Raman and IR Imaging Theory and Applications 2-Day Virtual Conference

Molecular Spectroscopy in Practice

A Dual Nanostructured Approach to SERS Substrates Amenable to Large-Scale Production 

An Inside Look at Near-Infrared Spectroscopy

Information-Based Classifications to Distinguish Characteristics of Raw Agricultural Materials 

Survey of Key Descriptive References for Chemometric Methods Used for Spectroscopy

Raman Spectroscopy as a Tool for Rapid Feedback of Perovskite Growth Crystallinity and Composition 

Analytical chemists are continually striving to advance techniques to make it possible to observe and measure matter and processes at smaller and smaller scales. Professor Vartkess Ara Apkarian and his team at the University of California, Irvine have made a significant breakthrough in this quest: They have recorded the Raman spectrum of a single azobenzene thiol molecule. The approach, which breaks common tenets about surface-enhanced Raman scattering/spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS), involved imaging an isolated azobenzene thiol molecule on an atomically flat gold surface, then picking it up and recording its Raman spectrum using an electrochemically etched silver tip, in an ultrahigh vacuum cryogenic scanning tunneling microscope. For the resulting paper detailing the effort [1], Apkarian and his associates are the 2021 recipients of the William F. Meggers Award, given annually by the Society for Applied Spectroscopy to the authors of the outstanding paper appearing in the journal 

. We spoke to Apkarian about this research, and what being awarded this honor means to him and his team. This interview is part of an ongoing series with the winners of awards that are presented at the annual SciX conference. The award will be presented to Apkarian at this fall’s event, which will be held in person in Providence, Rhode Island, September 28–October 1.

What is the significance to the scientific community of obtaining a Raman spectrum from a single molecule?

Improvement of spatial resolution has driven advances in optical microscopy, which has a history of over 1000 years. As far as molecular matter is concerned, seeing its smallest constituent, namely atoms, would be the ultimate limit. We have demonstrated this experimentally for the first time, by showing optical microscopy with a spatial resolution of 1.6 Å, equal to the radius of a silver atom (2). Beyond seeing single molecules, it is possible to wire molecules to photons (3), to use single molecules as complete opto-electronic devices (4), and, more generally, to access the sub-nano frontier in science.

Were there any previous efforts that you found especially useful in advancing this work further, be it a particular scientific approach or merely motivation or inspiration?

Surface-enhanced Raman scattering/spectroscopy (SERS) was discovered in 1974 by noting that the Raman intensity of molecules adsorbed on a roughened silver electrode were ~1 million times stronger than anticipated. Despite the very large literature on principles and applications of SERS, the subject remains a source of fascination, and we are only now reaching a rigorous understanding because of experimental advances. Perhaps the most incisive of these advances is SERS combined with scanning tunneling microscopy (STM) carried out under ultrahigh vacuum (UHV) conditions and cryogenic temperatures (5 K), exemplified by the current paper (1). This was the first direct demonstration that the spectrum of a single molecule can be observed on roughened silver by STM imaging of the molecule, and then picking it up by the tip and recording its spectrum. The observations, which were rigorously analyzed, went against many tenets as stated in the last line of the paper’s abstract: "Tips decorated with asperities break the rules and give unique insights on Raman driven by cavity modes of a plasmonic junction".

Our motivation and inspiration were the years of indirect experimental observations that had generated significant folklore about the subject. Decisive experimental observations were necessary to break through. It took us ~10 years to build the instrument and reach the point where we could carry out the definitive measurements. There are now a handful of laboratories in the world that have built up the same capabilities, such as Zenchao Dong's group in USTC and Martin Wolf's group at the Max Planck in Berlin, and Nan Jiang’s group at University of Illinois, Chicago and the number is growing as the tools become more accessible.

What motivated you to focus on this specific approach to this challenging problem?

The concept that light can be super-focused on a plasmonic wedge (5) or cone was well-established theoretically (6), and the approach of tip-enhanced Raman had already been taken by several groups starting circa 2000. The definitive experiments required atomic control of tip morphology, sub-Å resolution in scanning, and Å-scale stability in the substrate, which could be achieved by combining precision optics and a UHV cryogenic scanning tunneling microscope. This required significant instrumental development, and our earlier reports of atom-resolved optical imaging involved photo-tunneling (7) and electroluminescence with submolecular resolution (8).



What was your process to devise a method to measure the spectrum of a single molecule?

The combination of optics with high collection efficiency and ultrahigh vacuum scanning tunneling microscope operating at 5 K under pristine conditions was necessary to learn the governing physics, and to be able to definitively image a single molecule and record its Raman spectrum.



How was your approach different from what others have previously tried?

Precision instruments were necessary to learn that general notions of tip-enhanced Raman scattering (TERS) were misguided. We developed TERS in the atomistic near-field, by recognizing that on atomically sharp silver needles, the photon converts to charge.



Do you think that your method may be broadly used in laboratories, or will it be a specialized research project for some time to come?

I expect it to be broadly used, and already many new efforts are starting around the world.

Any word whether your findings were incorporated in other efforts? What sort of feedback did you receive

I find it somewhat ironic that previously the work was rejected twice. One criterion used to that end was that the described work could not be duplicated elsewhere. The body of the work carried by the team is now being recognized and adapted, as evident by the citations of the subsequent works (2-4).



Were there any particularly difficult challenges you encountered in your research?

The effort, which involved significant instrument development and the creation of a dedicated laboratory, required significant marshaling of resources and a long-time commitment, was only possible through Center type support provided by the National science Foundation (NSF). We first proposed the work to NSF in 2007, and created the Center for Chemistry at the Space-Time limit (CaSTL) for the expressed purpose of observing molecular motions with atomistic resolution. The Center had a 10-year lifespan, and delivered on its lofty promise of creating the Chemists' microscope.

Can you elaborate on the strengths that each member of the team bought to the project?

Joonhee Lee has been the team leader (project scientist) from inception to sunset of the CaSTL Center. He has led all aspects of the work, from construction of the instrument to execution of experiments to theoretical analysis, and now has started his own group at the University of Nevada to continue along the same general line of research. Nick Tallarida and Laura Rios were graduate students at the time. They were engaged in the execution of the very intensive experimental work and modeling, both in this project and several other joint publications on related work. The Mars mission is in Nick's good hands who is now at NASA, Jet Propulsion Laboratory (JPL), and the education of next generation scientists is Laura's good hands, as she is now on the faculty at the California Polytechnic Institute.



What are the next steps regarding this research?

This work has already led to a significant body of developments reflected in (2,3,4) and a series of papers in the making, addressing the sub-nano scale in microscopy, molecular devices, molecular electronics, with the key development being the confinement of the photon. Just like the confinement of the electron, which seeded nanoscience, the atomically confined photon (attained at the apex of an atomically sharp STM tip) has opened up the frontier of picoscience, another order of magnitude in miniaturization of devices.



What does being awarded the William F. Meggers award mean to you?

A tremendous amount of dedication and effort was expended by all team members in carrying out the work that appears on several pages of the journal. The cryo-UHV-STM-SERS/TERS work is complex and demanding. The experiments usually demand 24/7 operation over weeks on end. It is therefore satisfying to see an appreciation of the fruit of that labor by the Meggers Award.

(1) J. Lee, N. Tallarida, L. Rios, and V.A. Apkarian, 

(2) J. Lee, K.T. Crampton, N. Tallarida, and V. Ara Apkarian, 

(3) K.T. Crampton, J. Lee, and V.A. Apkarian, 

(4) J. Lee, N. Tallarida, X. Chen, L. Jensen, and V. A. Apkarian, 

(6) A. J. Babadjanyan, M. L. Margaryan, and Kh. V. Nerkararyan, 

(7) J. Lee, S. Perdue, D. Whitmore, and V. A. Apkarian

(8) J. Lee, S. M. Perdue, A. Rodriguez Perez, and V. A. Apkarian, 

Articles

Analytical chemists are continually striving to advance techniques to make it possible to observe and measure matter and processes at smaller and smaller scales. Professor Vartkess Ara Apkarian and his team at the University of California, Irvine have made a significant breakthrough in this quest: They have recorded the Raman spectrum of a single azobenzene thiol molecule. The approach, which breaks common tenets about surface-enhanced Raman scattering/spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS), involved imaging an isolated azobenzene thiol molecule on an atomically flat gold surface, then picking it up and recording its Raman spectrum using an electrochemically etched silver tip, in an ultrahigh vacuum cryogenic scanning tunneling microscope. For the resulting paper detailing the effort [1], Apkarian and his associates are the 2021 recipients of the William F. Meggers Award, given annually by the Society for Applied Spectroscopy to the authors of the outstanding paper appearing in the journal Applied Spectroscopy. We spoke to Apkarian about this research, and what being awarded this honor means to him and his team. This interview is part of an ongoing series with the winners of awards that are presented at the annual SciX conference. The award will be presented to Apkarian at this fall’s event, which will be held in person in Providence, Rhode Island, September 28–October 1.

Recording the Raman Spectrum of a Single Molecule 

Surface-enhanced Raman scattering (SERS) is used to improve Raman scattering, often allowing the detection of single molecules. It generates molecularly specific fingerprints of analytes and, with carefully controlled experimental conditions, can be highly quantitative. Roy Goodacre, a professor of biological chemistry at the University of Liverpool in the United Kingdom, first used this technique to achieve whole-organism fingerprinting of bacteria and then explored SERS in a variety of other applications, including within biotechnology, disease diagnostics, quantitative detection, imaging, food security, and more. Goodacre is the 2021 winner of the Charles Mann Award for Applied Raman Spectroscopy. This interview is part of an ongoing series of interviews with the winners of awards that are presented at the annual SciX conference, which will be held this year from September 26 through October 1, in Providence, Rhode Island.

Has your work with SERS solved some of the challenges commonly associated with Raman spectroscopy for quantitative analysis, such as native sample fluorescence and standardization of Raman intensity?

That’s a great question, and many people believe SERS to lack reproducibility. In fact, SERS can be made highly quantitative, which addresses the second half of your question in terms of standardization. Our group have developed SERS for quantitative analysis and embedded design of experiments in order to search the possible experimental landscape in terms of type of metal ions to reduce, how to perform the reduction (which dictates the “capping” agent on the nanoparticle surface), as well as conditions used to initiate aggregation (in terms of salts as well as controlling the pH of the environment). When you do this, you can come up with nice bespoke protocols that lead to high levels of standardization.

In terms of fluorescence, we very rarely see this. This is due I believe (I’m no physicist) to energy transfer occurring during the excitation. In fluorescence the molecules in the system absorb energy and are in the electronic excited state. After non-radiative decay fluorescence is seen as the emission of light at a longer wavelength to the incident radiation. However, in SERS which uses metallic nanoparticles, a type of FRET mechanism occurs where the light is not emitted but this energy is transferred to the metal. I might have that a bit wrong, but anyway SERS effectively quenches the normal fluorescence process. This is why a lot of people still use Rhodamine 6G which is a highly fluorescent dye to illustrate that their SERS conditions work. There are possibly more SERS spectra of this molecule in the literature than your readers have had hot diners!

In your paper “Recent developments in Quantitative SERS: Moving towards absolute quantification” (1) you and your colleagues critiqued the development of quantitative SERS from simple univariate assessment of single vibrational modes to multivariate analysis of the whole spectrum for improved quantification. What knowledge did you and your team gain from this study?

This was actually a review that I wrote along with my colleagues Professors Karen Faulds and Duncan Graham (from the University of Strathclyde in Scotland), rather than a primary paper. I’m glad you ask about this, as reviews are really good ways to synthesize knowledge and thus to refine concepts and to formulate new ideas. In this review, we specifically looked at how fellow scientists had developed SERS to be quantitative and thus used to provide reproducible and robust values for absolute levels of key analytes (or what we may term determinands). There are a number of things that can affect quantification, and these include (among others) variation in laser fluence during the experiment as well as the number of aggregated nanoparticles with associated molecules within the SERS/Raman collection voxel. At the end of this process, the CCD within a Raman spectrometer will give readouts of energy.

SERS is no different from using, say, liquid chromatography-mass spectrometry (LC–MS) to quantify drugs in athletes’ blood and urine during the Olympics. LC–MS only provides absolute levels of these drugs and their metabolites when the detector (the MS in this case) is calibrated appropriately. SERS is no different, one needs to calibrate the system, and once you do this then absolute quantification is readily achieved.

What we learned and reported in this review was that several groups had come to the conclusion that two general experimental strategies can be used to calibrate the whole system so that absolute quantification (for example, µM or ng/mL of a target in, as an example, blood or urine) could be accomplished. Rather interestingly, both these approaches have been borrowed from other fields including mass spectrometry!

The first method involves spiking the sample with a stable isotope of your molecule. This can be 13C or 15N, but what is particularly useful is the use of deuterium (2H or D) as the reduced mass changes (for example, substitution of C–H with C–D) cause very large shifts in vibrational frequencies. This method has been termed isotope dilution surface-enhanced Raman scattering (IDSERS) and the use of isotopologues of the target molecule overcomes any competitive co-adsorption or co-association with the metal surface, since the natural isotope and stable isotope have very similar physical properties. This also overcomes any laser power fluctuations as both analyte and internal standard are recording simultaneously. The isotope standards (isotopologues) can then be used as subtractions or as ratios if key peaks are clearly seen within the spectra for both natural and stable isotope. Alternatively, one can use multivariate analyses such as linear regression techniques like partial least squares (PLS) which uses the full spectrum, or multivariate curve resolution (MCR) which unmixes spectra to generate a series of ‘pure’ spectra as well as corresponding concentration values.

The second method is to use the standard addition method (SAM) where the standard here is the analyte you want to quantify. In SAM the standard is the natural isotope and so will have the same SERS spectrum as the molecule within the sample. The standard is then ‘spiked’ into the sample multiple times at different known levels. The areas of diagnostic peaks are then calculated and plots of these peak areas against the concentration of the spiked standard will yield a straight line. The key thing here is that the zero-level spike will still have a peak from the target molecule and so the “0” spike concentration will intercept the 

-axis. The slope can then be used to calculate the level of the molecule in the sample with high precision and accuracy.

These methods both give absolute quantification of targets and are excellent methods “borrowed” from other areas of analytical chemistry.

What are some of the newest uses of SERS for clinical diagnostics?

Well, let’s look at what effect the last 18 months or so have had on the SERS field. Many people have shifted their work towards detecting whether someone has Covid-19 or not. Some of these studies have been based on profiling a body fluid. For example, saliva has been used in a recent study by Carlomagno and colleagues (

https://doi.org/10.1038/s41598-021-84565-3

), where multivariate analysis was then used for identification of SERS patterns that are associated with infection. Alternatively, the virus can be measured directly using PCR-based approaches or via antibody detection and these methods are coupled with SERS where the read out is SERS of a specific target dye. These methods, including lateral flow devices, feature in a recent review by Saviñon-Flores 

I guess what this pandemic has achieved is to focus people’s minds on diagnostics. This proves that this area, and especially point of care diagnostics, is important and has allowed mass screening of populations. And of course, all these methods of diagnostics are embedded within excellent analytical science.

Do you see SERS being implemented in practical clinical diagnostics? And is quantitative analysis important?

In addition to Covid-19 detection discussed above, other developments of SERS for clinical application are highlighted in our review in 

) (1). I think one of the most interesting of these includes SERS methods for measuring drugs and their metabolites (xenometabolites) directly in human biofluids (for example, blood and urine). Because no chromatography is used, these methods are very rapid, with results generated in a few minutes. Here, quantification is very important and the measurement of drugs and their xenometabolites also allows one to calculate pharmacodynamics. In addition, SERS can also be used to know if someone has taken an illicit drug several days ago, because drug metabolites are generally cleared from the body at a slower rate than the drug itself.

The speed of analysis here is a huge advantage because many manufacturers are producing handheld and portable Raman instruments that can be taken to the patient, rather than shipping a sample to a central testing facility. I see the future of SERS being within diagnostic point-of-care applications.

In another paper, you described how SERS was used for simultaneous detection of multiple novel psychoactive substances (NPS) as an alternative to various hyphenated methods such as gas chromatography (GC) or liquid chromatography with mass spectrometry (LC–MS) (2). What advantages does the use of SERS have over these hyphenated methods for quantitative analysis of NPS?

The main advantages are similar to those articulated above. SERS can be made portable and is rapid, which means that the methods can be used for on-site for testing. This could be for testing someone for illicit use of these substances.

An alternative application would be to develop and deploy SERS within drug testing centers in large events like music festivals to check the authenticity of NPS. Unfortunately, some of these psychoactive drugs are impure or may contain NPS fentanyl analogs or indeed fentanyl itself. These can be lethal and there have, unfortunately, been several cases in the US, so authentication of drugs prior to use is really important.

What challenges do you think that SERS will have in sample presentation for quantitative detection and discrimination of a range of NPS?

The main challenges will be sample processing in order to extract NPS from body fluids and then ensuring that the NPS associates with the metal surface of the nanoparticles because the analyte needs to be close to or bound to the surface. Neither of these are straightforward, but we can again borrow approaches that we use in our mass spectrometry-based metabolomics. If we want to know the actual concentration in a biofluid then one can adjust for any variance in the extraction efficiency by spiking in isotopes of the NPS target molecule(s) directly into the sample being measured before any sample preparation has taken place. This means that any variation in sample preparation is accounted for as the NPS and its isotope equivalent have the same chemistries and so will be extracted with highly similar efficiencies. Any extraction and measurement errors can then be accounted for by using the ratios of the isotopes in the naturally occurring NPS and comparing this with any stable isotopes in the NPS spike. This approach is also discussed in our review published in 

For the discrimination of a wide range of NPS what we reported in (2) is that the SERS spectra contain vibrational features that reflect their core chemical structures. When we performed unsupervised cluster analysis using PCA (principal components analysis) we found that the NPS samples grouped into three distinct clusters that were identified as methcathinones, aminoindanes and diphenidines. This is potentially very exciting and could be exploited in the future. Clandestine chemists generate new NPS by modifying a designer drug by the addition of chemical groups to a core chemical structure, and that core reflects the psychoactive activity of the drug. Being able to identify the core structure of an NPS is as important as having a method that gives specific spectral fingerprints that allow for the identification of individual NPS.

Is there a specific direction you see this technique developing into over the next few years? In other words, are there unmet challenges to this technique that if resolved would bring broader applications?

SERS, at the moment, is mainly used within research laboratories. However, the realization that this method can be made to be robust, reproducible, highly quantitative, and that it is inherently portable suggests to me that it is only a matter of time before this technique can be translated from the laboratory to the clinical environment.

I’m not the only one to think this. Inspired by employing SERS for therapeutic drug monitoring, Professor Alois Bonifacio (from the University of Trieste in Italy) led a large-scale multi-instrument interlaboratory study, as part of the EU COST Action BM1401 Raman4Clinics to address whether equivalent data can be collected in different laboratories. This study was published last year in 

https://doi.org/10.1021/acs.analchem.9b05658

) and shows that SERS can indeed be standardized, and that once standard operating procedures (SOPs) are adopted, then SERS is robust enough to allow for the quantification of target molecules. Our group was proud to be part of this study which involved 15 different laboratories throughout Europe. Our study was one of the first multi-center SERS studies.

Of course, central to SERS is the instrument itself. In another study also published last year in 

https://doi.org/10.1021/acs.analchem.0c02696

) Professor Thomas Bocklitz (from the University of Jena in Germany) lead another Raman4Clinics study that was designed to address how Raman spectrometers can be configured to provide robust results and thus help improve inter-laboratory studies using Raman spectroscopy. This was also a large-scale round robin study involving 15 institutes within seven European countries and compared 35 Raman spectroscopic devices with different configurations. We were also involved in this study, and it led to a series of recommendations that we believe are worthy of future investigations to improve multi-laboratory studies.

I believe that both these studies are very important and show that SERS has great potential for adoption within clinical environments.

What are your next steps in your own work with Raman spectroscopy and SERS?

Lots, but I will just mention one very exciting area that we are developing.

Within the University of Liverpool’s Centre for Metabolomics Research (CMR) we are establishing a variety of Raman-based methods for chemical image analysis of tissues, cells, and bacteria. We have capabilities in spontaneous Raman spectroscopy from Renishaw, stimulated Raman spectroscopy (SRS), and coherent anti-Stokes Raman spectroscopy (CARS) from Leica. We also have a combined Optical Photothermal IR and Raman system from Photothermal Spectroscopy Corp.

Our group is very excited by these techniques, and we hope to combine vibrational spectroscopic imaging with mass spectrometry-based metabolomics. For those interested in the CMR please visit: 

https://www.liverpool.ac.uk/liverpool-shared-research-facilities/facilities/multi-omics/cmr/

Within these pages there are a few case studies, and to whet your reader’s appetites we have recently published papers on “metabolism in action” where we have been developing stable isotope probing using Raman and infrared imaging for understanding metabolic flux at the single bacterial cell level. This is really pushing the resolution limits of these platforms, and we’re very excited by the results.

Before we close out this interview, I would like to take this opportunity to thank FACSS for the Charles Mann Award. I was really shocked, absolutely thrilled and honored to find out about being given this award. There have been some exceptional scientists that I have looked up to (and indeed still do!) in the Raman field who have been bestowed this award, and I am amazed to be in their company. It’s a wonderful reflection of the work our fantastic research team has achieved over the last 20 years, and they are as deserving as I. Thanks to them all!

I'm also really hoping that I will be able to travel to Providencein September to meet up with friends and colleagues and of course to make new connections. I've certainly missed the international conference scene. I'm reminded of going to ICORS 2018 in Jeju and meeting Professor Luis Liz-Marzán (from CIC biomaGUNE in Spain) during a session or two on SERS. This chance meeting led to a substantial review by nearly 70 authors on SERS. A review that, while only being published two years ago, has already been cited over 500 times. If you’re really interested in SERS, then this tour-de-force in 

 on the “Present and Future of Surface-Enhanced Raman Scattering” (

(2) H. Muhamadali, A. Watt, Y. Xu, M. Chisanga, A. Subaihi, C. Jones, D.I. Ellis, O.B. Sutcliffe, and R. Goodacre, 

Roy Goodacre received his degree in microbiology from the University of Bristol (Bristol, England) and was awarded his PhD in 1992 in mass spectrometry applied to microbiological problems (also from Bristol). He is Professor of Biological Chemistry at the University of Liverpool and leads a team of around 20 researchers in the Department of Biochemistry and Systems Biology within the Institute of Systems, Molecular and Integrative Biology (ISMIB). Additionally, he is the Chair of the Institute's Research and Impact Committee, Deputy Head of Department, and a co-director of the Centre for Metabolomics Research. His research interests include analytical biotechnology and disease diagnostics, detection, imaging, food security, as well as systems and synthetic biology. He has more than 30 years of expertise in mass spectrometry (MS)-based metabolomics, Raman spectroscopy, and advanced multivariate data analysis.

https://en.wikipedia.org/wiki/Roy_Goodacre

https://scholar.google.com/citations?user=Dszi0U8AAAAJ&hl=en

Roy Goodacre, a professor of biological chemistry at the University of Liverpool in the United Kingdom, first used SERS to achieve whole-organism fingerprinting of bacteria and then explored SERS in a variety of other applications, including within biotechnology, disease diagnostics, quantitative detection, imaging, food security, and more. Goodacre is the 2021 winner of the Charles Mann Award for Applied Raman Spectroscopy. This interview is part of an ongoing series of interviews with the winners of awards that are presented at the annual SciX conference.

Quantitative Analysis Using Surface-enhanced Raman Scattering (SERS): Roy Goodacre, the 2021 winner of the Charles Mann Award for Applied Raman Spectroscopy

To supervise the quality of new cement, it is necessary to constantly update the monitoring and testing methods of the cement composition. The molecular layer deposition (MLD) method is an X-ray fluorescence (XRF) analysis method that can scrutinize a glass frit of an unknown dilution ratio. In this study, the method of preparing a glass frit of cement from phosphogypsum for XRF analysis by the MLD method was reviewed, and the working curve suitable for XRF analysis of the cement from phosphogypsum was established. The proposed analysis steps of a glass frit can be widely used in factories that produce cement from phosphogypsum.

The new cement product produced by the production process of sulfuric acid integrated with cement from phosphogypsum (1,2) has been widely used in the market. Phosphogypsum is the solid waste that is produced by sulfuric acid decomposing the phosphate rock in the process of phosphoric acid production by phosphorus chemical enterprises. Because of the harmful impurities and high purification cost of phosphogypsum, its effective use has become a major problem (3). Nowadays, the accumulation of phosphogypsum in China has reached 500 million tons, and nearly 80 million tons of phosphogypsum are newly produced each year. The utilization of large-scale solid waste resources of phosphogypsum can reduce the environmental and safety risks brought by the massive accumulation of phosphogypsum, which is significant for the protection of the Yangtze River Economic Belt and the safety of China’s grain production. Nowadays, phosphogypsum is used to produce cement clinker, which is an effective way to consume phosphogypsum (4).

The production of sulfuric acid with the co-production of cement from phosphogypsum is an effective measure to solve the problem of utilizing phosphogypsum. This project digests a large amount of phosphogypsum and balances the consumption of sulfuric acid in the production of ammonium phosphate, which is a sustainable method (5). When producing cement clinker from phosphogypsum, gypsum instead of limestone is used to prepare raw meal, and a reducing agent coke is required. The resulting prepared raw meal is called raw meal with phosphogypsum. During the cement production, the addition of an appropriate amount of reducing agent carbon promotes the decomposition of calcium sulfate in the raw meal into calcium oxide for producing cement clinker and sulfur trioxide for producing sulfuric acid. Adding the reducing agent carbon increases the difficulty of X-ray fluorescence (XRF) analysis of the raw meal, so it is necessary to explore the preparation method of the glass frit, which is suitable for raw meal with phosphogypsum.

The molecular layer deposition (MLD) method is a new XRF analysis method, which overcomes the drawbacks that the XRF analysis is subject to. The chemical method preparation of standard samples and the prepared glass frit reduces the dependence on the preparation equipment quality, the flux quality, and the technical level requirements of personnel (6).

In this experiment, the MLD method was used to analyze the raw meal with phosphogypsum. However, because of the presence of carbon, the high temperature burning of the sample causes a large amount of sulfate decomposition loss in the preparation of the glass frit, resulting in a large error in the analysis of the sulfur content. Therefore, the method of preventing pyrolysis of sulfate during the fusion process was studied, and a suitable method for preparing the glass frit for raw meal with phosphogypsum samples was proposed. At the same time, the corresponding standard samples were prepared, and the analytical method was established. It is known from our work that the glass frit preparation method proposed in this study to analyze raw meal with phosphogypsum has good accuracy, and the analysis time is greatly reduced.

For this study, we utilized a S8 Tiger X-ray fluorescence spectrometer from Bruker; a high-temperature furnace (working temperature: 600–700 °C, and 950–1000 °C) from Platinum; ammonium nitrate; and, for the analysis, special reagent lithium tetraborate and lithium metaborate was used in a 33:67 ratio along with 30% of the release agent ammonium iodide.

 refers to the amount of carbon dioxide, moisture, fixed carbon in the coal, and organic compounds lost (volatilized) in the sample at high temperatures. For the loss on ignition of conventional cement samples, it is only necessary to weigh the mass of the sample before and after burning it at 950 °C to 1000 °C. Calcium sulphate in the sample will decompose largely at 950 °C, which is because of reducing carbon in raw meal with phosphogypsum. Therefore, calculating the burning loss of the raw meal with the phosphogypsum sample requires the removal of the reducing agent carbon before reaching the decomposition temperature of the calcium sulfate. To cure the sulfur dioxide produced by the small amount of the decomposed calcium sulfate, it is also necessary to add a sulfur dioxide absorbent. In this study, the carbon was removed by oxidation with ammonium nitrate at 600 °C, and the sulfur dioxide is solidified with lithium borate (7).

For this experiment, 1.2 g of raw meal, 1.0 g of ammonium nitrate, and 6.0 g of flux (high-temperature treated) were mixed in a platinum crucible of known mass. The crucible was placed on the electric furnace and heated until the oxidant had completely evaporated. Thereafter, the crucible was transferred to a high temperature furnace at 600 °C and calcined for 10 min. Finally, the furnace was heated at 950 °C for 12 min. After cooling to room temperature, the total mass of raw meal and platinum crucible was weighed, and the loss on ignition of the raw meal was calculated according to equation 1. Three parallel measurements were made and the results of the loss on ignition are shown in Table I:

 is the mass before the raw meal being burned; and 

 is the mass after the raw meal is burned at 980 °C.

Two drops of the release agent were added to the glass frit in which the burning loss was measured. The crucible and the fused glass bead were placed in a high temperature furnace at 980 °C until the melt was completely molten. Then, the crucible was taken out and shaken to drive out the bubbles and make the melt completely uniform, and the platinum mold was reheated for 5 min in a high-temperature furnace. Finally, the sample was poured into a platinum mold and cooled to form a glass frit. The sample preparation process for the glass frit is shown in Figure 1.

FIGURE 1: Illustration of the process of sample preparation as described in the text.

The working curve of the MLD method shows the relationship between the fluorescence intensity and the concentration of sample, which relates to the parameters such as the MLD coefficient, curve slope, and matrix correction coefficient between elements (8). In this study, the MLD coefficient was calculated by preparing the raw meal with phosphogypsum glass frit with different dilution ratios; the matrix correction coefficient α

 between the elements is calculated by the theoretical calculation method (9), and only the main components of silica, aluminum oxide, ferric oxide, calcium oxide, and sulfur trioxide were subjected to matrix correction. The intensity–density conversion calculation is performed using the Lachance-Traill mathematical correction mode, that is shown in equation 2

 represents the concentration of the chemical component to be tested; 

 represents the concentration of coexisting chemical components; 

 represents the slope of the working curve; 

 represents the intercept of the working curve, also known as the background equivalent concentration (BEC); α

 represents the coefficient of influence of the coexisting chemical component to be tested; and 

indicates the intensity of the chemical component 

 to be measured in the fused glass bead when the dilution ratio of the sample to be tested is the same as that of the calibration sample (CS).

The intensity measured by the S8 Tiger instrument was the net intensity minus the background, at which time 

 = 0. In the experiment, four calibration samples were used to make the working curve (10). The mixed sample of gypsum and clay standard samples were used as the calibration samples. The gypsum ratios in the four calibration samples were about 100, 95, 90, and 80, respectively. The standard values of the four calibration samples are shown in Table II.

 was calculated by a regression analysis that measures the intensity of the calibration sample melted using the standard dilution ratio (11). The intensity of the fuse converted to the intensity of the standard dilution ratio by using equation 3 (12–14). The results of intensity that the three calibration samples measured three times in parallel at the standard dilution ratio are shown in Table III

 represents the standard intensity of the glass frit; 

 represents the measured intensity of the glass frit; 

 represents the dilution ratio of the glass frit of the sample to be tested; and 

 represents the standard dilution ratio of the glass frit (

In the XRF analysis experiment, three crystals are used to measure the elements, which are the LiF200 crystal, the PET crystal, and the XS-55 crystal, respectively. The angle of the element in the spectrum is related to the atomic number; and the intensity of the element is proportional to the concentration. The analytical angle of each element in the raw meal is shown in Figure 2. The MLD coefficient calculated with raw meal and the slope 

 values calculated with the calibration samples are shown in Table IV. The concentration value of the prepared calibration samples were calculated according to the relevant parameters of the curve in Table IV, and the values are shown in Table V. Compared with the standard value (Table II), the deviations of the measured value (Table V) of the calibration samples are less than the limit specified by the standard, and the deviation of the results obtained in three parallel determinations is within the allowed range of the standard. Therefore, this method has excellent accuracy in determining the element content in cement.

For this study, 1.2 g of raw meal with phosphogypsum, 1.0 g of oxidant, and 6.0 g of flux were added in a platinum crucible and stored evenly with a thin glass rod. The crucible was put on the electric furnace and baked for about 20 min until the oxidant was completely evaporated. After that, the crucible was transferred to a high temperature furnace at 600 °C and burned for 10 min. After cooling to room temperature, two drops of the release agent were added into the crucible. Finally, the crucible was placed in a high temperature furnace at 950 °C for 12 min before the crucible was taken out and shook to drive the bubbles, and a platinum mold was placed in a high temperature furnace for 5 min. The sample was poured into the platinum mold until cooling, and then the glass frit was made.

Measurement of Glass Frit and Conversion of Sample Concentration 

The prepared glass frits were put into the instrument to measure the intensity of each measurable element in the sample, and the mass percentage of the sample chemical components (element oxides) were determined by an iterative method according to the equation 2. The sum of the mass percentage of each chemical component in the sample was then calculated. The statistical results show that the total of the nine oxides determined in the raw meal with phosphogypsum was 99.78% (calculated on a burning basis). If the sum of the mass percentages of the chemical components in the sample was less than 99.78%, which indicates that the actual dilution ratio of the sample glass frit is greater than the standard dilution ratio, then we would have needed to set a dilution ratio greater than the standard dilution ratio and calculate the melt strength under the standard dilution ratio by equation 3, and then the sum of the concentration of the sample is calculated by using equation 2.

The above steps are supposed to be repeated until the total sample concentration is 99.78%. Conversely, the sum of the mass percentage is greater than 99.78%, which implies that the dilution ratio of the sample glass frit is less than the standard dilution ratio. Then, a dilution ratio that is smaller than the standard dilution ratio is set. The actual dilution ratio of the glass frit and the content of each chemical component in the sample could be obtained by the same method as above described. The commercial MLD calculation program automatically calculates the dilution ratio of the glass frit and the chemical content of the sample, according to the intensity result of the glass frit.

Accuracy of Sample Results with Different Dilution Ratios

To start off, 16 fuses were prepared according to the fuse method described above. The melt dose was fixed at 6 g, and the mass of raw meal varies from 0.6–1.5 g. The intensity value was measured by the instrument and the calculated dilution ratio was shown in Table VI. The concentration values were calculated using the MLD calculation program and the statistical standard deviation of samples were shown in Table VII. In routine measurements, each sample was subjected to two parallel tests, and the standard deviation was calculated (

 = 2). The results were compared with the statistical standard deviation. Table VI shows the standard intensity of samples with different dilution ratios that is corrected by equation 3, and Table VII shows the concentration values calculated by the MLD method. The deviation values of the obtained results are all less than 0.1, which shows that the precision of this method is excellent.

Loss of Sample Components During Calcination

Because of the particularity of the raw meal with phosphogypsum, the loss of the sample was reduced by adjusting the different preburning temperature without adding the oxidizing agent ammonium nitrate. The specific steps are as follows: 1.2 g of the raw meal and 6.0 g of the flux were placed in the crucible. Then, the crucibles were placed in a high temperature furnace with different temperatures and times. The temperature range was 500 °C to 800 °C, and the time range was 10 min to 20 min. The two parallel tests were performed, and the resulting data is shown in Table VIII. The temperature test of the sulfur content loss in the raw meal was carried out, and it showed that the sulfur content loss was the smallest when the temperature was 600 °C.

We can conclude from Table VIII that the carbon may not be completely removed by combustion at lower temperatures. Therefore, to further reduce the loss of sulfur, carbon was eliminated by adding different amounts of ammonium nitrate at a temperature of 600 °C. The influence of the amount of different oxidants on the experimental results is shown in Table IX. The results in Table IX show that about 1.0 g of ammonium nitrate can completely remove carbon from the sample as much as possible, and then the sample is melted at 950 °C, the sulfur element in the melt is stable and will not cause the loss of sulfur dioxide volatilization.

To summarize our findings, Table VIII and Table IX show that when calcination is performed without an oxidizing agent, the mass of sulfate sulfur in the raw meal is always low at a temperature that carbon can be removed. Thus, it is necessary to add 1.0 g of ammonium nitrate to the sample and calcine the sample at 600 °C before melting it.

The carbon in the mixture of raw meal with phosphogypsum and lithium borate salt is completely oxidized and removed at 600 °C under the coexistence of ammonium nitrate. When heated to 950 °C, the glass frit mixture of constant mass is formed, and sulfate in the mixture will not decompose.

In the range of dilution ratio of 5 to 12, the MLD coefficient was used to accurately convert the intensity of each element to the intensity at the standard dilution ratio. Accurate analysis results were obtained by preparing the samples with arbitrary dilution ratio, and the results showed that a XRF analysis of an MLD method could be applied to analyzing chemical components in raw meal with phosphogypsum.

(3) M.Y. Tan, L.X. Xiang, G.L. Li, et al. 

(4) Z.Q. Yang, Q. Chen, Q.C. Guo, et al. 

(5) H.G. Ding, R.B. Ding, X.Z. Yang, et al. 

Comprehensive Utilization SP&BMH Related Engineering

. S. J. Zhuo, Ed. (East China University of Science and Technology Press, Shanghai, China, 2006), pp. 1–108.

(9) China Patent CN103969273, 2013. https://patents.google.com/patent/ CN103969273B/en?oq=CN103969273.

(11) China Patent CN105486708, 2014. https://patents.google.com/patent/ CN105486708A/en?oq=CN105486708.

(12) J.J. Lu, Y.B. Liu, J.X. Ren, and W. Han, 

(14) P. Dai, Y. B. Liu, R. Yan, and Y. Z. Zhang, 

 is with the Key Laboratory of Interfacial Reaction and Sensing Analysis at the University of Shandong and the University of Jinan in Jinan, China, and with the China Building Materials Test & Certification Group Co., Ltd, in Beijing, China. 

 is with the Key Laboratory of Interfacial Reaction and Sensing Analysis at the University of Shandong and the University of Jinan in Jinan, China. 

, are with the China Building Materials Test & Certification Group Co., Ltd, in Beijing, China. Direct correspondence to: 

Phosphogypsum can be used as an intermediary material to produce cement clinker. To monitor the quality of phosphogypsum cement, a novel molecular layer deposition X-ray fluorescence (XRF) analysis method using a glass frit was developed.

An X-ray Fluorescence (XRF) Analysis of a Molecular Layer Deposition (MLD) Method Used in Producing Cement from Phosphogypsum

In this study, we examined the frequently reported “selective fluorescence quenching effects” of Fe

 ions on carbon dots. Instead of the believed “quenching effects” by Fe

, the strong reduction of the fluorescence of carbon dots is likely because of the inner filter effect, which is caused by the strong ultraviolet (UV) absorption of Fe With the addition of Fe

 ions, the UV absorption increases dramatically, compared to other metal ions. When Fe

 ions coexist with carbon dots (CDs) in the solution, most of the excitation light is absorbed by Fe

 ions, decreasing the excitation light obtained by CDs. This phenomenon leads to the “fluorescence quenching.” This finding helps us gain a better understanding of the fluorescence quenching effects of metal ions on carbon dots and understand the quenching effect of metal ions on other fluorescent materials.

Heavy metal ions are critically important to certain biological functions, but they are also considered environmentally hazardous and detrimental to human health if not handled and treated with the necessary precautions (1–7). As a result, methods of identification and quantification are necessary. Carbon dots (CDs) have attracted considerable attention as photocatalysts (8), sensors (9–10), printing inks (11), in drug delivery (12), and in bioimaging materials (13), because of their unique optical, electronic, and chemical properties. Because of the unique properties of CDs, metal ions demonstrate different fluorescence quenching effects on them. Particularly, Cu

ions have special fluorescence quenching effects on CDs, as summarized in Table I (14–24). However, there are still some important questions that need to be addressed. Why can Fe

 ions selectively quench many types of carbon dots? And why is there no wide range of linear relationships between the quenching effects and the Fe

 concentration, especially at relatively high concentrations (CFe

As shown in Table I, regardless of the raw materials, the preparation methods of CDs or the ion quenching conditions used, Fe

 ions have special selective fluorescence quenching effects on nearly all CDs. The selective fluorescence quenching of CDs by metal ions is because of the strong binding energy between the ions and the electron-rich nitrogen and oxygen groups on the surface of CDs (2,4–5,7,9–10). This theory can explain most cases; however, when it comes to Fe

For example, when it comes to electron capacity, which occurs in the order:

 is stronger than that of others, the fluorescence quenching effect of Ag

(17–18,23,25). This observation conflicts with the general assumption that electron capacity order determines quenching effect order. Furthermore, the ability of the Hg

 ion to obtain electrons is stronger than that of the Fe

 ion; however, in most cases, the fluorescence quenching effect of the Hg

 ion is also much lower than that of the Fe

 ion, which is also in conflict with the current general assumption.

In this study, we attempted to obtain a valid explanation for the selective fluorescence quenching effects on CDs.

All chemicals used were of analytical reagent grade, except garlic and grass samples. The compounds lithium chloride (LiCl), sodium chloride (NaCl), sodium hydroxide (NaOH), potassium chloride (KCl), silver nitrate (AgNO3), mercury (II) chloride (HgCl

), were purchased from Sinopharm Chemical Reagent Co., Ltd. The stock solutions of metal salts were prepared using distilled water.

To confirm and further investigate the quenching mechanism in Table I, four kinds of CDs, namely CD-A, CD-B, CD-C, and CD-D, were made from different raw materials such as grass, glycine, glucose, citric acid, ethylenediamine, and garlic, according to previous studies (18–20,25).

The ultraviolet-visible (UV-vis) absorption spectrum was obtained with a Cary 60 UV-vis spectrophotometer (Agilent Technologies). The photoluminescence (PL) spectra of CDs were obtained with a Cary Eclipse fluorescence spectrophotometer (Varian).

The QY of CDs was measured according to an established procedure (26). Quinine sulfate in 0.1 M of H

 (the literature QY is 0.54 at 360 nm) was chosen as a standard. Absolute values were calculated using the standard reference sample that had a fixed and known fluorescence QY value. To minimize the absorption effects, the absorbance in the 10 mm fluorescence cuvette were kept under 0.1 at the excitation wavelength (360 nm). The QY was estimated to be 6.8%, 4.2%, 4.6%, and 7.3% for CD-A, CD-B, CD-C, and CD-D, respectively.

The experiment was performed at room temperature in distilled water. In a typical run, CD dispersion was diluted with distilled water to make sure the UV absorbance was lower than 0.1. The metal ions were also prepared in aqueous solution with distilled water. Subsequently, 10 mL of the as-prepared CD solution and 10 mL of the metal ion solution was mixed in a 25 mL glass bottle. The PL spectra were recorded after reaction for 5 min at room temperature.

Figure 3 demonstrates that the CD-C material in aqueous solution has a single UV-vis absorption peak at 345 nm, and very bright blue luminescence under the illumination of UV (365 nm) light, even at a very low concentration (Absorbance <0.1). The CD-C aqueous solution can be seen in the inset of Figure 1. Excitation-dependent PL behavior was also observed as the literature (18), which is common in some fluorescent carbon materials (Figure 1) (27–30). When the excitation wavelength is changed from 300 nm to 400 nm, the emission wavelength demonstrated hardly any shift. This behavior contributes to the surface state affecting the bandgap of CDs (31). In the fluorescence spectra in Figure 1, CD-C has optimal excitation and emission wavelengths at 345 nm and 440 nm, which are different from the literature (19).

FIGURE 1: Fluorescence emission spectra (slit ex = 2.5 nm, em = 5 nm) and UV-vis absorbance spectra of CD-C; Inset: Photograph of CD-C under the illumination of UV (365 nm) light (left) and white light (right).

FIGURE 3: UV-vis spectra of the metal ions and the quenching samples with CD-C. (a) UV absorbance of 1 mM metal ions. (b) UV absorbance of the quenching samples with 1 mM metal ions. (c) UV absorbance of quenching samples with different concentrations of Fe

 concentration (0.005–1.000 mM) and the increased absorbance of the samples at 345 nm.

We investigated the fluorescence quenching effects of various metal ions on CDs in distilled water (Figure 2). A series of metal ions (Li

), each at a concentration of 1 mM were used in the experiment. As shown in Figure 2, the fluorescence quenching effects of the Fe

 ions were much stronger than that of the other ions for all four of the synthesized CDs. According to previous work, there must be a unique mechanism that has yet to be accounted for.

FIGURE 2: Fluorescence quenching effects of 1 mM metal ions on CDs in distilled water corresponding to (a) CD-A, (b) CD-B, (c) CD-C, and (d) CD-D, respectively (ex = 345 nm, em = 440 nm).

Subsequent experimentations revealed that the selective quenching effects of Fe

 on CDs were likely caused because of the inner filter effect, which occurred because of the strong UV absorbance of the Fe

 ions (Figure 3a). When the CDs solution was mixed with Fe

, the UV absorbance of the samples would dramatically increase compared to other metal ions (Figure 3b). To investigate the Fe

 fluorescence quenching effects on CDs, CD-C was used in the quenching experiments because of its high QY.

, 1 mM) and their corresponding quenching samples are demonstrated in Figure 3. Figure 3a shows the UV absorbance of the metal ions aqueous solutions; apparently, only Fe

 solutions have strong UV absorbance, whereas the other metal ions have hardly any absorption. As shown in Figure 3b, the addition of the Fe

ions would greatly enhance the UV absorption of the samples compared to the original CD solution. The absorbance of the samples intensified as the concentration of Fe

 ions increased (Figure 3c). The increased absorbance is as high as 0.81 (1 mM Fe

, 345 nm), and the excitation light could likely be absorbed by Fe

 ions to some extent, decreasing the excitation light obtained by CDs and contributing to the quenching effects.

Finally, Figure 3d shows there is a highly correlated linear relationship between the Fe

 concentration and the increased absorbance of the samples at 345 nm (which is the optimal excitation wavelength of CD-C). This indicates that it is possible to detect Fe

 by UV absorbance when using CDs as sensors. In comparison, there is no good linear relationship between Fe

 concentration and its absorbance (Figure 4b).

 ions “quenching effects” on CD-C in distilled water. (ex = 345 nm, em = 440 nm, slit ex = 2.5 nm, em = 5 nm). (a) “FL quenching” spectra of CD-C with different concentrations of Fe

The “selective fluorescence quenching effects” of Fe3+ ions on carbon dots are examined to gain a more comprehensive understanding of the interactions of metal ions with a variety of fluorescent materials.

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