Marilyn Jacox, PhD, was a pioneer in the infrared (IR) and electronic spectroscopies of free radicals and small molecular ions in matrix isolation. Before Jacox's recent passing, Ingeborg Iping Petterson had the pleasure of speaking with her about her career in spectroscopy. Jacox shared her personal story, reflections on her extensive career, and insight and advice for young scientists, especially women, who are just starting their careers.

Marilyn Jacox was born in 1929 in Utica, New York. Her father was a master baker, and her mother was a homemaker. Her parents possessed great intellectual ability but lacked the educational opportunities to become scientists themselves. Both came from single-parent homes with modest finances, and her father left school in the seventh grade to find a job.  

Her parents supported her interest in school and getting good grades, and eventually her budding science career. "They played very different roles, but one of the big things was that I was accepted," said Jacox. "My parents' dream for me when I was small was that I would continue in music, get a respectable office job, marry, and raise a family. When I began to have a successful science career, my father was intensely proud of his little girl, and my mother just plain accepted me."  

Figure 1: Marilyn Jacox outside her home near Gaithersburg, Maryland, in April 2013.			

Jacox grew up in the Depression, a time when most women in the United States did not drive, and many did not even write checks. Those were tasks for the male head of a household. The three generally accepted professions for women then were teaching, nursing, and secretarial business positions. Jacox started out studying business in high school because she thought that would give her the best prospect for a future job. She concluded that the business practices that she learned in her mandatory "Introduction to Business," course and typing skills she learned served her well throughout her career.  

She joked that her friends in school teased her that she only received good grades because she studied easy subjects. "But when I switched focus in my junior year and began doing college preparatory courses, my grades went up!" she recalled. She also remembered a young woman with whom she rode the bus, whose dream of studying chemistry inspired her to do the same. "I knew then that a college preparatory curriculum would help me." 

Figure 2: Jacox looking at a photograph of herself from 1973, when she was working at the National Bureau of Standards.			

As a young child, Jacox was passionate about music. She took voice and piano lessons from several different music teachers, including various women living in her neighborhood. Then a family moved into a house down the block, and the man was a professional musician. "He became my music teacher for most of the remaining time during which I studied music, and that made an enormous difference in my progress," Jacox said. "I translated that into 'If a young woman is seeking an education in science, she should go get the best education and best teachers she can.'" 

Her first scientific mentor, John C. Keller, her undergraduate supervisor from the Department of Chemistry at Utica College of Syracuse University, was indeed an excellent scientist. "In this world, it makes a lot of difference who your teachers are, and he was the best," she said. "I think this point is very important to pass along to young women who are highly motivated in science."  

Oscar K. Rice from the University of North Carolina, with whom she worked after her graduate study at Cornell University, was another important mentor. "He was a wonderful person in every sense, a superb scientist yet very unassuming," she said. Jacox talked about many of her mentors in her autobiographical article, "On Walking in the Footsteps of Giants" (1). 

Another important influence on Jacox's career was her long-time collaboration with Dolphus "Dick" Milligan, which began when Jacox became a Fellow at Mellon Institute of Industrial Research in Pittsburgh. The director of the Mellon Institute had a mandate to encourage new research, and she and Milligan were appointed at this opportune time. Although her first research studies were on the IR spectroscopy of molecular solids, soon she began to collaborate with Dick in his studies of free radicals. 

Jacox felt that her work with Milligan played an important role in her professional development. "He had information and experimental ideas left over from his Berkeley thesis, and he wanted to test them," she recalled. "In the beginning of our collaboration the ideas were his. I learned from him, and after collaborating for a modest period of time, sometimes we didn't know who had the idea first, and alternated first authorship of our publications." 

When considering the obstacles she faced professionally, Jacox named three areas of challenge. "I sum it up when I tell people about the rough times for me as a woman, as a government worker, and as a scientist doing matrix isolation of small molecules," she said. 

Jacox observed gender bias firsthand early in her career, when she was seeking an academic position after completing postdoctoral research in 1958 at the University of North Carolina. Despite the national excitement about science following the launch of Sputnik by the Russian space team, her résumé received a favorable response only from women's colleges. "I had never really liked the idea of separate men's and women's colleges, because in the real world you need to be able to deal with both men and women," she said. When she saw that coed schools would not hire a woman, she broadened her job search and eventually landed an appointment as a Fellow at the Mellon Institute. But that did not mean equal pay; her long-term collaborator, Dick Milligan, another Fellow, earned more. "This gender-based disparity was widespread," she recalled.   

The gender bias was not limited to pay, however. "The culture of the organization when I arrived was like something out of the 'Mad Men' television show," she said. "The guys went to a local pub for lunch, and would go golfing together. These were very 'guy things' to do, and the culture was very male dominated. It is much less so now." 

Jacox recalled a few specific examples, including one when she and Dick Milligan were new at the Mellon Institute. "Our work was advancing at a healthy rate, and he was friends with a next-up manager who was planning a cocktail hour at his home, to which he invited Dick, but not me," she recalled. Since Dick was married with two small children, she often gave him rides to work functions so that he could leave the family car with his wife. This time, too, she drove Dick to the party and waited for him in the car.  

That was around 1959. But another example occurred more than a decade later, many years after Jacox and Milligan had both moved to the National Institute of Standards and Technology (NIST), in Gaithersburg, Maryland, which was then called the National Bureau of Standards (NBS). In early 1972, a Canadian scientist they knew was in town and gave a seminar. Some of the older male scientists arranged for him to stay at the Cosmos Club in downtown Washington, DC, and arranged a dinner in his honor there the evening after his seminar. This time, both Milligan and Jacox were invited. "We went home in the evening, changed, and Dick arrived at the Cosmos Club just before I did," Jacox recounts. "He went to the front door, and even though Dick was black, there was no problem at all; Dick was directed to the room where the dinner was held." Not so for Jacox. 

It was a rainy day and she had parked about a block away, and by the time she made it to the front door, she was drenched. "An elderly man answered and told me that ladies could not come in the front door, but had to use the side entrance," she said. Before she could catch her breath, he took a second look, decided to have mercy, and let her in. "I never forgave the Cosmos Club for that injustice," she said. "I have since been invited to join, but have replied 'Thanks, but no thanks'." 

Although Jacox was pleased to see a lot of progress of women in recent years, she didn't expect sexism to go away. "Young boys go through a stage where they band together and build tree houses and don't like girls; some guys grow up and some stay in that psychological state," she said. "It's not likely to change. Where it matters most is in organizations with limited funding resources. The guys have their own special ways of communicating among themselves and getting themselves at the top of the list." 

As a result, Jacox said, women must step up to make names for themselves in the scientific community. "Stand on your feet and present your stuff — in academia and also in industry," she declared. "Too many young women shy away from that kind of thing. If you don't go to the meetings or present results you are not going to get very far in science."  

A good, solid publication record is vital, she continued. "Young women need to get participating in the scientific community, and they need to see the research through to its publication." 

When Jacox published, she felt it was important to use her full name, so that people knew she was a woman. She remembered discussing this with another woman scientist at the Mellon Institute. "She and I never agreed on how to handle our names," Jacox recalled. "When she applied for meetings she put her name on her papers with just her first and middle initial. I preferred, then and now, always to use my full first name. Using just your initials doesn't help other women."  

On Working in Government, and the Federal Women's Award

Jacox moved from the nonprofit Mellon Institute to NIST in 1962. Although Jacox felt that the situation for women scientists was generally a bit better at NIST, working at a government research institute also presented challenges. In Jacox's view, government workers often do not receive the respect they deserve, and that can lead to resource limitations. "Frankly, since the late 1970s, government has been perceived as a part of the problem." she said. "This public mindset has been exploited by both political parties, and government employees and their funding have not been totally respected because of where the work has been done." 

Within government, there was also a lack of recognition of the achievements of women. But in 1960, Julia Lee, a vice president at Woodward & Lothrop, a major department store in downtown Washington, DC, decided to do something about that. "Julia used her clout as a corporate vice president to establish a Federal Women's Award, given to six women each spring," Jacox explained. Awardees were drawn from all of the federal government agencies, nominated by their personnel departments. Jacox received the award in 1973. "It was nicely arranged," she said. "That award has not been given since about 1975, but the women who did receive it had this common bond, and we have stayed in contact." 

Jacox's career is important not only because of her leading role as a spectroscopist at a time when few women were prominent in the field, but also because she did pioneering work using IR spectroscopy to study the structure of small molecules trapped in matrix isolation. Matrix isolation of unstable molecules in solid noble gases was developed in the early 1950s (2) and Jacox was one of the first scientists to use this technique extensively to study the IR spectra of free radicals (3). 

"Scientifically, there wasn't much known [at the time] about spectra of free radicals, and Dick [Milligan] and I had a tool to observe these spectra, because the IR spectrum is to a molecule what a fingerprint is to you," she said. "Herzberg's two first spectroscopy volumes had not been available for very long when we began our work, so for many small molecules much was still unknown." 

Jacox noted that one of the challenges with her work arose because she didn't see spectra of molecules in the gas phase, but rather frozen in solid argon or neon. The interaction energy between an argon or a neon atom and a molecule is minimal but non-zero, implying that small spectral shifts occur. "People used that as a reason to question my data," she said. 

To address that problem, Jacox started a database comparing the positions of gas-phase band centers with the positions of the corresponding absorption maxima observed in various inert, rigid matrix environments. She found that the infrared absorptions of small polyatomic free radicals trapped in solid argon or neon usually appear within 1% of corresponding gas-phase band centers. "When the argon- or neon-matrix position of a free-radical absorption has been identified, this generalization becomes a useful tool in the search for the gas-phase absorption," she explained. "Through the years, this generalization was key to my scientific survival." The compilation grew and matured into the "Vibrational and Electronic Energy Levels" section of the NIST Chemistry WebBook, available on the internet (4). 

"Basically, my measurements are qualitative," she continued. "In order to be able to see free radicals at all you've got to have a spectrum, and you have to prove what the carrier of the spectrum is. That's the part I most enjoy." She noted that as analytical chemistry has grown enormously over the past 50 years, there has been a greater drive for quantitative analysis. Although matrix isolation measurements are not well suited to that, they interface very well with quantum chemistry. 

"It's been fun to interact with quantum chemists," she said. "It used to be that vibrational fundamental calculations for free radicals were very approximate. Recently, computers and computational procedures have developed that enormously." 

A split in the field of molecular spectroscopy also occurred, Jacox observed, between chemical spectroscopic analysis and studies of the nature of the chemical bond. "As a physical chemist, I'm biased toward studying the properties of chemical bonds," she said. "Even though computational chemists are getting to the point where, jokingly, I say that they are putting me out of business, refinements in computational procedures need to be checked by comparison with experimental data." 

In talking about all the scientific problems in this area yet to be solved, Jacox sounded like a scientist in mid-career, not someone in her eighties. And in fact, she continued to do research at NIST, as a scientist emerita until her passing.  

"Calculations are not so well developed for excited electronic states," she said. "Even more complexity is required to study crossings of potential surfaces, which are ubiquitous." 

She also commented that another question far from being pinned down is the relationship between the details of spectra and chemical reactivity. "There are lots of applications in which that could be important, ranging from the chemistry of interstellar matter to various industrial and environmental chemical processes," she said. "Interaction with quantum chemical calculations will continue to become more important to the field." 

Jacox experienced a lot of satisfaction in her career. "Each publication is a milestone for me," she said. "I don't have a perfect track record, but I have a respectable one, and it's very interesting to watch where the results of your work go." 

When Jacox was hired at NIST (then called NBS), the Free Radicals Program had recently finished. "It revolutionized chemistry at NBS," she said. "They were at the end of a generation of chemists, and it inspired the hiring of many new, talented people." Indeed, Jacox, and her friend and colleague Dick Milligan, were two of the talented scientists hired to continue research into free radicals, which was still a hot topic, even though the formal program had ended. 

What Jacox called her own "Mount Everest," however, involved moving beyond free radicals to the spectroscopic study of small molecular ions, which are often several orders of magnitude more reactive than are free radicals. "I had ideas about how molecular ions could be produced and stabilized, and how I could study their spectra," she said. "These ideas worked. I have been studying primarily the spectra of molecular ions for 25 years now." Mass spectrometry, she pointed out, gives primarily the empirical composition, not structure. "If you are clever enough, sometimes you can pin a given fragmentation pattern to a specific structure [using mass spectrometry] but most of the time the structural information isn't there." 

During Jacox's time at NIST, the laser soon began to be important, and Jacox envisioned that laser scientists would pick up where matrix measurements left off. Gradually more and more complicated molecules would be discovered, she thought, and the field and tools would develop in such a way that it would become possible to study larger and larger species. Instead, she watched as funding agencies shifted priorities and as a result the scientific community moved in the new direction, heavily focused on the properties of nanostructures.   

Jacox also lamented that most of the citations of her work are to the older part of her work, rather than on her later studies. "I had hoped to see where the molecular ion results would lead during my lifetime, but I am beginning to think I will need to be more than 100 years old to see that!"  

Jacox conceded that pursuing a career such as hers was not easy, and thus would recommend it only to young scientists who are truly motivated. "Too often young people are getting their degrees in science, only to face a difficult job market," she said.  

Her advice is essentially the same for young women considering a career in her field. "For a young woman graduate student, I would recommend my field if she is good at it and really enjoys it, but if she doesn't have that to begin with I would not try to point her in that direction," she said. But once a student decides to pursue a scientific path, and has committed to a graduate advisor, she should see it through to her degree, Jacox believed. "The advisor invests time, interest, and intellectual capital in a graduate student, and if that person drops out, that can be very disappointing to the advisor." That same dedication to completing work should continue throughout a woman's career, she added. "More generally, if a woman is committed to a project that yields publishable results, she should see it all the way to publication. She should also begin to participate in meetings as soon as she can." 

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Before Marilyn Jacox's recent passing, Ingeborg Iping Petterson had the pleasure of speaking with her about her career in spectroscopy.

Marilyn Jacox, a Pioneer in Infrared Spectroscopy

With the increasing proliferation of counterfeit drug products, there is an incentive to screen drugs for legitimacy. One method is to examine the tablet itself, which is usually a destructive operation. Another method that has been explored is to characterize the packaging, which enables nondestructive screening of the product. Raman microscopy has been found to be a useful tool, and it is often the tool of choice for these measurements. Raman mapping of tablets enables not only the identification of components, but also the mapping pattern can enable matching manufacturing sites of different tablets. The study of packaging materials can provide an identification of coating and a depth-profile measurement of thickness. In addition, unlike Fourier transform infrared spectroscopy, which can only measure surface materials, Raman microscopy can provide spectra of dyes and pigments, even if they are printed below a surface film.

In this column installment, we present results of tablet matching measurements and characterization of packaging. The examples show how these measurements aid in the detection of counterfeit drugs.  

Two tablets of a well-known drug product were acquired, one from a pharmacy in the United States and one via the mail from an internet pharmacy. Initially, maps of the entire tablets were produced using a DuoScan Raman imaging system (Horiba Scientific) with a 10× objective and a 200-μm pixel. Although maps were acquired in reasonable amounts of time, there did not seem to be any variability in the data. That is, on the 200-μm scale, the tablets were homogeneous, at least for this product. So, smaller maps were collected to make chemical and textural comparisons. This work was performed on an Evolution Raman microscope (Horiba Scientific), an 800-mm focal length system, using a 600-grooves/mm grating, and a 532-nm laser. Maps were acquired in the fingerprint region (200–1800 cm

). LabSpec multivariate analysis software (Horiba Scientific) was used to create the Raman maps, using either the classical least squares (CLS) algorithm when identifiable species could be seen in the data set, or the multivariate curve resolution (MCR) algorithm when the chemical factors were not apparent. As the results will show, operating on the two data sets independently produced verification of the conclusions.   

In reality, it was Witkowski of the Food and Drug Administration (FDA) Forensic Chemistry Center in Cincinnati, Ohio, who originally proposed the use of Raman mapping for identifying chemical species and morphological distribution as a means of differentiating counterfeit from authentic tablets, and for differentiating counterfeits from various manufacturing sites (1). Since that work was done, developments in mapping hardware have improved data collection times and multivariate software provides better quality images resulting from the use of scores mapping rather than intensity between cursors, which is subject to much more noise.  

Figure 1: Left: Raman maps of tablet purchased from a pharmacy in the United States produced with fingerprint spectra (top) and CHâOH spectra (bottom). Right: Spectral loadings used to create the maps on the left. The red species is presumably the active drug, having bands near 1600 cm-1 and above 3000 cm-1 that indicate unsaturation. The reference spectrum shown in dark green is that of a cellulose paper filter.			

Before data collection, the tablets need to have the coating (usually opaque and pigmented) removed and the surface flattened. The maps shown in Figures 1 and 2 were acquired with step sizes between 5- and 15-μm, using the DuoScan Raman imaging system to distribute the laser intensity over each pixel in the map. The SWIFT imaging option (Horiba Scientific) was used to further reduce the acquisition times by eliminating stop and start operations at each data point. After the acquisition of a map, the entire hyperspectral cube was baseline-flattened to improve the quality of the multivariate results. Then we either surfed the file to identify regions of homogeneous spectra that could be used with the CLS algorithm or the MCR algorithm was invoked to "find" the spectral components in the file. Figures 1 and 2 show maps for each of the two tablets based on the fingerprint spectra and then the CH–OH spectra. The loadings ("spectra") used to produce the maps are also shown with reference spectra of the excipient presented for comparison. Because the identity of the active pharmaceutical ingredient (API) was not known, its spectrum could not be shown for comparison. However, APIs tend to be aromatic with large intensities above 1500 cm

; the loadings shown in red in Figures 1 and 2 are presumed to be API and one can see that the red factors show the same spectra from the two tablets. That is, the counterfeit tablet did contain the active ingredient.  

Figure 2: Left: Raman maps of tablet purchased over the internet produced with fingerprint spectra (top) and CHâOH spectra (bottom). Right: Spectral loadings used to create the maps on the left. The red species is presumably the active drug, having bands near 1600 cm-1 and above 3000 cm-1 that indicate unsaturation. The reference spectrum shown in green is that of lactose. Note that use of the MCR algorithm in the CH region identified the presence of a third species (shown in grey) whose spectrum is similar to that of Mg stearate.			

Note that for the internet sample, whose results are shown in Figure 2, the CH–OH map has more morphological detail than the fingerprint map. Also, the use of the MCR algorithm revealed the presence of a hydrocarbon like magnesium (Mg) stearate. If we go back to the fingerprint map and examine the spectrum in the region where Mg stearate appeared in the CH region, then that component can be added to the factors, producing the map shown in Figure 3. Surprisingly, in this case the CH–OH region provided more information than the fingerprint region, as originally examined. We believe that this is because the fingerprint region will be dominated by the unsaturated API, whereas in the CH region, all species have similar intensities.  

Figure 3: Left: Raman maps of tablet purchased over the internet produced with fingerprint spectra. Right: Spectral loadings used to create the maps on the left. The red species is presumably the active drug, having bands near 1600 cm-1 and above 3000 cm-1 that indicate unsaturation. Both excipient spectra (grey = hydrocarbon and blue = lactose) are heavily contaminated with the API probably because the materials do not segregate on this length scale. The reference spectra shown represent lactose in green, and Mg stearate in black.			

Examining the exterior packaging of drug products could provide a more rapid and nondestructive means of screening products for authenticity. The packaging of four samples was examined. To measure the inks and papers, it was necessary to use Raman spectroscopy instead of FT-IR because in FT-IR the radiation will not penetrate any coating present on the paper. Figure 4 shows the spectra of the blue and blue-green pigments on each package, after baseline subtraction. Band frequencies in the bottom spectrum are labeled to assign these spectra as resonance-enhanced. Under such circumstances, overtone and combination bands are strong, and CH stretches are usually nonexistent. For example, 2 × 1343 ≈ 2677 cm

. (Note that the matches are usually not exact because of anharmonicity of the vibrations.)  

Figure 4: Raman spectra of green or blue inks from pharmaceutical packaging of a drug product and three possible counterfeits of that product. Spectra were excited with the 633-nm laser line and detected on an Evolution Raman microscope (Horiba) equipped with a 600-grooves/mm grating. All spectra exhibited a fluorescence background, presumably from the pigment itself; spectra were baseline-corrected for the display.  			

The three bottom spectra in Figure 4 appear to be quite similar. Although the bands in the top spectrum do coincide with bands in the other samples, some of the spectral bands in the bottom three samples are absent; in particular, small bands at 1269 and 1487 cm

  are missing. In the absence of information on the pigments, one can presume that the unsaturated cores of the molecule that provide the color are quite similar, with some small (unsaturated) modifications which would account for small spectral differences.  

Figure 5: Raman spectra of white regions from pharmaceutical packaging of a drug product and three possible counterfeits of that product. Spectra were excited with the 633-nm laser line and detected on an Evolution Raman microscope (Horiba) equipped with a 600-grooves/mm grating. Spectra were baseline-corrected for the display. 			

Figure 5 shows spectra acquired from the noncolored regions of the same packages. There is a one-to-one correspondence between Figures 3 and 4; that is, the top spectra on each figure come from the same sample, and likewise for the other spectra. Again, one can see that the top spectrum is an outlier.  

Figure 6: Raman spectrum of white region of one of the packaging samples (top, red) superimposed on a reference spectrum of polypropylene (middle), and calcite (bottom). 			

The goal of these measurements was to determine if the packaging materials could be differentiated, and obviously that is documented. But since we have been looking at spectra for so long, we always like to challenge ourselves and see if we can guess what is present. Clearly, that is a frightening idea for the novice who is looking for quick answers to a problem. So, at this point we want to show how current commercial products can alleviate the need for highly experienced spectroscopists. We looked at the spectra of the white packaging and recognized the presence of calcite and polypropylene. Figure 6 shows the bottom spectrum of Figure 5 superimposed on our reference spectra of calcite and polypropylene. It is easy to see that most of the spectral features and intensity can be accounted for by these two species. However, the interesting thing was to determine what the BioRad KnowItAll software (2) (BioRad Laboratories) and database could do with this spectrum. Figure 7 shows the results of a mixture analysis performed in that software. The top part of the figure shows the components that were used to fit the spectrum; note that although we were aware of the calcite and polypropylene we were not aware of the polystyrene until the mixture analysis pointed out its presence. Each of these spectra are shown, as well as the measured spectrum and the residual from the fit. Note that because of the tendency of polymers to orient, the relative intensities of the various bands will vary from measurement to measurement, so most of the features in the residual spectrum are not of concern. In addition, the weighting column at the top of the figure shows the relative contributions that were used to fit the measured spectrum. The bottom part of Figure 7 shows, in more detail, the measured spectrum superimposed on the composite spectrum.  

Figure 7: Results of the mixture analysis in the KnowItAll software. The bottom of the figure shows the measured spectrum (in black) superimposed on the composite spectrum (in red) which is constructed using the pure spectra shown in the top of the figure			

In the first part of this column installment, we showed that Raman images constructed from 

 maps of tablet surfaces can provide information both on the composition of the tablets and the components' distribution pattern, both of which can help determine if a sample is authentic or not. In addition, differences can aid in determining if counterfeit samples have a common origin. In the second part of this column, we showed that spectra of the packaging material, which can be recorded without any sample destruction, can also aid in the identification of counterfeits. In addition, for those who want to be able to identify material from the spectra, we showed how the use of commercially available software can provide identification, even for a mixture sample. 

(1) F. Adar, E. Lee, and M. Witkowski, "Single Point Analysis and Raman Mapping of Dosage Formulation as a Means for Detecting and Sourcing Counterfeit Pharmaceuticals," presentation at Pittcon 2007. 

(2) Bio-Rad Laboratories, Inc., Philadelphia, Pennsylvania, 2014. 

 is the Principal Raman Applications Scientist for Horiba Scientific (Edison, New Jersey). She can be reached by e-mail at 

 is the applications manager for the Americas for the chemical and biological product line at Smiths Detection. Prior to holding her current position at Smiths, Pauline worked as a pharmaceutical R&D scientist at Purdue Pharma in Ardsley, New York. Pauline holds a B.A. degree from Marist College, an M.S. degree from John Jay College of Criminal Justice, and a PhD from the Graduate Center of the City University of New York. 

 is an Associate Professor of Forensic Science and Chemistry at John Jay College and the Graduate Center, CUNY. He has been in the academic world for more than 15 years after completing 23 years in a municipal crime laboratory. His research interests are in the applications of light and electron microscopy along with UV–vis and vibration spectrometry to the analysis of micro and ultramicro samples. 

Molecular Spectroscopy Workbench

Results of tablet matching measurements and characterization of packaging are presented.

Raman Microscopy for Detecting Counterfeit Drugs — A Study of the Tablets Versus the Packaging

Laser ablation molecular isotopic spectrometry (LAMIS) involves measuring isotope-resolved molecular emission. Measurements of several key isotopes (hydrogen, boron, carbon, nitrogen, oxygen, and chlorine) in laser ablation plumes were demonstrated. Requirements for spectral resolution of the optical detection system could be significantly relaxed when the isotopic ratio was determined using chemometric regression models. Multiple applications of LAMIS are anticipated in the nuclear power industry, medical diagnostics and therapies, forensics, carbon sequestration, and agronomy studies.

Laser ablation molecular isotopic spectrometry (LAMIS) is a technique that uses optical spectra of transient molecular species produced in ablation plumes in air or buffer gases (Ar, Ne, He, and N

) for rapid isotopic analysis of solid samples (1–8). This technique is similar to laser-induced breakdown spectroscopy (LIBS), but optical emission spectra in LAMIS are measured at longer delays after an ablation pulse than what is used in LIBS. Molecular emissions yield relatively easily detectable isotopic information. Therefore, LAMIS adds a supplementary function of isotopic measurements to the well-established benefits of LIBS (real-time elemental analysis at atmospheric pressure, minimal sample preparation, chemical mapping and depth profiling at high spatial definition, and laboratory and field operation possible at a standoff distance to the sample). The LIBS and LAMIS techniques can be accomplished on the same instrument. 

Molecular radicals are generated effectively when the ablation plume cools down, resulting in an increase of the molecular emission in the plasma afterglow. Several mechanisms contribute to the formation of molecules in the plume such as radiative association of neutral atoms, associative excitation or ionization, recombination of molecular ions, fragmentation of polyatomic clusters or nanoparticles, and vaporization of intact molecules from the ablated surface. Molecular spectra are advantageous for isotopic analysis because the isotopic shifts in molecular emission are considerably larger than in atomic spectra. The difference in isotopic masses has only a small effect on electronic transitions in atoms, but significantly affects the vibrational and rotational energy levels in molecules (1). A compact spectrometer can be sufficient to resolve molecular isotopic spectra; therefore LAMIS measurements can be performed in the field (4,5). The ability to measure isotope abundance using a portable spectrometer with modest spectral resolution is a significant merit of LAMIS, along with no sample preparation and data collection at ambient pressure. 

The qualitative and quantitative studies of isotopes of the light elements are both very important for their use in many fields and for their very extensive scope, with many applications in different fields of their chemistries and materials science. This article describes measurements of isotopes of hydrogen, boron, carbon, nitrogen, oxygen, and chlorine. All of these elements are ubiquitous in nature as well as in practice, with carbon being the basis of many millions of compounds. Variation of the natural isotopic ratio 

C in different materials ranges from 0.96% to 1.15% (9). These variations can be measured using LAMIS. Fractionation in stable isotopes of C, N, and O can be particularly indicative of a range of diverse biotic and abiotic processes relevant to the ecology, biosphere, and geochemistry, both organic (fossil) and inorganic. Fertilizers enriched typically above 40% in 

N are broadly used to track the efficiency of plant uptakes, fertilizer losses, and nitrogen turnover in soil.  

B isotope led to the development of multiple boron-loaded materials for neutron shielding in nuclear reactors and spent fuel storage pools, as well as for screens and curtains in nuclear medicine centers. Enrichment from 50% up to 99% in 

B is often used. Many neutron detection devices are also based on highly borated (

H) scintillator materials. Heavy water nuclear reactors require deuterium enrichment to 99.85%. Water-

O is used as a precursor in the radiopharmaceutical industry. Deuterated drugs and stable isotopic markers are increasingly used in the fields of medicine, pharmacology, nutrition, and physiology for tracing biochemical processes. Medical multinuclear magnetic resonance imaging (MRI) scanners require compounds enriched in nuclear magnetic resonance (NMR)-active isotopes (

O). To assess quality, specifications, or aging of these materials, their isotopic homogeneity or distribution, and  degree of isotopic enrichment can be rapidly tested by LAMIS in open air or inert gas flush without using laborious and expensive mass spectrometry (MS). The feasibility of standoff LAMIS analysis is particularly important for the nuclear industry.  

Natural geochemical heterogeneity in isotopic composition reflects the complex history of our planet. Isotopic ratios of B, C, O, and Cl are particularly variable in nature because of chemical reactivity, high solubility, and volatility associated with the compounds consisting of these elements. Boron and chlorine isotopic fractionation is used to trace the records of evolution and weathering reactions because of interactions of rocks, soil, and sediment materials with water. Biomediated alteration of the 

Cl ratio is distinctive in microbial reduction of anthropogenic perchlorates, biphenyls, halocarbons, and other chlorinated compounds. Simultaneous measurement of multiple stable isotopes is also increasingly used to constrain the carbon cycling in ecosystems better. LAMIS can facilitate the isotopic analysis directly in the field. Numerous applications of LAMIS are anticipated in the nuclear power industry, medical diagnostics and therapies, forensics, carbon sequestration, and agronomy studies.  

A flash-lamp pumped Nd:YAG laser operating at 1064 nm with a pulse energy of 50–150 mJ and a pulse duration of 4 ns was used to ablate the samples. The laser beam was focused onto the sample with a fused-silica lens to a spot diameter of ~100 μm, either in open air or in a cylindrical quartz chamber flushed with inert gas. Inert gas flow removed ambient air when the rare isotopes of nitrogen and oxygen were to be measured. A second lens was used to collect the laser-induced plasma emission in the direction perpendicular to the ablating laser beam. Two spectrometric configurations were interchangeably used. In one of them, collected light was focused onto a fiber-optic cable coupled to a compact echelle spectrograph (EMU-65, Catalina Scientific) fitted with an electron multiplying charge-coupled device (EMCCD). In the other configuration, collected light was focused onto the entrance slit of a Czerny-Turner spectrograph fitted with an intensified charge-coupled device (ICCD). The spectrum acquisition gate widths and delays were optimized to detect molecular emission, while minimizing intensity of atomic and continuum radiation. The collected spectra were averaged over several tens or hundreds of laser shots (see further text and notes in the figure captions). The spectral averaging, background correction, normalization, and final chemometric regression analysis of spectral data were performed using proprietary software designed at Applied Spectra for multivariate calibration and quantification of LIBS spectra. 

Substances with known isotopic content were obtained from commercial sources in the form of powder, then mixed in appropriate proportions and pressed by a 7-ton press into 1-cm-diameter pellets. Isotopically enriched water was frozen into ice tables, which were kept cold by a Peltier thermoelectric plate during ablation for the LAMIS analysis. Samples of borated aluminum materials were provided by Ceradyne, Inc. 

Our previous measurements of boron isotopes 

B were performed using three chemically pure substances, isotope-enriched 

, and natural BN (2,7). The results indicated that LAMIS can be calibrated using multivariate partial least squares regression (PLSR) for quantitative measurements of isotopic ratio with high precision represented by relative standard deviation of only ±0.45% (2σ = 9‰), and this can be further improved (7). Abundances of individual isotopes in material composition can be measured at least down to ~1% (

N can be detected in subnatural concentrations, below 0.3%). High spectral resolution was found unnecessary for isotopic determination (2). However, an ambiguity remained about whether similar results could be obtained on practical samples with possible spectral interferences from other chemical elements.  

The pertinence of LAMIS measurements to industrial applications was tested on neutron absorber materials. Two samples of aluminum alloyed composites with different boron isotope ratios were ablated and analyzed using a 320-mm IsoPlane spectrograph fitted with a PI-MAX4 ICCD detector (Acton/Princeton Instruments). One of these samples was a Boral core (Ceradyne) that consolidated aluminum alloy with 56% boron carbide of natural boron isotope abundance. The other sample was BorAluminum (Ceradyne), a regular unalloyed aluminum (grade 1100) with 4.5% added elemental boron, enriched in the lighter isotope 

B up to 95%. The ablation spectra of these samples recorded in the 591–608 nm spectral interval are presented in Figure 1 (continuum background subtracted). They are different because the isotopic composition is different. These spectra belong to moderately resolved rotational lines of boron monoxide in the vibrational band (0

  emission system. The spectra were acquired using an 1800-grooves/mm (gr/mm) grating with the ICCD delay of 10 μs and a gate width of 100 μs. Spectral intensity was accumulated over 1000 laser pulses (analysis time 100 s at 10 Hz).  

Figure 1: Isotope-specific molecular spectra of BO (AâX; 0-3) emission formed during ablation of two aluminum-based composite materials containing different 11B/10B ratios. The upper spectrum (BorAluminum) is shifted up for clarity. Lower spectrum: Boral.			

  emission was used at both the (0-2) band (4,7) and the (0-3) band (2,7,10). A choice of the spectral interval was driven by quality of the spectra for multivariate quantification with different chemometric procedures. The data in Figure 1 illustrate a significant isotopic shift between spectra of Boral (80% 

B), requiring only modest spectral resolution. Another important factor is that this spectral interval does not have any visible interferences from atomic aluminum or chemical impurities in the samples. When the ablation spectrum was recorded at the usual acquisition delays for LIBS (<1 μs), several spectral lines of Fe, Ti, and Mn atoms coming from impurities in borated aluminum were observed within this range. Provided that the interfering lines are sparse, they do not necessarily preclude the isotopic analyses. Thus, time-gated attenuation of atomic emission is deemed essential for LAMIS measurements in practical analyses.  

A possibility of LAMIS applications for geological analysis was examined using two tourmaline samples of different color (greenish "blue grass" and black minerals). The regular LIBS spectra of these samples indicated that a spectral region from 572 to 585 nm had the least amount of atomic interferences. Aimed at further decreasing atomic emission, the acquisition gate delay was set at 3 μs for LAMIS measurements. However, two spectral lines of atomic iron were persistent in the spectrum of one of the tourmaline samples. The positions of these interfering lines could be masked from the spectrum to determine the isotopic composition using multivariate PLSR described in the literature (2,7). Further quantitative work is needed to determine the limits of isotopic precision and sensitivity in LAMIS of geological samples. However, measuring boron isotopes in real geological samples using LAMIS has already been established as a credible prospect. Our previously attained precision of 9‰ (7) is reasonably adequate because natural variation of the 

B ratio in the terrestrial environment is more than 90‰ (9). 

B content by LAMIS will be useful in other applications. For instance, the 

B isotope is added to semiconductor grade silicon as a doping agent that must be controlled. LAMIS can monitor nuclear facilities remotely, delivering a laser beam through a small window and collecting optical emission either by a telescope or an optical fiber cable.  

Carbon and nitrogen isotopes can be determined simultaneously using spectra of CN molecules in LAMIS measurements. This possibility was briefly mentioned in our earlier publications (1,4,5). The carbon isotope 

C is the most important one for studying biochemistry, but measuring only one isotope is often insufficient. Several key isotopes can follow the biological processes better. To demonstrate simultaneous isotopic measurements, we ablated natural graphite and several solid substances enriched in 

N. The spectra presented in Figure 2 include the B

  vibrational band progression: (0-1), (1-2), (2-3), (3-4) of the CN radical. The rotational structure of these bands is barely resolved in our measurements, but isotopic shifts between isotopologues of 

N are clearly observed. Two pellets of isotope-enriched benzamide with 

N atomic fractions 14% and 99%, respectively, were used as ablation targets. The latter was ablated in a helium flow to preclude nitrogen entrainment from air. Another sample was 99% enriched with 

C amorphous carbon. The spectra were acquired using a 10-μs timing gate delayed after the laser pulse only by 1.5 μs because CN molecules are promptly formed in laser ablation.  

Figure 2: Emission spectra of CN vibrational band progression (BâX; ÎÎ½ = +1) formed during ablation of benzamide pellets enriched in 13C and 15N isotopes. Spectra were averaged over 100 laser pulses.			

Isotopic shifts in the bands with changing vibrational quantum number Δν = +1 shown in Figure 2 are all larger than 0.3 nm and are easily resolvable using a compact optical spectrograph. These shifts are about 10 times greater than the isotopic shift in the (0-0) band head of the same CN B

  transition at ~388 nm (1). The opposite change in a vibrational quantum number Δν = –1 produces another CN band progression around 359 nm (Figure 3). The isotopic differences in these bands are well resolved, but their rotational structure is not. An advantage of using the bands (1-0), (2-1), and (3-2) near 359 nm shown in Figure 3 compared to those shown in Figure 2 is that the spectral peaks with Δν = –1 attributed to the minor isotopes 

N occur on the other side of and apart from the sharp edge of the 

N (1-0) band head. This is important for quantitative calibration. The progression with Δν = +1 requires multivariate calibration because the bands from minor isotopes can be hidden within unresolved rotational lines of the major 

N bands. In contrast, spectral data of the progression with Δν = –1 can be quantified using either univariate or multivariate calibration.  

Figure 3: CN vibrational bands (BâX; ÎÎ½ = â1) formed during ablation of natural graphite and enriched benzamide pellets. Spectra were averaged over 100 laser pulses.			

The results of calibrating LAMIS spectra of a CN molecule using a multivariate PLSR procedure for quantitative determination of 

C content in solid organic samples are presented in Figure 4. Artificial reference samples were prepared by physically blending powders of natural and 

C atomic fraction was assumed as 1.07%, and an additional five blended samples were made nominally at 1.2%, 1.5%, 2.0%, 5.0%, and 9.9%. The CN B→X (Δν = –1) spectra were collected in the interval 353–362 nm, similar to those shown Figure 3. The error bars in Figure 4 correspond to standard deviation of a single measurement within 10 replicates, while each measurement was made by accumulating spectra from 100 laser pulses. The natural 

 C atomic fraction was recalculated from these measurements as 1.09 ± 0.14%. Averaging over 10 measurements (each one made of 100-pulse accumulations) reduces a random error of the average down to ±0.044%. It is obvious from data presented in Figure 4 that LAMIS can measure subnatural abundances of 

C with sufficient precision. However, the decanoic acid powders used to prepare our reference samples consisted of microcrystals and it was difficult to fully homogenize a blended material. Better homogenized standards will improve precision and accuracy of LAMIS measurements.  

Figure 4: Calibration plot with a linear fit and 95% confidence interval for the 13C isotopic content calculated using the PLSR model.			

We tested LAMIS in analyzing the soil samples, in which the total carbon content was 12.26%, and the nitrogen content was 0.27%. For initial experiments, we spiked this soil with 

C-enriched decanoic acid in different proportions. Several spectral lines of iron appear as interferences in the region of CN B→X Δν = –1) bands, but iron lines were well resolved from carbon features. An algorithm that automatically fits a Lorentzian profile to every interfering spectral line and digitally subtracts them from the spectrum can be applied as a remedy. Such a procedure can work relatively well if the interfering lines are no more than three times stronger than the main spectrum.  

A similar multivariate PLSR calibration was performed for measurements of the 

N isotope using reference samples prepared by blending powders of natural and 

N atomic fractions in prepared reference samples were 0.37%, 1.0%, 2.0%, 5.0%, 10%, 20%, and 99%. In these experiments, the CN B→X (Δν = +1) spectra were collected within the range 414–422 nm, similar to those shown Figure 2. The results are displayed in Figure 5 with the error bars corresponding to standard deviation of a single measurement, each made by signal accumulation from 100 laser pulses. Again, quantification precision and accuracy were limited by microscale isotopic inhomogeneity of the reference samples that were prepared. Inaccuracies in preparing these reference mixtures to be equal to the nominal isotopic content also increased the calibration errors.  

Figure 5: Calibration plot with a linear fit and 95% confidence interval for the 15N isotopic content calculated using the PLSR model.			

During laser ablation of carbon-containing samples, both CN and C

 radicals are often generated in comparable quantities. Hence, both of these species can be used for measuring the carbon isotopic ratio. Utilization of diatomic carbon molecules C

 for LAMIS measurements was described previously (1,5,8). Rapid spatial and temporal evolution of the plasma plumes during laser ablation may cause kinetic fractionation effects altering stoichiometric and isotopic equilibrium between the plasma and the sample. Kinetic isotope fractionation was indeed observed at early stages of plume evolution (gate delays <2 μs) in C

 emission (8). At longer delays, the measured isotopic ratio was close to the real ratio in the sample. These results suggest that carbon isotopes 

C can behave differently in laser ablation plumes, and the change in ratio is related to the time and spatial distance above the sample. For the purpose of isotopic analysis, it is important that the isotopic ratio measured at longer delays (>2 μs) equilibrates and approaches the real ratio in the sample roughly at the same time when molecular emission is also reaching its maximum. 

To demonstrate LAMIS for hydrogen and oxygen isotopic measurements, we ablated ordinary water ice (H

O. As in earlier work (1,6), we recorded emission of hydroxyl radicals at the A

 (0-0) transition in the ablation plumes. The major features of OH emission are R

 branch head at ~309 nm. These rotational branch heads are suitable for the D/H ratio determination because the isotopic shifts between OH and OD features are large and can be measured at low resolution. However, isotopic shifts between 

OH are smaller, especially for the lines with low rotational quantum numbers. The isotopic shifts increase with increasing quantum numbers in the band tails.  

The hydroxyl isotopologue spectra recorded in wavelength region 311.8–314.0 nm using an IsoPlane spectrograph (Princeton Instruments) with a 3600-gr/mm grating are presented in Figure 6. The ICCD gate duration was 50 μs and delayed 5 μs after the laser pulse. Argon flow was applied to displace air. The most intense features contributing to the shown spectra belong to OH rotational lines of Q

 branches with quantum numbers from J = 13.5 to J = 18.5. Smaller contributions come from P

, and other minor branches of the (0-0) band, and also from the second vibrational band (1-1). These results demonstrate that hydrogen and oxygen isotopes can be determined simultaneously using spectra of hydroxyl isotopologues 

OD recorded with a compact spectrograph. Quantitative isotopic calibration for ice analysis can be accomplished using multivariate PLSR as previously realized for water vapor (6).  

Figure 6: Spectra of 16OH, 18OH, and 16OD molecules in the AâX (0-0) band formed during laser ablation of ice. Spectra were averaged over a series of 10 replicates, each made of 100-pulse accumulations.			

Isotopic analysis of ice is the main tool in paleoclimatology and glaciology studies, but measuring 

O ratios in rocks and minerals is important for geochemists as well. Aluminum oxide is a common constituent in many minerals. As a result, molecular spectra of AlO are easily detectable in laser ablation of rocks. We observed five band progressions of AlO B

  (Δν = 0,±1, ±2) emission within a broad interval 445–545 nm from several rocks including feldspar, tourmaline, and rock salt. However, this spectral range is generally contaminated by many other spectral lines, and each particular experiment will require choosing a smaller specific segment of the spectrum. We ablated natural and isotope-enriched Al

 pellets, flushed with helium flow to illustrate that Al

O can be resolved in LAMIS. The spectra of AlO B→X (Δν = +1) are presented in Figure 7, indicating that AlO emission can be used to measure the oxygen isotopic ratio 

Figure 7: Emission spectra of AlO vibrational band progression (BâX; ÎÎ½ = +1) formed during ablation of Al2O3 natural and 18O-enriched pellets. Spectra were acquired with a 20-Î¼s delay and averaged over 100 laser pulses.			

Calcium oxide is another common constituent of rocks and minerals. Several emission bands of CaO A

  (2-0, 1-0, 0-0, 0-1) were clearly observed within 765–960 nm during ablation of calcite (CaCO

) in air. Molecular structure and spectrophysical properties of CaO are similar to those of SrO. Emission of the latter was previously used for LAMIS detection of three strontium isotopes (

Sr) (3–5). Our numerical simulation of the Ca

O spectra predicted that CaO emission can be used to infer oxygen isotopic information. Additionally, laser ablation of CaCl

  band progression with Δν = –1. We recorded the spectra of CaCl B→X system using two spectrographs, one of which was 1250-mm Czerny-Turner spectrograph (Horiba JY) and the other one was an echelle spectrograph EMU-65. The results are displayed in Figure 8. The natural isotopic ratio of chlorine 

Cl is 24.2% to 75.8%, respectively. The isotopic shifts because of these two isotopes are apparent in the spectra shown in Figure 8. While the large high-resolution spectrograph recorded a superior spectrum, the compact echelle spectrograph yielded sufficient resolution for LAMIS measurements.  

Figure 8: Spectra of CaCl band progression (BâX; ÎÎ½ = â1) formed during ablation of natural CaCl2 pellets. Spectra were acquired with a 20-Î¼s delay and averaged over 20 laser pulses.			

We demonstrated several possibilities of using LAMIS for rapid optical analysis of rare isotopes of hydrogen, boron, carbon, nitrogen, oxygen, and chlorine. A significant advantage of this technique is that solid samples can be analyzed in their original unaltered condition, without preparation. Precleaning of the sample surface can be completed by laser ablation before or during the analysis, which may be considered as depth profiling. Isotopic two- and three-dimensional mapping is possible. Quantitative measurements can be realized using multivariate regression models that will relate the spectral intensities of the isotope-specific molecular spectra to the original abundances of isotopes in the sample. Quantification is based on measuring spectra of known reference samples. Calibrating the LAMIS response for quantitative determination of C and N isotopes is presented here, while calibration for H and B isotopes was described earlier (2,6,7). Accuracy and precision of determination depends on isotopic homogeneity of the reference standards, but the analyzed samples can be heterogeneous. LAMIS can yield a map of the isotopic distribution.  

 (1) R.E. Russo, A.A. Bol'shakov, X. Mao, C.P. McKay, D.L. Perry, and O. Sorkhabi, 

 (2) X. Mao, A.A. Bol'shakov, D.L. Perry, O. Sorkhabi, and R.E. Russo, 

 (3) X. Mao, A.A. Bol'shakov, I. Choi, C.P. McKay, D.L. Perry, O. Sorkhabi, and R.E. Russo, 

 (4) A.A. Bol'shakov, X. Mao, C.P. McKay, and R.E. Russo, 

 (6) A. Sarkar, X. Mao, G.C.-Y. Chan, V. Zorba, and R.E. Russo, 

 (8) M. Dong, X. Mao, J.J. Gonzalez, J. Lu, and R.E. Russo, 

 (9) T.B. Coplen et al., US Geological Survey, Water-Resources Investigations Report 01-4222 (2002).  

(10) B. Yee, K.C. Hartig, P. Ko, J. McNutt, and I. Jovanovic, 

 is with Applied Spectra, Inc., in Fremont, California. 

 is with Applied Spectra, Inc., and the Lawrence Berkeley National Laboratory at the University of California in Berkeley, California. Xianglei 

 are with the Lawrence Berkeley National Laboratory at the University of California. Direct correspondence to: 

Peer-Reviewed Article

This article describes measurements of isotopes of hydrogen, boron, carbon, nitrogen, oxygen, and chlorine using laser ablation molecular isotopic spectrometry (LAMIS).

Laser Ablation Molecular Isotopic Spectrometry for Rare Isotopes of the Light Elements

How does one compute the mathematical certainty of wavelength standards used for calibrating instrumentation used for molecular spectroscopy measurements? This question becomes of major importance when the technique used for measurement requires the collection of large databases for use in qualitative searches or quantitative multivariate analysis. Wavelength drift over time within a single instrument or wavelength differences between instruments create errors and variation in the accuracy of measurements using databases collected with different wavelength registrations. The loss of integrity over the wavelength axis of data collected over time is a significant problem in large database creation and usage. So, what are the techniques and mathematics used to compute uncertainty, and the optimum methods for maintaining wavelength accuracy within instrumentation over time, when considering measurement condition changes?

Wavelength precision and accuracy, more formally termed repeatability and reproducibility, within a single instrument and between multiple instruments over time are essential for optimum transfer of quantitative calibrations, and for the continuous use of databases in qualitative search techniques. To maintain the integrity of data, it is essential that the wavelength axis be stable within instruments and identical across different instruments over changing measurement conditions. This is a difficult task for the basic commercial instrument designers and manufacturers in terms of making spectrometers highly precise and accurate on the wavelength axis. Questions arise as to the engineering tolerances required for high-precision spectrometers in terms of mechanical, optical, and electronic components. Also essential to the precise wavelength alignment are the exact procedures required to properly calibrate the wavelength axis of the spectrometer based on measurements of known reference standards (1,2).  

Different Approaches to Alignment of the Wavelength Axis

Because of the inherent uncertainties of standard reference materials (SRMs), one may ask if they are precise enough to be useful for aligning the wavelength axis of spectrometers over time (3). For example, National Institute of Standards and Technology (NIST) SRM 1920a reported a published uncertainty of ±1 nm (4,5). With large uncertainties in published wavelength peak positions, referred to as 

, is the most reasonable approach to ensure that wavelength accuracy is within inherent NIST uncertainties when using SRMs, but strive to be far closer in agreement for instrument-to-instrument matching? This column demonstrates the state of the art in commercial spectrometers today and proposes the question of whether there might be improved methods for spectrometer wavelength alignment for enhanced repeatability and reproducibility. 

A seminal paper on the use of rare-earth oxides for use as wavelength standards in near infrared (NIR) spectroscopy is familiar to many spectroscopists (4). One important wavelength standard made for use in the NIR region is SRM 1920a, consisting of a mixture of rare-earth oxides. This SRM has known stability problems related to variation in wavelength scale and, as such, has been replaced with SRM 2036 glass (6). SRM 1920a is not a particularly stable material and wavelength registration changes based on temperature have been published as (±0.15 nm). In the certification of SRM 1920a, NIST also experienced challenges with their secondary Cary model spectrometer and their manual method of peak selection resulting in the large reported uncertainty (4). In calibrating this SRM, NIST reportedly used the precise NIST (formerly the National Bureau of Standards [NBS]) spectrometer to measure three wavelengths (that is, 1012.9, 1260.7, and 1535.5 nm). The Cary instrument was reported to exhibit a deviation of ±1 nm during the test measurement period. For the NIST spectrometer, emission reference lamps included the use of neon (Ne) at 703.24 nm, xenon (Xe) at 881.94 and 979.97 nm, mercury (Hg) at 1013.97 and 1529.58 nm, and krypton (Kr) at 1816.73 nm. The use of such atomic line spectra for wavelength registration produces the lowest uncertainty (7).  

For the longer NIR wavelength region, 1,2,4-trichlorobenzene was used to mark the 2152.60 nm position (8). Toluene (used for infrared at 3290.8, 3422.0, 3484.0, 5147.2, 5381.8, and 5549.0 nm) and carbon disulfide (2224.0 nm) have also been recommended for wavelength calibration in early published work (9,10). The task of selecting appropriate standards for certified peak wavelength positions is critical for improved 

-axis (wavelength) alignment and calibration of spectrometers. Wavelength alignment technology is dependent on selecting a suitable set of reference peak wavelengths and stable materials to align each spectrometer system using best available measurement practices. If wavelength precision must approach ±0.05 nm and accuracy across the entire spectral region must be better than ±0.2 nm for 95% confidence interval, a series of technological improvements must be implemented in the future.   

So, what if a master instrument, with its own idiosyncratic scanning features, can be duplicated? One might analyze a paired reference standard with the master instrument and align the newly manufactured instrument to the set of wavelength peak position values found using the master instrument. This approach was used in the early development of NIR instruments in an attempt to make them more similar (11–13). The problem with this approach is the entire world may have a different set of nominal wavelength values (measurands) than the master instrument. Spectrometers change over time and conditions and wavelength alignment drifts. A common spectrometer may demonstrate inherent nonlinearity in its wavelength axis, which does not allow accurate alignment throughout the complete spectral range. So to consistently align the wavelength axis with other instrument types, and even to align a single instrument over time, requires not only repeatability, but also accuracy indicated by agreement with reference standard peak positions (measurands). These reference peak positions are provided by a set of known physical standards (physical in the sense of primary lines that are universally reproducible). Such standards may be primary emission standards (that is, atomic lines), physical standards of mixed rare earth glasses, or even organic liquids at standard pathlength and temperature. The measurands for calibrating wavelength scales would optimally be obtained using first principles whenever possible because this approach is universal and based on sound physical science and metrology, which by definition is fixed to a specific known uncertainty. The national laboratories around the globe use this approach as much as possible (14,15). There are other materials that may be used as physical standards, such as etalons. The use of etalons for wavelength alignment was successfully implemented for a process diode array spectrometer (16–20). The instrument was extremely precise, but too expensive for market acceptance because the spectrograph had originally been designed for the harsh conditions of an Earth-orbiting satellite. 

To determine a unique set of peak wavelength values based on an arbitrary master instrument does not serve our purposes adequately over time. One would ideally expect high precision with universal accuracy so that databases now and in the future may be aligned with any instrument technology and be carried reproducibly into the future. In addition, using a first principles approach allows one to standardize the data based on known wavelength positioning at any time. By using first principles, wavelength alignment may be defined electronically and, thus, no physical instrument or object is required to transfer the technology required for wavelength axis calibration. What is required to make all this work are repeatable and well-characterized peak wavelength standards, either emission sources, pure compounds, or solid rare-earth oxide glasses with known peak wavelength positions, stability, and well-defined uncertainty. Requirements are for glasses, crystalline polymers, and pure chemical solutions with an uncertainty of better than ±0.1 nm for multiple wavelengths. For infrared and Raman systems the uncertainty should be better than ±0.1% and better than 0.05 cm

  (wavenumber) (21). If a spectrometer is repeatable, but not reproducible (accurate), then database and calibration transfer becomes an issue of trying to align the wavelength axis to some unknown or poorly defined system.  

NIST uncertainty is a true measure of uncertainty, unlike the typical manufacturer report as either precision or accuracy. For example, NIST uncertainty has been calculated from equation 1 (4):  

 = twice the largest standard deviation of measurement of multiple emission lines (measurands) versus the spectrometer measured values over a period of 31 days (or some designated period); 

 = twice the standard deviation of the uncertainty in the peak selection method used; and 

 is the maximum variation in the standard because of temperature changes over a specified range and conditions. (Note: The number 2 (twice) is referred to as the 

 value also known as the coverage factor; see reference 14). 

In testing SRM 1920a, the reported error for multiple wavelengths using emission lines for the NIST spectrometer was ±0.265 nm for all the data points measured over 31 days. The NIST actual reported uncertainty for SRM 1920a is equal to equation 2. 

This value was rounded up to ±1 nm considering the added uncertainty of the Cary spectrometer measurements and material stability over the lifetime of the SRM (4). 

A more detailed description of uncertainty computations and rationale with respect to wavelength SRMs is found in other published work (6,14,22). There are significant complexities to the question of uncertainty in reference materials, so the reader is referred directly to the three citations referenced above. One may also obtain more detailed information on the history of the international metrical committees responsible for defining the technical details for uncertainty associated with measurement results; the reader is referred to appendices C and D in reference 14 for historical details.  

Let us begin with a set of measurement data as shown in Table I. This is real data measured on a series of commercially calibrated NIR spectrometers using an identical sample to measure a variety of performance characteristics. For this column, we refer to the wavelength measurement data across the A–G instruments consisting of seven commercial instrument models manufactured by four companies. These will remain anonymous for the purpose of this analysis. Please note that within the models used for this experiment, there were dispersive monochromators, diode-array based instruments, and Fourier transform near-infrared (FT-NIR) systems. 

Table I: Commercial instruments using identical SRM 1920a material. (Wavelength measurement comparison using different instrument models from different manufacturers.)			

This table demonstrates the instrument models A through G as individual columns, with the different nominal wavelength values (measurands) for the SRM 1920a standard material along the first column. The results of a set of standard spectra are shown in each column demonstrating the peak positions as determined using a five-point cubic spline, peak interpolation method. No data processing or smoothing was applied to the spectra, but after the manufacturers' recommended calibration the reference material was measured using three replicates. For Tables I and II a single spectrum was selected for identification of peak position. For Table III each of the replicate spectra are used for computing the reported parameters. The reported wavelengths are shown as compared to the NIST nominal wavelengths (measurands) for a nominal bandwidth of 10 nm. The last two rows show the mean difference and standard deviation between the measured values and the measurand values for each peak, respectively, for the complete set of instrument measurements at each wavelength. One may quickly observe that relatively large differences between the measurand and measured wavelengths are present in the commercial world of NIR spectrometers.  

Table II: Results of commercial instruments on SRM 1920a for each nominal wavelength (measurand 10 nm bandwidth values as top row)			

From Table I it may be observed that there is significant variation from commercial NIR instruments and published nominal wavelength positions for this reference standard. Let's review these results from a measure of uncertainty viewpoint. 

What are the techniques and mathematics used to compute uncertainty, and the optimum methods for maintaining wavelength accuracy within instrumentation over time, when considering measurement condition changes?

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Vol 29 No 6 Spectroscopy June 2014 Regular Issue PDF