The identification and characterization of biological molecules is central to the biotech industry, particularly in drug development, bioprocess optimization, and advanced analytics. Yet, despite revolutionary advances, many scientists still struggle with the limitations of traditional tools like mass spectrometry (MS) and nuclear magnetic resonance (NMR).

In this episode from the Smart Biotech Scientist Podcast, David Brühlmann meets Tom Rizzo, Professor Emeritus at EPFL and Co-Founder and CSO at ISOSPEC Analytics, a start-up that spun off from his laboratory, where they aim at simplifying molecular identification.

Key Topics Discussed

• Fundamentals of analytical chemistry tools: mass spectrometry vs. NMR.
• Tom Rizzo’s scientific journey from early inspiration to academic research.
• Combining laser spectroscopy and mass spectrometry at EPFL.
• Transition from academia to industry and founding ISOSPEC Analytics.
• Overview of cryogenic infrared ion spectroscopy and its advantages.
• Key differences from traditional analytical techniques.
• Integration of the new method into biotech workflows and LC-MS systems.
• Recap of insights and outlook toward applications in biomarker discovery.

Episode Highlights

• The limitations of conventional methods in biomolecular analysis and the need for innovation [02:51]
• How Tom Rizzo's early fascination with science shaped his academic path and focus on spectroscopy [03:31]
• The story behind combining mass spectrometry and laser spectroscopy, and building a novel machine for biophysical research [04:52]
• Why Tom Rizzo transitioned from academia to ISOSPEC Analytics and the desire to see laboratory techniques applied in practice [09:17]
• The fundamentals of cryogenic infrared ion spectroscopy: what sets it apart from traditional IR and MS methods [11:14]
• Key advantages of CIRIS, including its sensitivity, specificity, and the ability to distinguish isomers by their unique molecular "fingerprints" [14:25]
• Integration into existing biotech workflows, compatibility with LC-MS setups, and ongoing efforts toward commercial instrumentation [17:29]
• Reflections on the challenges and lessons of bringing scientific innovations to real-world industry settings [17:43]

In Their Words

Mass spectrometry is an incredibly sensitive tool. NMR is a fantastic method for analyzing and identifying molecules, but you need a lot of material to acquire an NMR spectrum. A mass spectrometer, on the other hand, detects ions—and because you can detect ions, it’s incredibly sensitive.

You can essentially detect individual ions by doing spectroscopy inside a mass spectrometer. We were able to measure well-resolved infrared spectra with the sensitivity of mass spectrometry. And that’s what makes it really unique.

Episode Transcript: Cryogenic Infrared Ion Spectroscopy: From Mass Spec Limitations to Molecular Precision - Part 1

David Brühlmann [00:00:34]:
Welcome to The Smart Biotech Scientist. I’m your host, David Brühlmann. Today’s guest is Professor Tom Rizzo, former Dean of the School of Basic Sciences at EPFL in Lausanne, Switzerland, and now Chief Scientific Officer at ISOSPEC Analytics.

We’re diving into cryogenic infrared ion spectroscopy—a game-changing analytical technique that could revolutionize how you identify unknowns, characterize glycans, and solve those frustrating structural puzzles in your bioprocess development.

If you’ve ever struggled with molecular identification using traditional mass spec alone, this conversation will open your eyes to what’s possible today. Let’s get started.

Welcome, Tom—it’s great to have you on today.

Tom Rizzo [00:02:35]:
Thanks, David, for having me. I’ve listened to a few of your podcasts and really enjoyed them, so it’s an honor to be here.

David Brühlmann [00:02:42]:
The pleasure’s mine, Tom. To start, share something you believe about biomolecular analysis that most people would disagree with.

Tom Rizzo [00:02:51]:
I don’t like to be too controversial, but I’d say there hasn’t been a fundamentally new commercially available technology for the analysis of biological molecules in the past 10 years—probably not since the introduction of ion mobility into mass spectrometry.

David Brühlmann [00:03:08]:
All right—now we’re getting into the nitty-gritty of analytics! I’m excited to unpack that. But first, I’d love to hear your story, Tom—what got you started in science, what sparked your passion for chemistry, and what were some pivotal moments throughout your long and distinguished academic career?

Tom Rizzo [00:03:31]:
This will date me a little bit, David, but I grew up in the ’60s, and I grew up in New York. In 1964–65, there was a New York World’s Fair not too far from where we lived. My parents would take us almost every other weekend to this World’s Fair. All of the big technology companies had buildings with presentations and demonstrations—there was IBM, there was Bell Labs, there was DuPont—and I was just fascinated by it all.

I remember very, very distinctly the DuPont exhibit, where they did chemistry demonstrations. I was fascinated by this. In fact, many years later, I performed those same demonstrations in front of my chemistry class at university, and even in my kids’ elementary school classes. That really sparked my interest in science.

I knew I wanted to be a scientist, but I wasn’t sure whether it would be chemistry or not. In fact, I thought I would be a physicist—I thought my mathematics background was pretty good—so I was planning to go into physics. But I took an Advanced Placement course in chemistry in my last year of high school. Part of the course involved being given small vials with unknown samples, and we had to determine what was in them. This was really the start of my interest in analytical chemistry, if you will.

The way we were graded was that the teacher would give you a piece of paper with tabs on it, and each tab had a different answer. If you pulled the first tab and got it right, you received the highest grade. If it took two pulls, your grade went successively lower. I set up a little laboratory in my room at home to run these chemical tests. That really grabbed and sparked my interest in chemistry.

So I decided to study chemistry at university, but I was oriented more toward the physical side of chemistry. When I went on for a PhD, I pursued physical chemistry, but I also did a minor in physics. I’ve always kept that interest in physics. But deep down, my interest in analytical chemistry was there from the early days.

When I started my PhD, I was doing something very “physics-y,” in a sense. I was really looking at molecules by how they interacted with light. The whole field of spectroscopy fascinated me. I really liked the tools of physics. I thought lasers were cool—that’s what I pursued.

By the time I finished my PhD, I asked myself: how large a molecule could we get interesting information from using spectroscopy? This led me to a postdoc at the University of Chicago with Don Levy, a distinguished professor who developed techniques to put large molecules into the gas phase and measure their spectra. We measured the first spectrum of an isolated amino acid in the gas phase at low temperatures, and that was kind of my start.

When I took my first faculty position at the University of Rochester, my goal was to combine mass spectrometry and laser spectroscopy as a new way to study biological molecules. But I had no reputation in mass spectrometry at all. I was trying to get my grants funded, and no one wanted to fund this kind of work. I got close at the National Institutes of Health and close at the National Science Foundation, but no one would fund it.

I had to step back, since I needed to get tenure as an assistant professor. I returned to studying the physics of smaller molecules and managed to do well enough to earn tenure. Shortly afterward, I was approached about taking a position at the École Polytechnique Fédérale de Lausanne (EPFL). I applied, got the position, and we packed up our family and moved to Switzerland. There, I continued my work looking at the chemical physics of laser-excited molecules.

But I always had in the back of my mind the experiments I wanted to do as an assistant professor—combining laser spectroscopy and mass spectrometry—I just never had the funding.

An opportunity came at EPFL, where I had spent two years as a department head. I said I would never do that job again—it was really uninteresting. But the president of the institution changed and asked me to take over as department head in chemistry again. I agreed, on one condition: that I would finally be able to do the experiment using laser spectroscopy and mass spectrometry, which I had never been able to fund.

This was in the year 2000. I told him, if you give me the money to build this machine, I’ll serve another term as department head. He agreed. I wrote up a project and obtained 400,000 Swiss francs to build a new machine.

That was the beginning of the end for me, in the sense that ever since, I’ve been involved in the spectroscopy of biological molecules in combination with mass spectrometry.

Most of my work over the years has been curiosity-driven research. I was fascinated by what we could learn about large molecules through spectroscopy at low temperatures. Early on, I wasn’t really thinking about practical applications or commercial products. But toward the end of my academic career, I began reflecting on my scientific legacy.

I developed a real passion for seeing the techniques we’d developed in the lab make their way into practical use—to become part of a commercial instrument that could help people analyze biomolecules or even diagnose disease. That became very motivating for me.

I had a couple of postdocs in my group who shared that same passion. So, about a year before my formal retirement from EPFL, we founded ISOSPEC Analytics, and I took on the role of Chief Scientific Officer. When I officially retired a year later, I maintained my affiliation with ISOSPEC and left EPFL.

Honestly, it turned out to be a great way to retire. When you leave an institution after 30 years, from one day to the next you go from being “somebody” to being “nobody,” right? But at the company, I felt my expertise and experience were still highly valued. It was a wonderful transition from academia to industry.

And the world of startups is completely different from academia—there’s so much new to learn, and I found that very stimulating.

David Brühlmann [00:10:48]:
That’s inspiring, Tom. So let’s unpack the technology itself and dive into the details of what you’re developing. You’re working on a novel infrared spectroscopy method—but infrared spectroscopy has been a cornerstone analytical technique for decades.

Can you explain the fundamental breakthroughs you and your team have achieved? How is your approach different from traditional IR methods?

Tom Rizzo [00:11:14]:
There are several key differences. You’re right—infrared spectroscopy has been around for decades and has been a cornerstone of analytical chemistry. But traditional infrared spectroscopy, especially in the condensed phase, has some major limitations.

In the gas phase, infrared spectroscopy can be extremely detailed and precise because molecules are isolated. But for biological molecules—typically studied in solution or in complex matrices—the spectra are often broadened and distorted by environmental interactions. The vibrational modes are influenced by their surroundings, so the spectral bands become broad and less uniquely characteristic of a specific molecule.

If you can instead bring large molecules—like peptides or sugars—into the gas phase and remove them from that solution-phase environment, the spectra become much simpler. The lines sharpen significantly.

Then, if you cool those gas-phase molecules to cryogenic temperatures, the spectra become even simpler and sharper, because thermal motion is minimized.

Cryogenic spectroscopy can also be done in rare-gas matrices, where you can achieve very sharp spectra—but that’s a labor-intensive process and not practical for analytical applications. So our goal was to make this simplification accessible in a practical, analytical way: by cooling isolated ions to cryogenic temperatures in the gas phase.

And perhaps the most important innovation is that we can measure infrared spectra with the sensitivity of mass spectrometry.

Mass spectrometry is incredibly sensitive. NMR is also a fantastic tool for analyzing and identifying molecules, but it requires much larger sample amounts. In contrast, mass spectrometry can detect individual ions.

By doing spectroscopy inside a mass spectrometer, we can essentially perform spectroscopy on single ions—and obtain well-resolved infrared spectra with the unmatched sensitivity of mass spectrometry. That’s what makes our approach truly unique.

David Brühlmann [00:13:33]:
Just to make sure I fully understand—your method—is it using mass spectrometry, or is it something separate?

Tom Rizzo [00:13:41]:
It’s actually performed inside a mass spectrometer. We have a cryogenic cell integrated into the instrument. You can separate ions using liquid chromatography, ion mobility, or any technique that couples to mass spectrometry. Once the ions are separated, we send them into the cryogenic cell and measure their infrared spectra. The molecules are mass-selected—they could also be mobility-selected or LC-selected—but it’s all done inside the mass spectrometer.

David Brühlmann [00:14:12]:
Ah, I see. Could you recap the main advantages over traditional MS-based methods? Are the differences at the sample, matrix, or quantity level?

MS/MS is used very widely for the identification of molecules, and it’s a very effective technique. However, it has some drawbacks. For example, when you have different isomers—molecules that have the same mass but differ only very slightly—MS/MS can struggle. The position of a hydroxyl group on a molecule, for instance, or in the case of sugars, which have enormous isomeric complexity, distinguishing just the linkage between two sugars can be extremely difficult. Molecules that are so similar can fragment in similar ways, making it hard to distinguish isomers by their MS/MS spectra.

There was an interesting paper within the last year by a group in Nijmegen. Often, to interpret MS/MS spectra, you use calculations to predict what the fragmentation products would be. They compared these predictions with actual measurements of fragments made using spectroscopy, and found that, in a large majority of cases, the calculations were simply wrong. It’s just too difficult to calculate fragmentation patterns accurately enough to be certain about assignments using MS/MS techniques.

By contrast, the infrared spectrum of a molecule cooled to very low temperatures provides an absolute, distinguishing fingerprint. No two molecules will have exactly the same spectrum if measured at high enough resolution. A cryogenic infrared spectrum, using the technique we call CIRIS—cryogenic infrared ion spectroscopy—can even distinguish subtle differences, like whether a hydroxyl group is pointing up or down. That small difference is enough to shift the vibrational frequencies of a molecule and differentiate one isomer from another.

This makes the technique extremely sensitive, and it also gives a high degree of certainty in identification. It introduces a completely new data dimension. Earlier, you asked me about my controversial statement regarding biomolecular analysis, when I said there are no new technologies. What I meant is that there hasn’t been a new data dimension for analyzing molecules—until now.

With a cryogenic infrared spectrum, you add that new dimension. In addition to retention time, mass, and fragmentation patterns, you now gain an independent, orthogonal measurement. For every mass, you get a cryogenic infrared spectrum—a barcode or fingerprint of that molecule. It’s truly a new dimension in biomolecular analysis.

David Brühlmann [00:17:01]:
So, in layman’s terms, it’s almost an orthogonal method—another dimension of data that complements traditional mass spec.

Tom Rizzo [00:17:12]:
Exactly. You get all the information from traditional methods, plus this new dimension. And we’ve optimized the technique so that it doesn’t significantly increase measurement time—you get this extra layer of data essentially for free.

David Brühlmann [00:17:29]:
How well does this integrate into traditional workflows? Many biotech companies use LC-MS setups—would this method be compatible or disrupt their processes?

Tom Rizzo [00:17:43]:
If I can digress for a moment: one lesson I’ve learned moving into industry is that having the best technique isn’t enough. People have established workflows, and changing those is very difficult. Going into a pharmaceutical company and telling scientists to do things differently can be a tough sell.

So our goal is to integrate as seamlessly as possible. We don’t yet have a commercial instrument, but we’re collaborating with Agilent to bring our technology to the market. In the meantime, we have a purpose-built instrument in our lab.

The idea is that you can continue your existing workflow—run LC-MS as usual—but inside the mass spectrometer, a laser scans as the peaks elute. You analyze the mass and measure the infrared spectrum simultaneously. The perturbation to the workflow is almost invisible. On your screen, you’ll see the new infrared spectrum plotted alongside your regular data.

Modern lasers make this feasible. When I was a PhD student, lasers were huge, complicated, and required a physics degree to operate. Now, the laser is fully integrated inside the instrument—there’s nothing to adjust. It’s completely transparent and doesn’t impede the workflow. It simply adds this new dimension of data.

David Brühlmann [00:19:31]:
That wraps up part one with Tom Rizzo. We explored the fundamental science behind cryogenic infrared ion spectroscopy and how it addresses real analytical challenges in the lab.

In part two, we’ll dive into practical applications for biomarker discovery, therapeutic development, and how this technology is becoming accessible for biotech companies like yours.

If you found this episode valuable, please leave us a review on Apple Podcasts or wherever you listen. Thank you for joining us on your journey to bioprocess mastery. For additional bioprocessing tips, visit us at www.bruehlmann-consulting.com.

Stay tuned for more inspiring biotech insights in our next episode. Until then, let’s continue to smarten up biotech.

Disclaimer: This transcript was generated with the assistance of artificial intelligence. While efforts have been made to ensure accuracy, it may contain errors, omissions, or misinterpretations. The text has been lightly edited and optimized for readability and flow. Please do not rely on it as a verbatim record.

Next Step

Book a free consultation to help you get started on any questions you may have about bioprocess development: https://bruehlmann-consulting.com/call

About Tom Rizzo 

Tom Rizzo earned his PhD in Physical Chemistry from the University of Wisconsin–Madison in 1983 after completing his undergraduate studies at Rensselaer Polytechnic Institute. Following postdoctoral work at the University of Chicago, he joined the University of Rochester and later became a Professor of Chemistry at the École Polytechnique Fédérale de Lausanne (EPFL), where he also served as Dean of the School of Basic Sciences.

His research combines laser spectroscopy, ion mobility, and mass spectrometry for biomolecular analysis. After retiring from EPFL in 2023, he became Chief Scientific Officer at ISOSPEC Analytics, a start-up focused on biomarker discovery. His honors include the Bourke Award, Ron Hites Award, and an ERC Advanced Grant, and he is a Fellow of the APS and AAAS.

Connect with Tom Rizzo on LinkedIn.

He is also a biotech technology innovation coach, technology transfer leader, and host of the Smart Biotech Scientist podcast—the go-to podcast for biotech scientists who want to master biopharma CMC development and biomanufacturing.  

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