Analytically Speaking Podcast, Episode 8

Estimation of the Fluorescence Band Parameters

Smoothing of the Time-Resolved Spectral Matrix Obtained by a Streak Camera

Clay and Soil Organic Matter Drive Wood Multi-Elemental Composition of a Tropical Tree Species

Three-Dimensional Printing and the Art of Making Small Working Prototype Spectrometers

Thibaut Van Acker has won the European Rising Star Award for 2023. He will receive the award on February 1 at the European Winter Conference on Plasma Spectrochemistry 2023 (EWCPS 2023) in Ljubljana, Slovenia, where he will give a keynote talk, titled, “Pushing the Limits of Elemental Mapping via Laser Ablation – ICP-mass spectrometry with a Nanosecond 193 nm kHz Laser.”

The European Rising Star Award for Plasma Spectrochemistry promotes outstanding contributions of an upcoming young scientist at the beginning of his/her career (first 5 years) within the field of plasma spectrochemistry. The European Rising Star Award for Plasma Spectrochemistry honors and promotes outstanding contributions of upcoming younger scientist at the beginning of their career within the field of plasma spectrochemistry.

The European Rising Star Award for Plasma Spectrochemistry is an important opportunity for researchers working in European labs to promote their work and share it with the community at the EWCPS.

Van Acker is from the Atomic & Mass Spectrometry – A&MS research unit of Ghent University (Belgium), and his work is focused on both fundamental aspects of laser ablation – ICP-mass spectrometry and analytical method development to explore the capabilities of the technique for high-resolution elemental mapping applications in challenging interdisciplinary contexts. Based on a number of hardware improvements, Thibaut has brought elemental mapping – that is, the revelation of the two- and even three-dimensional (3D) distribution of elements across a sample – to another level. This can currently be accomplished at a pixel acquisition rate up to 1,000 Hz and a laser spot size down to <1 μm.

So far, Thibaut is (co-)author of 23 publications in peer-reviewed international journals, and his work has been presented in more than 30 lectures at international conferences. He has also been involved early in his career in the mentorship of Bachelor, Master and PhD students and was awarded the Junior Postdoctoral Research Fellowship granted by the Research Foundation Flanders (FWO), which is securing the start of his individual carrier.

Articles

Thibaut Van Acker Wins the European Rising Star Award for Plasma Spectrochemistry

Alexander Gundlach-Graham has won the JAAS Emerging Investigator Award for 2023. He will receive the award on February 3 at the European Winter Conference on Plasma Spectrochemistry 2023 (EWCPS 2023) in Ljubljana, Slovenia, where he will give a keynote talk, titled, “Simultaneous Imaging of Elements and Biomolecules by ICPMS and Atmospheric Pressure Ion Source-MS.”

The JAAS Emerging Investigator Award recognizes early career researchers, within 10 years of their PhD, who have made a significant contribution in the area of atomic spectrometry, in their independent academic career. This award is presented annually. The winner is selected by a panel of 

Gundlach-Graham’s group focuses on the development and application of atomic mass spectrometry (MS) to address current measurement challenges in environmental and bioanalytical sciences.

Gundlach-Graham and his group investigate the use of single-particle inductively coupled plasma time-of-flight mass spectrometry (sp-ICP-TOF-MS) for analysis of inorganic nanomaterials in complex matrices such as biological fluids and environmental samples. They place specific emphasis on the development of novel sample introduction approaches, calibration strategies, and NP classification strategies with sp-ICP-TOF-MS. Accurate quantification of nanoparticles in terms of composition and particle-number concentration is critical for assessing routes of nanomaterial exposure and supporting nanotoxicological studies.

Gundlach-Graham and his group also investigates new high-power nitrogen-sustained microwave inductively coupled atmospheric-pressure plasma (MICAP) sources for atomic mass spectrometry. This research is motivated by persistent limitations of the conventional argon-sustained ICP, such high cost of operation, incompatibility with ambient (air-based) aerosol sample introduction, and argon-based polyatomic interferences that complicate mass-spectral analysis. Here, Gundlach-Graham and his group explore the combination of the MICAP source with mass spectrometry to develop a next-generation argon-free ICP-MS.

Alexander Gundlach-Graham Wins the JAAS Emerging Investigator Award

Katerina (Kate) Mastovska has been named the Deputy Executive Director and Chief Science Officer of AOAC International, a nonprofit organization that serves to ensure the safety of foods and other products that impact public health. She will join the organization on January 30.

Mastovska has been an active member of AOAC International since 2004 and received the association’s highest scientific honor, the Harvey W. Wiley Award, in 2021. She has extensive experience in research chemistry—working for the University of Chemistry & Technology in Prague, the U.S. Department of Agriculture, and founding her own independent consulting business, Excellcon International. In 2009, she joined Covance Laboratories, where her responsibilities grew rapidly. Following the acquisition of Covance Food Solutions by Eurofins Scientific, Mastovska continued to serve in roles of increasing importance, most recently as the Chief Scientific Officer of the Eurofins US Food Division.

AOAC International, founded in 1884, is an independent, third party, not-for-profit 501(c)(3) association that develops voluntary consensus standards. The 

 database is used by food scientists around the world to facilitate public health and safety and to promote trade.

In addition, two current AOAC staff members, each with almost 20 years of experience at AOAC, Dawn L. Frazier and Deborah McKenzie, have been promoted.

Frazier has been promoted to the position of Deputy Executive Director of Engagement. Previously she served as the Senior Director of Membership, Marketing, and Communications. Her responsibilities include leading and implementing the organization’s engagement strategy, which includes developing and maintaining relationships with key stakeholders and partners, as well as overseeing outreach and communication efforts — all to achieve the organization’s strategic plan.

McKenzie has been promoted to the new role of Deputy Assistant Executive Director and Chief Standards Officer. McKenzie was previously the Senior Director of Standards and Official Methods. Her responsibilities include overseeing implementation and execution of voluntary consensus standards processes and the official methods. She and her team coordinate and administer associated activities with standards development and method approval programs.

Katerina Mastovska Named AOAC International Chief Scientific Officer

Heidi Goenaga Infante has won the 2023 European Award for Plasma Spectrochemistry. She will receive the award on February 3 at the European Winter Conference on Plasma Spectrochemistry 2023 (EWCPS 2023) in Ljubljana, Slovenia, where she will give a plenary talk, titled, “A Journey of Continued Contributions to Plasma Spectrochemistry: Focus on Metrology at the Nanoscale.”

The European Award for Plasma Spectrochemistry, which promotes analytical plasma spectrochemical developments and applications in Europe, is awarded for a single outstanding piece of work or for continued important contributions in the field. It is sponsored by Agilent Technologies, and the winner is selected by an independent panel of five prominent European scientists in plasma spectrochemistry and one representative from Agilent Technologies. The winner receives a prize of EUR 6000.

For more than 25 years, Goenaga Infante has been working in elemental and speciation analysis using a wide variety of inductively coupled plasma–mass spectrometry (ICP-MS) methodologies. Over the past 12 years, she has made substantial contributions to novel fractionation analysis techniques in the areas of elemental speciation, metallomics, and nanomaterials characterization. She has worked to reduce uncertainty and improve the sensitivity of speciation measurements, push the boundaries of analytical technology to enable multi-element species quantification and characterization in very small samples, such as cells, and to develop high-accuracy measurements for nanomaterial quantification in complex samples using complementary hyphenated ICP-MS approaches. 

Goenaga Infante has also developed novel calibration strategies using tissue-matched calibrants and internal standardization (including isotope dilution analysis) for quantitative elemental imaging of tissue relevant to critical diseases. This work is currently being extended to single-cell inorganic metrology.

For the past 15 years Heidi's research activities have been carried out at LGC, where she is the chief scientist within the National Measurement Laboratory (NML) and as one of only four LGC science fellows. She is also a principal scientist and team leader of the Inorganic Analysis team of 15 doctoral and postdoctoral scientists, and the primary research supervisor of several PhD students from overseas universities and international metrology institutes.

Heidi Goenaga Infante Wins the 2023 European Award for Plasma Spectrochemistry

Offering the high spectral resolution of conventional Raman spectroscopy combined with reduced acquisition time, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses has been accepted as a mature and straightforward technology for label-free bioimaging. In a recently published paper, Philippe Leproux and associates have introduced the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). Leproux spoke to 

 about combining the techniques, as well as discussing the potential of the results.

Your paper (1) states that coherent Raman imaging has been extensively applied to live-cell imaging in the last two decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. What advantages does this particular technique offer compared to other label-free spectroscopic procedures?

It combines a high spatial resolution (~300 nm laterally) and a high acquisition speed, making it compatible with the observation of biological specimens like living cells or fresh tissues. This is a major asset for a chemically-selective (spectroscopic) imaging technique.

Does label-free spectroscopy produce better results for bioimaging than labeled methods, in terms of spatial resolution, and definition of composition of cells? What are the biggest advantages of label-free methods?

Confocal and multiphoton microscopy approaches are very powerful for investigating biological cells with subcellular resolution. They are supported by a mature and reliable technology, and the myriad of available fluorescent molecules allows one to collect loads of structural and functional cellular information. However, the use of labeled samples implies that those samples were somehow altered (chemically or genetically) and that the observation be restricted to “what” was labeled (for example, organelle or protein). These drawbacks disappear with label-free methods, not to mention that sample preparation is drastically simplified.

You and your co-authors introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation, utilizing multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy. Would you give our readers a basic explanation of this statement and share with us what inspired the development of this methodology?

MCARS is a microscopy technique based on spectral measurement, which is mature today (2), but generally requires a complex subsequent data processing step under the supervision of an expert. Yet it provides datasets that, in principle, should be computed by means of chemometric tools. In light of this, we focused our efforts on developing an algorithm to perform the unsupervised (“automated”) analysis of MCARS data. By combining MCARS technology with such automated data processing, we could introduce a simple method for label-free cell imaging and segmentation, that we intend to be accessible to a broad range of users, especially from the biomedical field.

What is the meaning of hyperspectral cell imaging versus basic cell imaging methods?

“Hyperspectral” means that the collected datasets contain a high number of spectral channels (one thousand, typically). Comparatively, “basic” fluorescence imaging uses one spectral channel per label or fluorophore, but spectral measurement is also possible (in this case, the word “basic” should be removed).

While, as you state, chemometric methods such as multivariate curve resolution (MCR) are widely used in spectroscopy-based data analysis, to date, MCR has never been applied to the analysis of MCARS hyperspectral datasets. What is the advantage of applying MCR, and why do you think MCR has not been applied and published before your work?

The main benefits are the unsupervised analysis, and the fact that output results are directly readable and interpretable by biologists, chemists, or physicists. Before our work, few studies reported on the use of other chemometric methods for processing CARS data in the context of cell imaging. We believe that we could successfully apply MCR owing to the quality of our datasets, including the high spectral resolution.

What was the most surprising result in your work?

Rigorously, the assumptions behind MCR should make the method non-compatible with MCARS data. Nevertheless, we could demonstrate the validity of our work from the numerical point of view, and we are currently working on the theoretical aspects. As a precaution, we consider that our approach is qualitatively valid at the moment and that further investigations would confirm quantitative imaging. Anyhow, at the very beginning of this work, we were fairly surprised by the quality of the results obtained, in terms of both spectral unmixing and chemically-selective imaging!

Were there any memorable limitations or challenges that you encountered in your work?

The study of 3D tissue samples is highly challenging because of the quantity of data to be processed. In that case, the required (random-access) memory–typically a few terabytes–can be a limitation, and subsampling strategies need to be implemented.

Another challenge was to establish and keep an interdisciplinary approach in our work within a broad team, including cell biologists, computer scientists, and physicists. This type of approach, although in the era of time, is nevertheless sometimes tricky to handle (technical language, practices, objectives, etc.).

Can you please summarize the feedback that you have received from other researchers regarding this work?

Actually, the feedback primarily came from biologists who would like to apply our method to their research field. This was expected following the scope of the journal in which we published our work (1). However, researchers from other fields, such as materials science and earth science, have also expressed their interest in our work.

What discoveries do you hope to realize using this method of cell imaging, and what do you think its impact will be in cell research and diagnostics?

We are now starting to use this label-free method to study the biological mechanisms at the interface between living cells and calcium phosphate ceramic scaffolds, in the context of bone tissue engineering. We conduct this work in the frame of Σ-LIM laboratory of excellence (3). Furthermore, we are collaborating with our Japanese partners (the research group of Prof. Hideaki Kano at Kyushu University) on the subject of label-free brain imaging, with the expectation of building a whole brain atlas with single-cell resolution (4).

What are the next steps in this research?

First, we will maintain our efforts to develop biomedical applications of MCARS technology, as illustrated above. Moreover, in terms of data processing methodology, the next step is to perform multivariate curve resolution by using deep learning approaches, given the large amount of data available and with the aim of accurately modeling the physical phenomenon of vibrational signal generation. In that respect, we are already evaluating autoencoders as candidate artificial neural networks (ANNs) for such a purpose.

Boildieu, D.; Guerenne-Del Ben, T.; Duponchel, L.; Sol, V.; Petit, J-M.; Champion, É.; Kano, H.; Helbert, D.; Magnaudeix, A.; Leproux, P.; Carré, P. Coherent Anti-Stokes Raman Scattering Cell Imaging and Segmentation with Unsupervised Data Analysis. 

 10, 933897. DOI: 10.3389/fcell.2022.933897

Kano, H.; Leproux, P. Multiplex CARS Microscopy in the « Long-Pulse » Regime : Where Are They Now? Proc. SPIE 11973, 

Advanced Chemical Microscopy for Life Sciences and Transactional Medicine 2022

https://www.unilim.fr/labex_sigmalim/?lang=en

Murakami, Y.; Miyazaki, S.; Boildieu, D.; Rajaofara, Z.; Helbert, D.; Magnaudeix, A. ; Carré, P.; Leproux, P.; Honjoh, S.; Kano, H. Toward Whole Brain Label-Free Molecular Imaging with Single-Cell Resolution Sing Ultra-Broadband Multiplex CARS Microspectroscopy. SPIE 11973, 

 obtained his PhD in 2001 at the University of Limoges, France, on the design of optical power amplifiers based on rare-earth doped double-clad fibers. Since 2002, he is an Associate Professor at the University of Limoges in the fields of photonics and ICTs. His research activities at XLIM Institute mainly deal with nonlinear fiber optics – especially supercontinuum generation – and biophotonics, from the design of new optical fibers to the demonstration of innovative biology-oriented experiments, like MCARS microspectroscopy. Dr. Leproux is the co-founder of LEUKOS company, Limoges, France, a spin-off from the University of Limoges and the French CNRS manufacturing supercontinuum lasers. He authored or co-authored over 240 publications/conferences and 14 patents.

Offering the high spectral resolution of conventional Raman spectroscopy combined with reduced acquisition time, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses has been accepted as a mature and straightforward technology for label-free bioimaging. In a recently published paper, Philippe Leproux and associates have introduced the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). Leproux spoke to Spectroscopy about combining the techniques, as well as discussing the potential of the results.

Coherent Anti-Stokes Raman Scattering Cell Imaging and Segmentation with Unsupervised Data Analysis

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Spectroscopy’s 2022–2023 New Product Review

Acrylate polymers are derivatives of acrylic acid, but contain a plethora of different functional groups and are best discussed by themselves. In this column, we study the spectrum of an important polymer, polymethyl methacrylate (PMMA), which is otherwise known as plexiglass, and is found in windows, car parts, and paint. We then study the spectra of PMMA mixtures and copolymers in detail.

, is the founder and CEO of Big Sur Scientific, a maker of portable mid-infrared cannabis analyzers. He has over 30 years experience as an industrial infrared spectroscopist, has published numerous peer-reviewed papers, and has written three books on spectroscopy. As a trainer, he has helped thousands of people around the world improve their infrared analyses. In addition to writing for Spectroscopy, Dr. Smith writes a regular column for its sister publication Cannabis Science and Technology and sits on its editorial board. He earned his PhD in physical chemistry from Dartmouth College.

We have studied esters (1) and polyesters (2,3) in the past. We discuss the spectroscopy of the ester functional group in this article, amongst others. Thus, I briefly reintroduce you to the structure and spectroscopy of esters.

Esters are characterized by C=O, C-C-O, and O-C-C linkages, and are divided into the families of saturated or aromatic esters depending upon the nature of the carbon attached to the carbonyl carbon. Ester infrared (IR) spectra follow the Rule of Three (1). This means they have three big peaks at approximately 1700, 1200, and 1100 cm

 (going forward all peak positions will be in cm

 units even if not so noted). These correspond to the stretching of the C=O, C-C-O, and O-C-C units. Table I summarizes the Rule of Three group wavenumbers for esters.

The family of acrylate polymers is based on the parent molecule acrylic acid, whose structure is seen in Figure 1.

FIGURE 1: The chemical structure of acrylic acid.

Note that acrylic acid contains a vinyl C=C bond, and a carboxylic acid, functional groups whose structures and spectra we have studied in the past (4,5). The IR spectrum of acrylic acid is seen in Figure 2.

FIGURE 2: The chemical structure and IR spectrum of acrylic acid.

Recall (5) that any peak associated with the O-H bond in a carboxylic acid is broadened because of hydrogen bonding. Thus, the broad O-H stretching envelope seen in Figure 2 extends from approximately 3800 to 2000, and the bottom of the O-H wagging envelope is at approximately 930. Carboxylic acids also have strong C=O and C-O stretching peaks, which fall at 1702 and 1248 in Figure 2. The C=O stretch for acrylic acid falls below that for saturated acids, because the carbonyl group conjugates with the C=C bond, similar to how aromatic rings conjugate to C=O bonds (6).

The vinyl group in acrylic acid can be reacted to form a polymer, like many other vinyl containing monomers (4). Polymers made from acrylic acid derivatives are called polyacrylates. There are many different functional groups that can be attached to the acrylic acid frame, including esters, which is the focus of this column.

Perhaps the most common polyacrylate is polymethyl methacrylate, or PMMA. This polymer is economically important, and trade names for it include PlexiGlass and Lucite (7). Because PMMA is transparent, it can be used in place of traditional glass in uses such as windows, doors, and aquariums. The IR spectrum and structure of PMMA are seen in Figure 3.

FIGURE 3: The structure and IR spectrum of polymethyl methacrylate (PMMA).

Note that there is a pendant ester group and two CH

 groups in PMMA. The first methyl in the name denotes the CH

 refers to the fact that the acrylic acid monomer has a methyl group attached to the C=C bond. The major peaks in this spectrum are, not surprisingly, the Rule of Three peaks from the ester (1). Specifically, the C=O stretch is labeled A at 1730 cm

, and they are labeled together as B in Figure 3. Unfortunately, PMMA does not strictly follow the Rule of Three peaks for saturated esters listed in Table I. The C=O stretch is 5 cm

 too low, the C-C-O stretch is in the right place at 1191, but there is no O-C-C stretch between 1100 and 1030. There is an intense peak at 1149, which, for lack of a better assignment, I will call the ester O-C-C stretch. I have no idea why PMMA does not follow the rules for esters. As I have said in the past, all the peak position ranges I give you have exceptions, which is why there are few absolutes in IR spectral interpretation.

Copolymers of polystyrene and PMMA are common materials as they are also clear and rigid. Uses of these materials include as food containers, drinkware, and as additives to things like cement and concrete. A common name for these copolymers is styrene methyl methacrylate (SMMA). The structure and IR spectrum of SMMA are seen in Figure 4.

FIGURE 4: The structure and IR spectrum of polystyrene methyl methacrylate (SMMA).

Note that the structure of SMMA is a mixed molecule containing both saturated and unsaturated carbons. Hence, there are C-H stretches above and below 3000 (8). The styrene portion of the polymer contains a monosubstituted benzene ring, and as we have seen before (9) these have an aromatic C-H wag from 770–710, and an aromatic ring bend at 690±10. The monosubstituted benzene ring in SMMA exhibits these peaks at 757 and 695, respectively. The ring modes of the benzene ring in styrene, due to the stretching of the carbon-carbon bonds in the ring, are at 1601 and 1583. Recall (8) that these peaks are always sharp, are used to distinguish between C=C and aromatic carbon-carbon bonds, but vary in number and intensity depending upon the molecule.

The methacrylate ester part of SMMA will have three peaks because it is an ester (1). In Figure 4, the C=O stretch is at 1727, the C-C-O stretch is at 1197, and the O-C-C stretch is at 1074. These Rule of Three peaks are not as big as we are used to seeing, because the PMMA units in SMMA are diluted by the presence of polystyrene.

Figure 5 shows a comparison of the spectra of pure polystyrene and a polystyrene/PMMA copolymer. Note that the monosubstituted benzene ring peaks in the two spectra line up quite nicely, with the C-H stretches at 3060 and 3025, the ring mode at 1601, C-H wag at 756, and the aromatic ring bend at 698 in both spectra. The point here is that, even though polystyrene/PMMA copolymers have two repeat units in the same backbone, their spectra look like a simple mixture of the two. This indicates there is very little chemical interaction between the different repeat units in the polymer.

FIGURE 5: A comparison of the spectrum of pure polystyrene and a polystyrene-methyl methacrylate copolymer.

A molecule similar to PMMA is polyethyl methacrylate (PEMA), where the CH

 group attached to the chain in PMMA is replaced by an ethyl (CH

) group. This material is softer than PMMA, and is used to make artificial fingernails amongst other things (10). The structure and IR spectrum of PEMA are seen in Figure 6.

FIGURE 6: The chemical structure and IR spectrum of polyethyl methacrylate (PEMA).

Because PEMA has both methyl and methylene groups, there are three saturated C-H stretching peaks between 3000 and 2850 (11). However, this spectrum is dominated by the Rule of Three peaks for the ester present, with the C=O stretch at 1728, the C-C-O stretch at 1147, and the O-C-C stretch at 1064.

These three peaks in PMMA fall at 1730 cm

, respectively. Thus, the spectra of these two materials are similar, but not identical.

In this article, we have studied the structure and spectra of acrylate polymers. The name for the class comes from acrylic acid. We saw that the spectrum of acrylic acid has peaks from the C=C and -COOH functional groups. Polymers made from acrylic acid monomers often times contain pendant ester groups. We covered the structure and spectrum of the economically important molecule polymethyl methacrylate, its copolymer with polystyrene, and its related molecule polyethyl methacrylate. Their spectra were dominated by the Rule of Three peaks from the esters present.

(1) Smith, B.C. The C=O Bond, Part VI: Esters and the Rule of Three. 

(2) Smith, B.C. Infrared Spectroscopy of Polymers, VIII: Polyesters and the Rule of Three. 

(3) Smith, B.C. Infrared Spectroscopy of Polymers, IX: Pendant Ester Polymers and Polycarbonates. 

(4) Smith, B.C. The Infrared Spectroscopy of Alkenes. 

(5) Smith, B.C. The C=O Bond, Part III: Carboxylic Acids. 

(6) Smith, B.C. The Carbonyl Group, Part I: Introduction. 

https://en.wikipedia.org/wiki/Poly(methyl_methacrylate)

(8) Smith, B.C. Group Wavenumbers and an Introduction to the Spectroscopy of Benzene Rings. 

(9) Smith, B.C. Distinguishing Structural Isomers: Mono- and Disubstituted Benzene Rings. 

https://en.wikipedia.org/wiki/Poly(ethyl_methacrylate)

(11) Smith, B.C. More Theory and Practice: The Thorny Problem of Mixtures and More on Straight Chain Alkanes. 

, is the founder and CEO of Big Sur Scientific, a maker of portable mid-infrared cannabis analyzers. He has over 30 years experience as an industrial infrared spectroscopist, has published numerous peer-reviewed papers, and has written three books on spectroscopy. As a trainer, he has helped thousands of people around the world improve their infrared analyses. In addition to writing for 

, Dr. Smith writes a regular column for its sister publication 

 and sits on its editorial board. He earned his PhD in physical chemistry from Dartmouth College. He can be reached at: 

We study the spectrum of polymethyl methacrylate (PMMA)—otherwise known as plexiglass—and the spectra of PMMA mixtures and copolymers.

Infrared Spectroscopy of Polymers X: Polyacrylates

In the past, moving from a concept for an analytical instrument to actually producing a functioning instrument was a relatively tedious and lengthy process. The traditional approach was to draft a design and then go through a number of iterations, along with design reviews and sign-offs. Today, if one has a good designer who is well-versed in computer-aided design (CAD) engineering software, then it is possible to significantly reduce the time to develop a good-looking functional design. However, the time to go from the CAD design to a complete piece of hardware is typically the longest part of the process. It is not unusual for machine shop delays to be up to 6–8 weeks. Today, such delays can be eliminated by producing the designed parts on a desktop with a three-dimensional (3D) printer. This article shows, by example, how a 3D printer can speed up the process of making prototype hardware for a spectral measurement system, shortening the time to produce functional hardware from weeks to days—or less.

The traditional approach to the prototyping of the optics for a spectrometric measurement system is to build the prototype, starting with an optical bench and using off-the-shelf components, such as lenses or mirrors with associated mounts, slit assemblies, source and detector modules, and so on. This approach works for testing out the optical train and allows the optics to be optimized in terms of location, beam direction, and geometry. The next step of the transition to a facsimile of the intended instrument is a little more challenging because although optical components can be obtained from optics houses, such as ThorLabs, Newport, or Edmund Optics, the basic frame and shape of the instrument has to be built up from machined parts. Obtaining the right engineering resource, such as a machinist with experience of building instruments, is not so easy, and it is a skill that is disappearing. However, there is a good practical solution available today that can fill in the gaps and enable a good-looking prototype instrument to be produced, and that is three-dimensional (3D) printing. A 3D printer not only can generate good-looking and well-finished parts, but it can simplify assembly and speed up the prototyping process. This work is made possible by the use of a desktop 3D printer, where the transition from a computer-aided design (CAD) file to a finished part is as simple as a mouse button press, thus eliminating the need to wait for parts to be made at a machine shop. This article illustrates, by practical examples, the advantages gained from 3D printing of prototype parts.

Practical Examples of the Role of 3D Printing for Instrument Prototypes 

The following are actual examples of the implementation of 3D printing as used to evaluate prototype spectral measurement concepts. As an important note, these examples are now more than 10 years old, and in terms of the technology of 3D printing, they are by no means state of the art. The technology of 3D printers and printing substrates has advanced by leaps and bounds in recent years. However, the concept and rationale for its use remains the same.

The first assembly, shown in Figure 1, involved in situ measurements on a custom extraction device with a hydrophobic membrane supported on a metal mesh. The membrane was used as the extraction medium for the isolation and direct measurement of organic contaminants in water. An issue for the measurement was the need to measure everything in situ in the presence of a high degree of optical scattering from the supported membrane. The goal was to make the measurement without the need for any additional focusing optics.

FIGURE 1: (a,b) Measurement system for organic contaminants extracted from water, showing the various design elements listed and (c) the detector.

Here, the most important considerations were to rigidly retain and locate the membrane sampler, shown as C2 in Figure 1, to bring the infrared (IR) source (C) into close (and reproducible) proximity to the supported membrane, and to locate the exit of the IR radiation (D2) immediately in front of the quad detector (D). This design helps to concentrate the highly attenuated IR beam, passing though the membrane on to the four-channel detector (shown as D2 and D) without any supplemental focusing optics. The detector assembly provides four electronically independent channels representing the unique analytical wavelengths as defined by narrow bandpass filters mounted in front of the detector elements.

This design was simple and effectively inexpensive for a four-channel non-dispersive IR (NDIR) instrument. Figure 1a shows the system open with all relevant components shown and the membrane sampler location unoccupied, ready to receive a sample for analysis. Once the sampler is loaded, the lid and an actuation cam (B) is closed around hinge (A). In Figure 1b, the cam pushes the IR source assembly into the sampler (C2), making it ready to be measured by the quad detector (D2). The ability to reproduce the exact mode of measurement was invaluable, and it simplified the final instrument design, saving both time and prototyping costs. Figure 1c is a close-up of the four-channel detector.

The concept being prototyped here in Figure 2 is equivalent to the electronic nose system of measurement where gases and vapors are detected by the unique responses of individual channels made up of electrically conductive polymer membranes. In such systems, there are at least eight or nine (possibly more) individual channels that each give a unique set of responses for the compounds detected in the ambient air being sampled. In the example being prototyped here, the conductive polymer membranes are replaced by IR detectors that are responsive to specific wavelengths. Like in example 1, the wavelengths are defined for each quad detector by a set of narrow bandpass IR filters. Unlike the previous example, this system features four quad detectors, providing a total of 16 unique detection channels. The wavelengths selected cover a nominal spectral range from 2.5 μm (2500 nm or 4000 cm

). Three of the wavelengths represented common IR absorptions for C-H, carbon monoxide (CO) and carbon dioxide (CO

). The rest of the wavelengths selected were spread over the full range and included the statistically most relevant absorptions. Conceptually, this prototype can may be considered to be an improvement over the conductive polymer system because a correlation to known spectral absorption wavelengths can be established. One relevant thought here is that we are dealing with molecular vibration-based IR absorptions, and the olfactory system (smells and odors) responses are also linked to molecular vibrations.

FIGURE 2: An electronic nose simulation with a 16-channel ambient air monitor: (a) PCB layout, (b) integrated nose sensor unit, and (c) output results.

The assembly developed (Figures 2a and 2b) is designed to be a standalone ambient air monitoring system, with 16 independent channels, as described above. The key components of the system are exposed in the “semi-transparent” version designated (Figure 2a). The spectral measurements are obtained from four quad detectors (four channels each). In this particular case, each quad is equipped with its own IR source, with the electronics set up to monitor the output of the detectors providing a set of 16 signals, representing 16 different sets of wavelengths. The system monitors the air, which is drawn in by a fan assembly on the side of the measurement chamber. Then, 16 sets of absorbance data are generated and these are normalized and presented in a histogram form (Figure 2c). In this particular example, the 3D printing provides the frame of the overall assembly and the measurement chamber that is coupled to the fan used to transport the air into the chamber. The IR beams from the individual sources transverse the chamber, with a 50-mm optical path, which is sealed at both ends with IR-transmitting windows.

Again, in this case, the 3D printing provided a testing platform that enabled the overall measurement concept to be evaluated.

Ambient air monitoring is intended to measure and monitor low concentrations of gases and vapors in the air. For the nose simulation, a relatively short optical path was used for the measurement. Although this process was effectively multiplexed with the simultaneous measurement of 16 channels the sensitivity for individual species may have been limited by the optical measurement path. For the IR measurement of individual gases (vapors) at low concentrations, it is beneficial to extend optical path to a meter or more. When working with a small measurement system, a 1-m path (or greater) can be difficult to implement within a limited space. The popular “White Cell” has been used for such measurements; however, it can yield a relatively large measurement volume. The system shown in Figure 3 was devised to enable long path measurements, of one to two meters, while maintaining a small internal measurement volume. When optimized, the cell has 4x to 5x less volume than the comparable pathlength “White Cell.”

FIGURE 3: (a,b) Compact 1-m multipass gas cell showing various design elements listed, and (c) a 1-m standalone monitor.

The system shown in Figure 3a (A1/A2) is a packaged design for continuous monitoring applications where the main measurement component, a ring containing 13 mirrors (Figure 3b), is housed within a cylindrical enclosure chamber. In this case, the IR beam is injected into the chamber with an IR source mounted at A1, and is collected at A2 following multiple passages, with reflections from the mirrors mounted in the walls of the ring (Figure 3b). The beam is directed in and out of the enclosure with a pair of off-axis paraboloid mirrors (one in and one out). The ring in this case is 3D printed with the mirror recesses designed to provide an exact push fit for the mirrors. The dimensions are exact to provide a 1-m path following the multiple reflections. This design is unique insofar as it provides for a quick and easy assembly with all 13 mirrors pre-aligned with no additional alignment required.

A modified version of the ring mount is shown in Figure 3c. In this version, the ring also forms the external part of the enclosure, where the IR beam enters and exits on the horizontal axis. For this variant, the beam enters directly from the source and exits at a quad detector, both equipped with their own electronic interfaces. With the exception of these electronic components, the rest of the enclosure components are 3D printed, which are shown in gray and white. Note that the gray component is actually printed from a 50:50 polyamide:aluminum blend. The blend was preferred in this case because the aluminum helped improve the thermal characteristics of the overall package. The benefit provided in this case by the 3D printed part was that it simplified assembly, and it eliminated the need for optical alignment (by design).

In 2015, I gave a webinar on new technical advances in optical spectroscopy. At the end, a participant asked if a technology could be recommended for a Hadamard Transform spectrometer. The answer back was that a technology, such as the Texas Instruments (TI) digital light processing (DLP) chip, could be a potential solution to encoding spectral data for a Hadamard transform. It turned out that not only could the DLP enable Hadamard encoding, but it could also be applied to the design of a spectrometer and eliminate the need for a detector array. This was an unanticipated benefit because it enabled a spectrometer to be developed in a wavelength range that required an expensive array, and it enabled a higher pixel count than such high-cost arrays. A good example is the elimination of the costly extended InGaAs array from a long wavelength NIR spectrometer. In this case, a high-performance single element detector would be used in place of the array. In this case, the DLP provides the array function by collecting the dispersed NIR radiation from a planar grating, and with wavelength selection being made by addressing the individual or groups of programmable reflective pixels.

This resulted in the development of an NIR spectrometer platform based on TI’s DLP technology. It should be pointed out that not all spectral ranges benefit from the use of the DLP scanning. For example, the standard charged-coupled device (CCD) array UV-vis-NIR systems use relatively low cost, high resolution arrays and, in such cases, there is no gain from the use of a DLP. However, the benefits of DLP scanning for the long-wave NIR enables a spectrometer to be designed that covers the spectral range of 1300–2600 nm. An added benefit gained for this spectral range is that extended InGaAs requires cooling to obtain the best noise performance. Changing from an array to a single element detector significantly reduces the thermal overhead, as well as cost of implementation.

It is important to note that TI does not make instruments; it produces advanced computer chips. For the DLP technology to be evaluated, TI designed a functional optical bench and this design was made available to instrument developers and researchers. The evaluation system that was made available is shown in Figure 4. This system had a conventional design, which turned out to be difficult to assemble because each optical component required adjustment and alignment; these alignment components can be seen in Figure 4a.

FIGURE 4: (a,b) Conventional design and the (c) 3D printed optical bench design.

In a collaboration with TI, a new lens recipe was generated based on the use of seven spherical lenses (instead of six aspherical lenses), and this resulted in a significant cost reduction. The optical layout with the spherical optics is shown in Figure 4b. It was realized that an alternative approach to the assembly could be adopted if the optical bench could be fabricated as a single component without the individual optical subassemblies. Yes, this was made possible by a transition to a 3D printed part for the optical base. A 3D printed version of an assembled instrument is shown in Figure 4c, and the components that required alignment by the conventional approach (Figure 4a) can be seen on this assembly, but no alignment is required. All lenses are just inserted into slots and are pre-aligned and optimized.

Was this transition a success? The answer was yes and no. It was possible to make one-off prototypes that performed well. However, at the time, it was hoped that a product could be made directly with this simplified 3D printed baseplate. The main issues came down to limitations of the 3D printing materials in terms of making threaded parts and thermal conductivity. These issues limited the reproducibility of optical alignments, particularly for the grating. The timeframe for this work was late in 2018. We are four years further on now with significant improvements in 3D printers and 3D printing materials. With an optimistic view, it is anticipated that many of the original goals here for instrument manufacturing can be met.

The application of 3D printing has been described in context of a prototyping and concept-proving tool. The examples shown have been limited by both “age” of hardware and materials, and the ability to describe the end-application in depth because of commercial proprietary issues. Although the examples described may appear to lack style, they were all functional and proved a point. Important early limitations were the need to use special fastening aids to enable fixtures, such as screws to be used, the inability to make metal parts and the lack of materials to make optical components, such as lenses. The closest thing to metal parts were made from a 50:50 blend of polyamide and aluminum. Most of the other structural parts were made from selective laser sintering (SLA) grade polyamide.

Today, it is possible to purchase a desktop 3D printer for a couple of thousand dollars, and so conceptually concepts and applications can be tested in “real-time”. In other words, no need to wait for parts to come back from a machine shop or an external contractor. The implied ability to transition from conceived idea to functional prototypes and on to manufactured products can be realized today. Also, the material and fabrication limitations cited above, for the most part, have been addressed. For example, both metal parts and transparent small optical parts can be produced from commercial 3D printers. Examples of 3D printed micro-spectrometers operating in the visible spectrum have been published. However, as an important caveat, it is possible that spectral measurements that require optical transparency in certain wavelength regions may not be available, at least for now, who knows what might exist in a year or two.

It has been a while since I have written an article for 

. I have shared a long-term working relationship with the magazine, going back several decades. During that time, the nature and volume of my work in spectroscopy, and the science itself, have changed significantly. Normally, I write and document in the third person, but for this article, I have deviated from this and written in both the first and third person.

My background and training were in analytical chemistry, and over time became focused on spectroscopy. However, in the 1980s I started the transition to instrumentation, with some focus on optics, opto-mechanics, and spectral data handling. Coates Consulting was established in the mid-1990s, leading to a specialization on measurement technologies and small, low-cost instrumentation.

A few months back, the editorial staff contacted me to see if I could put together an article or two on technologies tied to spectral measurements, and in particular, on key technologies that are involved with new instrumentation, such as with the miniaturization or scaling down of spectrometers, and the changes in measurement platforms. The latter involves the move away from the laboratory and toward handheld or distributed measurements involving spectrometers designed as sensors. Having agreed to write the article, I was faced with the challenge of what material I could discuss and what concepts could be presented; in recent years, most of my instrumentation projects have been covered by non-disclosure agreements (NDAs) and confidentiality documents limiting what can be discussed in public.

I started writing this publication at the beginning of July. Over that first weekend, my attention was drawn to an article published in the New York Times entitled “3D Printing Grows Beyond its Novelty Roots,” by Steve Lohr. The case being made was that the technique of 3D printing had jumped from being a tool to make one-off and prototype parts to a full-blown means of making specialized manufactured mechanical parts, with a high degree of “accuracy” and reproducibility. In my opinion, 3D printing has been a game changer for evaluating spectral measurement concepts, and I have reflected on this aspect in this text.

My personal involvement with 3D printing started around 2012. At the time, it was used for making small numbers of prototypes for projects that involved unique or complex designs for spectral measurements. At issue was how to test out a concept without great expense and without risking going down blind alleys if the “guesses” were wrong. All the systems involved making IR measurements, in the near infrared (NIR) and mid-IR spectral regions. For some applications, it is possible to simulate the application on a laboratory instrument. Although that may work for some applications, the approach may not duplicate the exact sampling or data acquisition conditions and may give a false impression relative to the “success” of the application. This situation is where the benefits of 3D printing emerged. A complete measurement system could be designed (with a standard CAD package), assembled and tested, where the 3D printing included the enclosures and assembly structures as well as some of the optical components. For reference, the CAD package used for the projects outlined here was SolidWorks (published by Dassault Systemes).

The author is not writing this article as an expert in 3D printing; in fact, quite the opposite is true. The article is written from the perspective of a person experienced in the design and implementation of spectroscopic instrumentation and spectral sensing technologies. There is no doubt that many readers may well be more expert in 3D printers and the associated technologies, and so please do not write and complain! I have included several references useful for further information on this subject (1–6).

(1) Think 3D Team, 3D Printing Tutorial (PDF): Beginner’s Guide to 3D Printing, version 0.1. 

https://www.think3d.in/landing-pages/beginners-guide-to-3d-printing.pdf

(2) Workman Jr., J.; Coates, J.; Naranjo, D. Micromirror spectrophotometer assembly. U.S. Patent 10,054,483, issued August 21, 2018.

(3) Workman Jr., J.; Coates, J.; Naranjo, D. Micromirror spectrophotometer assembly. U.S. Patent 10,309,829, issued June 4, 2019.

(4) Baumgartner, B.; Freitag, S.; Lendl, B. 3D Printing for Low-Cost and Versatile Attenuated Total Reflection Infrared Spectroscopy, 

(5) Nesterenko, P.N. 3D Printing in Analytical Chemistry: Current State and Future. 

(6) Wang L.; Pumera. M. Recent Advances of 3D Printing in Analytical Chemistry: Focus on Microfluidic, Separation, and Extraction Devices. 

A 3D printer can shorten the time to make prototype hardware for spectrometers from weeks to days—or less.

The research interests of Beatriz Fernandez, who is an Associate Professor at the University of Oviedo in Spain, are mainly focused on the field of spatially resolved analysis by mass spectrometry-based techniques, particularly glow discharge (GD) sources and laser ablation inductively coupled plasma-mass spectrometry (ICP-MS). Fernandez’s research is mainly focused on imaging studies of biomedical interest tissues and cell cultures for detection of heteroatoms and proteins, with special efforts devoted to development of accurate strategies for absolute quantification, including the use of stable isotopically-enriched tracers (isotope dilution MS). Recently, at the 2022 SciX conference in Covington, Kentucky, Fernandez presented, as part of the Technical Program, a discussion regarding the determination of proteins in single cells by ICP-MS using metal nanoclusters as labels of specific recognition reactions. Fernandez spoke to 

What makes inductively coupled plasma-mass spectrometry (ICP-MS) an essential tool for quantitative ultratrace elemental and isotopic determinations in biological samples?

Nowadays ICP-MS is a well-established technique, both in research and routine laboratories, for analyzing very low concentrations of metallic and semi-metallic elements in a wide variety of samples, including biological samples. ICP-MS does not only allow for the detection of the elements present in the sample (their identification), but also allows for the determination of accurate and precise concentrations of the target analytes (their quantification). Compared to traditional techniques employed in clinical or biological laboratories, the possibility to achieve the determination of absolute concentrations of ultratrace elements makes the ICP-MS technique extremely attractive for the analysis of biological samples. Additionally, the analysis of liquid samples (including, but not limited to, blood, urine, and aqueous humor) can be done in an almost straightforward manner with minimum sample preparation. For the analysis of other type of samples, such as tissue sections or cultured cells, a previous digestion step is required for the analysis by conventional nebulization ICP-MS. However, we can find alternatives by using ICP-MS coupled to single cell (sc) or laser ablation (LA) for the direct analysis of individual cells (without the prior digestion).

How is measuring proteins in single cells related to elemental analysis using ICP-MS?

Due to the high temperature reached within the ICP and the atomization of the sample, ICP-MS technique can only be used to get elemental information from the samples. Therefore, for the analysis of target proteins in single cells, it is necessary to resort to an immunoassay using specific antibodies for the proteins of interest (antigen:antibody recognition reaction). Additionally, such antibodies have to be labeled with an easily detectable element by ICP-MS (not present in the native cells). Thus, it can be stated that elemental analysis in single cells is simpler compared to the analysis of proteins since the immunocytochemistry step is not required. In any case, it should be highlighted that for the analysis of single cells by ICP-MS is necessary to use sample introduction systems fit to purpose: for example, sc-ICP-MS to detect individual cell events in a cell suspension or LA-ICP-MS to identify the inner distribution of individual cells.

What are metal nanoclusters and how are they used to study specific immunochemical reactions?

Recent advances in nanotechnology have introduced a new class of nanoparticles, named as metal nanoclusters (MNCs). MNCs have typical sizes between 0.2 and 3 nm. This type of nanostructure is characterized for having sizes comparable to the Fermi wavelength of electrons and, therefore, they can exhibit molecule-like properties (fluorescence). In addition, MNCs are composed of a few to several hundred atoms of a unique metal, providing high signal amplification by MS detection. Concerning how are they used to study specific immunochemical reactions, the surface of the nanostructure can be tailored with selected groups for different chemistries. For example, carboxylic groups of the MNCs surface can be bioconjugated with specific primary antibodies (for the target proteins) by carbodiimide coupling to act as a label immunoprobe.

How do metal nanoclusters compare to other labeling techniques as metal tagged immunoprobes?

Recently there has been a burgeoning interest in applying water soluble MNCs for biomedical and biosensing applications. MNCs present excellent properties compared to alternative labels, such as metal chelates like tetraxetan (DOTA), polymers containing several metal chelates, quantum dots, or metal nanoparticles—mainly due to their combination of small volume and high number of metal atoms per label. On the one hand, their small size (below 3 nm) makes them of interest for labeling biomolecules such as antibodies, since they will have a lower risk of hindering the antibody recognition capabilities compared with larger tags. On the other hand, the high number of metal atoms per label (up to 1000 metal atoms can be possible per NC depending on the MNCs) provides a high amplification by MS detection. Finally, it can be also pointed out that their fluorescent emission as well as their catalytic properties offer the possibility of multimodal detection—by fluorescence, electrochemical techniques, and by MS detection.

What has been learned so far using metal nanoprobes to study immunochemical reactions in human cells?

The use of metal nanoprobes, such as MNCs, offers us the possibility of achieving molecular information by using the ICP-MS elemental technique. Furthermore, the high sensitivity of ICP-MS together with the high amplification provided by metal nanoprobes make affordable the analysis of proteins in individual cells at very low concentrations. For example, specific proteins in the attogram level can be detected and quantified in individual cells by using sc-ICP-MS and LA-ICP-MS. ICP-MS also shows another important feature for the analysis of human cells: by using state-of-the-art instrumentation (for instance, time of flight mass spectrometry) and several metal labeled nanoprobes it is possible to obtain the simultaneous analysis of different target biomolecules in individual cells.

What are the challenges or limitations involved with using this technique?

Here we can distinguish between the determination of proteins in individual cells by sc-ICP-MS or LA-ICP-MS. In both cases, the accurate and precise determination of the immunoprobe stoichiometry (such as the number of metal atoms per antibody molecule that will be detected by MS) is mandatory. Concerning sc-ICP-MS one of the main challenges is associated with the sample introduction system: a high transport efficiency is required to get representativeness of the cell population as well as preserving cell integrity during the transport to the ICP. On the other hand, the determination of protein concentration by LA-ICP-MS is also challenging, considering the lack of certified reference materials for biological samples. Laser-based techniques exhibit matrix effects and, therefore, calibration standards with a similar matrix to the sample matrix is required to get accurate and precise protein concentrations.

As experience with the technique advances, and mastery of the technique is achieved, what are the expected advances for clinical research discovery?

It is well known that cell populations in all biological systems are heterogeneous. Furthermore, genome, transcriptome, and proteome cell-to-cell variations can be the origin of different pathologies. The quantitative analysis of individual cells in a target population is currently a critical research area in order to evaluate cellular variability as well as to study the behavior of cells submitted to different external stimuli (for example, applicability of treatments to specific diseases). Traditionally, cellular samples have been analyzed by conventional nebulization ICP-MS after digestion of the cells, providing information of the whole population as an average (so, information regarding individual heterogeneity is lost). The combination of sc-ICP-MS and LA-ICP-MS for the analysis of individual cells allows a comprehensive quantitative study of the expression of endogenous proteins by cell populations.

Beatriz Fernandez is Associate Professor in the Department of Physical and Analytical Chemistry at the University of Oviedo (Spain). After her PhD, she joined the research group of Prof. Olivier Donard at the IPREM - CNRS (France) until July 2008. Afterwards she joined the University of Oviedo as Research Scientist. She is co-author of more than 80 original research publications and reviews, seven book chapters and one patent. Her research has been presented in more than 100 conferences and has been co-supervisor of six Doctoral Thesis and sixteen Master Thesis Projects. She is principal investigator of several research projects through which her research is funded. Direct correspondence to: 

Recently, at the 2022 SciX conference in Covington, Kentucky, Beatriz Fernandez, who is an Associate Professor at the University of Oviedo in Spain, presented, as part of the Technical Program, a discussion regarding the determination of proteins in single cells by ICP-MS using metal nanoclusters as labels of specific recognition reactions. Fernandez spoke to Spectroscopy about her presentation. 

Determination of Proteins in Single Cells by Inductively Coupled Plasma–Mass Spectrometry Using Metal Nanoclusters as Labels of Specific-Recognition Reactions

In a follow-up to my February 2020 column, I started a more systematic study of extractables and leachables. Following a suggestion from Mark Witkowski of the FDA, I looked at three sets of centrifuge vials that were exposed to the following liquids in an effort to evaluate the potential of Raman microscopy to identify compounds exiting in polymers under particular conditions: saline, phosphate buffer, water, saline treatment at 100 

C, ethanol, chloroform, pH 5, and pH 9. Although all containers were made of polypropylene (PP), they didn’t behave similarly. Compounds that were extracted from PP vials from different manufacturers were not always the same. Although the number of spectral types that are recorded is large, this article focuses on a few whose interpretation is interesting. The goal was to figure out when it makes sense to employ Raman microscopy for such identification. The characteristics considered were ease of sample preparation, the minimum quantity of material amenable to analysis, and the quality of the identification.

 is the Principal Raman Applications Scientist for Horiba Scientific in Edison, New Jersey.

The selection of solvents for generating extractables and leachables from these polypropylene vials was informed by two USP publications (1,2). Polished stainless steel slides were used as substrates for the analysis because most metals, including stainless steel, do not have a Raman spectrum. These slides virtually provided a background-free substrate for the spectra. For each vial, samples were prepared immediately after receipt and then approximately 10 days after receipt. After the droplets dried, a mosaic micrograph was recorded with a low magnification objective. Then, the regions of interest were selected and imaged at 50x or 100× magnification before spectra were recorded. Interestingly, it was the 638-nm laser line that provided the best results; there was often fluorescence at 532 nm, the signal-to-noise (S/N) using the 785-nm laser was significantly poorer, and the sensitivity in the CH/NH region was compromised. Because of differences in solubility, if there are more than one species in a sample, they tend to separate during precipitation so spectra of the different species are recorded separately. In all cases, spectra were recorded from multiple spots with similar morphology to ensure that a spectrum was representative of the sample. Although multiple spectra can be recorded with the mapping function (that is, in a two-dimensional [2D] area with evenly spaced points), we more often used a function where we could select points in the image. By selecting spots with the same morphology, the analysis time was significantly reduced compared to what would have been required for a map acquisition. To improve the S/N of the spectra, identical multiple spectra from a given data set were averaged together.

We used KnowItAll (KIA) (3) software (Wiley) for spectral interpretation. To optimize the relative intensities in the spectra for matching, spectra were collected with the instrument correction spectrum (ICS) function turned on. If a suitable match did not appear, we used standard rules for spectral interpretation to provide an identification of at least the class of molecule represented by the spectrum.

To demonstrate the functionality of our method, Figure 1 shows the spectrum of one of the vials, as identified as polypropylene (PP) in KIA. However, you may notice that I have not chosen the spectrum with the highest hit quality index (HQI). The first bands near 1620 and 1650 cm

 that are spectrum, with an HQI of 92.1, has extra not in my spectrum. The second spectrum with an HQI of 90.1, is identified as a copolymer of PP and polyacrylic acid (6%). For the moment, I am assuming that these tubes are pure PP, possibly containing some additives, so I have chosen the third entry. The ambiguity in even such a simple query indicates the reasons that some purist spectroscopists warn against solely using spectral data bases for identification.

FIGURE 1: (a) Raman microscope spectrum of a centrifuge vial (bottom black spectrum) with (b) the top three matches in KIA software (see text for discussion). Top matching spectra are shown in both (a) and (b).

Figure 2 shows mosaic images of deposits from centrifuge vials from the three vendors. Many of the deposits indicate the morphology of NaCl crystals, but with material of more disordered morphology adhering to the NaCl cubes. Of course, NaCl has no first-order Raman spectrum, so the spectra that we show were acquired from material clinging to the salt crystals. Figure 3 shows spectra of deposits from vials exposed to saline from each of the 3 suppliers, with the spectrum of PP shown at the bottom for easy reference. The startling observation is that each vial yielded different spectra. When a spectrum indicated the presence of elemental carbon, its two bands had been eliminated manually before plotting in Figure 3 or importing into the KIA software.

FIGURE 2: Low magnification mosaic micrographs of deposits from the saline-exposed centrifuge vials of the three sources studied here. From left to right: first, second, and third vendors.

FIGURE 3: Spectra of deposits from the saline solutions in centrifuge vials of the three vendors. Note that two different species were observed in the vial of the second vendor (second and third spectra from the top). The spectrum of one of the polypropylene vials is shown on the bottom as a reference.

Each spectrum was examined separately in the KIA software, and the results are displayed in Figure 4. None of the matches are as good as that for PP, but there still is ample information present. I should explain how I decide that a match is “good” when a match of the quality of the PP does not appear. The most important thing to understand is that the HQI represents the numerical overlap of the query spectrum with a spectrum from the data base. If there is a baseline, it will tend to overwhelm the HQI without really representing the species present. So the first thing to do is to subtract any baseline in your query spectrum and to enable baseline subtraction in KIA. I have also found that if there are broad bands in my spectrum, for instance from carbon, the searching algorithm finds carbon before anything else. Therefore, it is often helpful to remove the carbon contribution manually from the query spectrum. I will be the first to admit that doing so is not rigorous, but it is effective. That is because looking for a mixture spectrum with sharp bands and the carbon spectrum is not always straightforward because the carbon spectrum is so variable.

FIGURE 4: (a) Spectrum of extract from the first vial (black spectrum) exposed to saline with the “best” match spectrum being 3-hydroxybutanoic acid, found in IDExpert matching software (red spectrum). (bi) Spectrum of extract from the second vial exposed to saline (red spectrum) with the “best” match identified as a block copolymer styrene/isoprene as displayed in SearchIt (blue spectrum). (bii) Spectrum of a second extract species from the second vial (black spectrum) exposed to saline with the “best” match as displayed in ID Expert, a dye called sepisol fast blue (red spectrum). (c) Spectrum of extract from the vial of the third vendor exposed to saline (blue spectrum) with the “best” match being a mixture of poly (N-vinyl pyrollidone) and naphthalene 1,3,6 trisulfonic acid trisodium salt, as found in SearchIt (red composite spectrum).

So if a really good match does not pop up, the question is how to find the “best” match. What I do is to examine the listing in software, which is arranged in decreasing values for HQI while also examining the spectral pattern. I want to find a hit that has prominent bands in the same region as the prominent bands in the query spectrum. Unfortunately, this is not a simple selection of the spectrum with the highest HQI because of the reason explained in the previous paragraph.

So now let’s consider the results of the species found in the saline solutions from the three vendors’ vials. The first is a small molecule of 3-hydroxy butanoic acid. The second had two spectra—the first, a block copolymer of styrene isoprene, and the second, a spectrum of a dye, Sepisol Fast Blue. The spectrum of the deposit from the third vendor’s vial matched best to a mixture of poly (N-vinyl pyrollidone) and naphthalene 1,3,6 trisulfonic acid trisodium salt.

In Figure 4a, the match to 3-hydroxy butanoic acid is fairly good except there is no >C=O band in my spectrum at approximately 1750 cm

. In fact, this spectrum had the presence of the broad carbon bands near 1340 and 1600 cm

 that were interfering with my search so I eliminated them manually, and a small feature at ~1750 cm

 may have been buried in the broad carbon band that I subtracted. Figure 4a is an example that illustrates the hazards of this kind of operation. In addition, my spectrum also shows a broad doublet above 3000 cm

, which indicates the presence of residual water in the deposit.

Figure 4bi shows the best match to a copolymer of styrene isoprene, which was found in SearchIt but not IDExpert. In IDExpert, the sharp band at approximately 1000 cm

Extractables and leachable are typically studied using chromatographic techniques. This study explores what we can learn by using from using Raman microscopy for such studies.

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