in the analysis of organic and inorganic covalent materi-als. The IR spectrum of a material is a physical constant. Materials that differ in molecular structure give rise to dif-ferent IR absorption spectra. Therefore, the IR spectrum can be used to identify an unknown by comparison with known standard spectra. Also, chemical functional group information can be obtained by direct interpretation of the IR spectrum. With the introduction of the IlluminatIR™, light microscopists can add the chemical analysis capabili-ties of IR spectroscopy directly to a microscope already in their laboratory. In this discussion we will describe the IlluminatIR™ and give an example of its performance in an analysis relevant to forensic science.

System Description The IlluminatIR™ is the first Fourier-transform infrared (FT-IR) spectrometer system to interface to a light microscope frame. The optics and electronics of the spectrometer are contained in a compact enclosure that is mounted between the microscope frame and the observation tube mount (see Figure 1). The IlluminatIR™ optical interface takes advantage of the infinity corrected design of modern light microscopes. The output from the spectrometer is in the form of a parallel beam. An optical element and image software package allows the simultaneous observation of the specimen as IR spectral data is collected and electronically stored. The system includes a special CCD camera. The camera is sensitive to IR wavelengths. With this camera the analyst knows exact-ly where the IR beam is focused and therefore, the area of the sample that will be measured.

Specialized objectives are designed specifically for use in the IR region. These objectives are mounted in a standard one inch objective thread in a single or multi-position nosepiece. The ContactIR™ attenuated total reflection (ATR) objective allows analysis with little or no sample preparation. The spectrum of the microscopic specimen is recorded by contacting a diamond element at the end of the objective. The sample can be viewed through the diamond during this process. An all-reflecting objective allows spectra to be recorded in an incident mode. This mode is useful for particles or thin samples that can be transferred to reflective microscope slides.

Application Example The analysis of fiber evidence is important in many crimi-nal investigations including heinous crimes such as mur-ders or rapes. In violent crimes, trace fiber evidence from hair, clothing, carpet, or upholstery can be exchanged between the perpetrator and the victim or crime scene. The detailed analysis of fiber evidence can place a suspect at the scene of the crime. Light microscopy is an important analytical technique in the analysis of fiber trace evidence. Many fibers can be differentiated based on morphology alone. This is especially true of natural fibers such as cot-ton, wool, or silk. Synthetic fibers are more difficult to dif-ferentiate in this manner, but cross sections of multi-lobal fibers have been successfully used, and the birefringence of synthetic fibers from polarized light measurements also provides a basis for identification.

Figure 1.


IlluminatIR™Application Brief AIL-F03 (continued)

IR microspectroscopy can provide complementary information to such analyses and is typically faster to employ. In some cases IR spectroscopy provides identification when light microscopy alone cannot. A case in point is discussed here. Figure 2 is a polarized light photomicrograph of two types of synthetic fiber, one of which is ubiquitous in textile products. These fibers cannot be differentiated by morphology since both have a regular, cylindrical shape. Also, the birefringence of the fibers is the same and the fibers are not amenable to differentiation using chemical means in conjunction with the microscope. However, the fibers can be rapidly differentiated using IR spectroscopy.

Figure 3 shows IR spectra of two fibers shown in Figure 2. This data was recorded using the IlluminatIR™ and the ContactIR™ diamond ATR objective, simply by contacting the individual fibers. At first glance, the spectra look very similar.

However, if one analyzes the data in detail in the 1450 - 850 cm-1 region, differences become apparent. First, the amide III bands near 1270 cm-1 are shifted 10 cm-1, indicated by the vertical cursor in Figure 3. Other bands exhibit similar shifts. Secondly, additional absorption bands are observed in the top spectrum that are absent in the bottom spectrum, in particular the doublet near 950 cm-1 (Figure 3). By comparison with reference IR spectra, these fibers were identified as Nylon 6 and Nylon 6,6*. This identification is a good example of the inherent strength of IR spectroscopy to differentiate structurally similar materials. As Figure 4 indicates, Nylon 6 and Nylon 6,6 differ in structure only by alternating methylene groups between the amide functional group.

*Nylon is a registered trademark of DuPont, Inc.

Figure 2. Photomicrograph of unknown synthetic fibers

Figure 3. IR spectra of two fibers from Figure 2. From the top spectrum, the unknown fiber was identified as Nylon 6; from the bottom spectrum, the second unknown fiber was identified as Nylon 6,6

Figure 4. The molecular structures of Nylon 6 (top) and Nylon 6,6 (bottom)


IlluminatIR™Application Brief AIL-I01

Determination of Product Authenticity Using the IlluminatIR™ Infrared MicroprobeIntroduction Intellectual property in the form of copyrights, patents, and trademarks promote cultural development, foster innovation and growth and protect public health and safety.1 Theft of these goods results in substantial economic, social and developmental costs that have increased exponentially in recent years. Theft is also shown to decrease employment opportunities, diminish tax revenues for governments and reduce incentives to invest.2

The growth in intellectual property rights violations was fueled in part by the spread of enabling technology allowing for simple and low cost duplication of copyrighted products, as well as by the rise in organized crime groups that smuggle and distribute counterfeit merchandise for profit.3 Global access to the internet also plays a critical role, especially in regard to accessibility to counterfeit pharmaceuticals. The internet has become the primary tool for criminal organizations to advertise, communicate and conduct sales of counterfeit pharmaceuticals, as well as being the primary mechanism for consumers to find, order and make payments for counterfeit pharmaceuticals.4

Defense of intellectual property assumes it is possible to differentiate an authentic article from a counterfeit one. Frequently this is a relatively simple task. As counterfeit-ers become more experienced in their trade, though, differentiation becomes more and more challenging. In many instances, it is necessary to perform chemical analysis before a conclusion about authenticity can be made. The IlluminatIR™ Infrared Microprobe is ideally suited to perform this chemical analysis, enabling a rapid, reliable and non-destructive evaluation of the sample, i.e., within minutes an accurate conclusion as to a sample’s authenticity can be made. In this report a simple method to determine sample authenticity is proposed.

Experiment Design There are many different approaches to analysis that can be used when determining a sample’s authenticity. In some instances it is necessary to analyze the merchan-

dise itself. Frequently, however, analysis of the packaging used during shipment can determine whether a sample is authentic or counterfeit. Packaging is an integral part of the counterfeiting industry. So much so, in fact, that a bill was passed in the U.S. House of Representatives and signed into law in March 2006 called the Stop Counterfeiting in Manufactured Goods Act (H.R. 32). This rare piece of congressional legislation focuses almost exclusively on product packaging and labels, and shows why the ability to easily differentiate packaging materials can help to authenticate a genuine product from a counterfeit.

In this experiment, the IlluminatIR™ Infrared Microprobe was used to differentiate a genuine pharmaceutical product from an unbranded replicate. Differentiation was attempted simply by analyzing different aspects of each sample’s packaging. The genuine product contained 16 tablets within two sets of blister packs in a sealed box. The replicate product contained 16 tablets within two sets of blister packs within a sealed box. Seven different aspects of the packing were analyzed, as follows:

1. Outside of sealed box2. Inside of sealed box3. Glue used to seal box4. Outside of foil backing side of blister pack packaging5. Inside of foil backing side of blister pack packaging

(side exposed to tablet)6. Outside of polymer side of blister pack packaging7. Inside of polymer side of blister pack packaging

(side exposed to tablet)

Results and Discussion The infrared spectra from four of the seven aspects analyzed were different from each other in the two different samples making it possible to easily distinguish the genuine article from the replicate. These aspects include the outside of the sealed box, the glue used to seal each box, the outside of the foil backing side of the blister pack packaging and the outside of the polymer side of the blister pack packaging. The infrared spectra collected from each of these areas for each sample are different from each other and are displayed within Figure 1 through 4.


IlluminatIR™Application Brief AIL-I01 (continued)

Conclusion Public Health Laboratories (PHLs) face a myriad of analytical issues. These results show that it is possible to easily differentiate authentic from counterfeit articles using the IlluminatIR™ Infrared Microprobe. In this instance, simply testing the packaging used for storage and shipment was enough to provide the discrimination needed to determine authenticity. It is also possible to analyze the suspect item itself in a similar manner in order to reach the same end result. The IlluminatIR™ Infrared Microprobe is ideally suited to perform this chemical analysis, enabling a rapid, reliable non-destructive evaluation of the sample.

References1 http://usinfo.state.gov/products/pubs/intelprp/protecting.htm2 http://www.iccwbo.org/policy/ip/id2948/index.html3 http://www.ice.gov/pi/cornerstone/ipr/index.htm4 ICE Efforts to Combat Counterfeit Pharmaceuticals, Fact Sheet,

July 11, 2006

Figure 7. Inside of Box

Figure 1. Outside of Sealed Box

Figure 4. Outside of Polymer Side of Blister Pack Packaging

Figure 5. Inside of Polymer Side of Blister Pack Packaging

Figure 2. Glue Used to Seal Box

Figure 6. Inside of Foil Backing Side of Blister Pack Packaging

Figure 3. Outside of Foil Backing Side of Blister Pack Packaging


Product Overview IlluminatIR™

Changing the pace of microscopic identification IlluminatIR™ has been widely adopted as an easy and accurate method to identify the chemistry of microscopic unknown compounds. Samples can be microscopic quantities of crystals, solids, “specs”, “spots”, powders, films, fibers, paint chips, pastes, gels, and liquids of pharmaceuticals, foods, petrochemicals, plastics and many others.

Fast and easy as 1-2-3Powerful SynchronizIR software provides outstanding capability for microscopic analysis and allows non-FT-IR-specialist personnel to operate the instrument with minimal training while ensuring accurate and verifiable results• View your sample and capture images with the high resolution

digital camera• Rotate the infrared objective into place and collect your data

and generate results• Clean the diamond in seconds by wiping with ordinary solvents

making it ready to analyze the next sample with no delay

Reliable and accurate Simplicity in training, operation and service make the IlluminatIR™ the most productive and cost-effective instrument for identification of microscopic chemicals in a wide range of applications.• FT-IR spectroscopy is well established as a simple, precise

measurement technique that gives reliable results in seconds.• Smiths offers the most comprehensive set of libraries in the industry

which assures your results are accurate and agree with well accepted standards.

• Training, application support, and technical support are at your service

Identification microscopically IlluminatIR™ is an advanced infrared microspectrometer which will fit onto a research-grade, infinity corrected light microscope.



There are six configurations of the IlluminatIR™ available. The standard or basic configuration comes with a library of over 3,300 chemicals. Four configurations are available with additional libraries tailored to the drug analysis, pharmaceutical laboratories, forensic labs, and public health labs.

IlluminatIR™ StandardIlluminatIR™ Standard system is the base model for all IlluminatIR™ packages. It includes both the ARO and ATR objectives, as well as a sample prep kit and the SynchronizIR software platform.

Included with this system is the common chemicals library totaling over 3,300 spectra.

IlluminatIR™ DAIlluminatIR™ Drug Analysis brings the power of chemical analysis to light microscopy for the forensic drug chemist. Easily identify an illicit drug without requiring extraction of the drug from its cutting-agent matrix. Results are generated with ease and accuracy.

Spectral libraries are sold separately.

IlluminatIR™ PIIlluminatIR™ Pharmaceutical Industry (PI) brings the power of molecular analysis to light microscopy for the pharmaceutical scientist for the instant identification of microscopic unknowns. Applications include API, excipient and impurity identification, counterfeit analysis, reverse engineering, product distribution within a tablet and many more. Samples include tablets, powders, liquids, films, fibers, gels and many more.

Included with this system are the common chemicals and pharmaceutical excipients libraries totaling over 3,400 spectra.



IlluminatIR™ FLThe IlluminatIR™ Forensic Laboratory (FL) is aimed at helping to identify the types of unknowns that are common in the forensics laboratory such as white powders, fibers, and other trace evidence.

Included with this system are the common chemicals, explosives, forensic drugs, drug precursors, fibers and common white powders libraries totaling over 4,200 spectra.

IlluminatIR™ PHLIlluminatIR™ Public Health Laboratory (PHL) was developed as a comprehensive trace evidence system including a sealed cell kit for handling potentially dangerous materials.

Included with this package are the common chemicals, nerve & blister (WMD) agents, toxic industrial chemicals, explosives, forensic drugs, drug precursors, fibers, asbestos, and common white powders libraries totaling over 4,600 spectra.

IlluminatIR™ ATMSIlluminatIR™ Automated Thermal Microscopy & Spectroscopy (ATMS) system unites the power of three chemical analysis techniques (microscopy, spectroscopy and thermal analysis) to enable solid-state characterization of pharmaceutical compounds, active pharmaceutical ingredients (API), and their polymorphic and other solid-state forms.

Included in the package is the Linkam LTS350 hotstage, the common chemicals and pharmaceutical excipients libraries totaling over 3,400 spectra.


Software IlluminatIR™

SynchronizIR SoftwareSynchronizIR software is used to drive the IlluminatIR™ Infrared Microprobe for the analysis of microscopic particles of interest. In 2001, the IlluminatIR™ was introduced into the marketplace and became the only infrared spectrometer accessory capable of attaching to research grade, infinity-corrected light microscopes. This was possible because of the innovative design of a miniature infrared spectrometer capable of reliably collecting quality infrared spectral data from particles with dimensions as small as 10x10 μm2. This system now operates using SynchronizIR software – a full integration of analysis FIVE, the premier image-analysis software package – with the IlluminatIR™ Infrared Microprobe and its accessories.

A significant advantage of integrating an infrared micro-spectroscopy system with a complete image-analysis program is that, aside from being able to use the image-analysis features to select the appropriate particles for infrared analysis, it is also possible to use any image-analysis functionality to enhance the quality of the data generated. Some of these features are highlighted.

The optical and morphological features of the sample are observed using the light microscope.

Particles of the appropriate size and shape are selected, counted and measured automatically using SynchronizIR software.

Infrared spectra are collected from only particles of the appropriate grey-scale intensity, size and shape.


Software IlluminatIR™

MeasuringPerforming functions like counting particles, measuring dimensions, and calculating the distance between two lines is made simple with an easy-to-use, point-to-point interactive software interface.

DatabaseThe software systematically stores all images, spectra, analysis results, data sheets, graphs and other acquired data in an easily-retrievable format.

Extended Focal ImagingMultiple versions of the same image, each focused at a different position, can be combined to produce a single, wholly-focused image. This function allows clear imaging of samples with a depth that cannot be observed in complete focus using conventional techniques.

Particle AnalysisAutomatic separation of particles within a given image is possible using the integrated separator function. Users can set a specific “detection area” or region of interest. Many other parameters can be used to measure particles automatically, or carry out statistical data processing.

ReportThe click of a mouse creates your Report, which incorporates the sample’s image, spectra, library search results and data collection parameters. Reviewing and summarizing data into a single document has never been easier.


Technical Specifications IlluminatIR™

Description SpecificationSample Type Microscopic solid, gel, liquid, powders, fibers,

chips, etc.Sensitivity 4 cm-1

Range 4000 to 650 cm-1

Beam Splitter Options KBR or ZnSeSample Size 10 microns to 100 micronsSample Interface IR microscopic objectives: ATR, AROSample Viewing Research grade microscopeUser Interface (Software) SynchronizIR SoftwareOperating System Windows XP®

Computer Interface USB 2.0Size 8” x 16.5” x 6” (20 cm x 43 cm x 15 cm)Weight 21 lbs. (10 kg)Input Power 100 – 240 VAC, 50 – 60 Hz, 2.0 Amps maxPower Supply Output 12 VDCStandard Microscopes Olympus BX Series MicroscopesCompatible Microscopes Nikon, Olympus, Zeiss, and Leica Microscopes. Compatible Microscopic Techniques Bright field, Dark field, Polarized Light

Microscopy (PLM), Differential Interference Contrast (DIC), Fluorescence, Hot Stage Microscopy, Raman Microprobe Spectroscopy, Visible Light Micro Spectroscopy

Windows XP® is a registered trademark of the Microsoft Corporation.


Ordering Information IlluminatIR™

Product Applications Part Number IlluminatIR™ DA (OBX) Drug Analysis 006-2215

IlluminatIR™ Standard (OBX) Standard Configuration 006-2201

IlluminatIR™ PI (OBX) Pharmaceutical Industry 006-2209

IlluminatIR™ FL (OBX) Forensic Laboratories 006-2207

IlluminatIR™ PHL (OBX) Public Health Laboratories 006-2203

IlluminatIR™ ATMS (OBX) Automated Thermal Microscopy System

Pharmaceutical Polymorph Analysis

006-2211

Note: OBX indicates the IlluminatIR™ is configured for use with Olympus BX Series Microscope (microscope sold separately; see below).

Accessories Part NumberOlympus BX-41 PLM Microscope 006-2328Olympus BX-51 Microscope 006-2331Olympus BX-51 PLM Microscope 006-2329Olympus BX-61 Microscope 006-2302Olympus BX-61 PLM Microscope 006-2330

Options Part NumberGalactic GRAMS / AI with Spectral ID 040-4033IlluminatIR™ Desktop Controller 006-4126IlluminatIR™ Laptop Controller 006-4065


Notes IlluminatIR™

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