Troubleshooting Everywhere! An Assortment of Topics from Pittcon 2025

The Pittsburgh Conference, more commonly known as Pittcon, is the largest conference focused on analytical chemistry in the United States. It was held in Boston during the first week of March. With multiple technical oral presentation sessions held in parallel, it can be difficult to decide exactly which talks to attend. This year was no exception, but my choices did not disappoint; I walked away from the conference with new troubleshooting knowledge related to multiple topics including oligonucleotide analysis by mass spectrometry, and analysis of anthropogenic compounds in the environment. In this installment of “LC Troubleshooting,” I touch on highlights from these talks, as well as troubleshooting advice distilled from a lifetime of work in separation science by LCGC Award winner Christopher Pohl.

At the time of my writing this article, I have just returned from Pittcon 2025, which was held at the Boston Convention and Exhibition Center from March 2–5 2025. As usual, I experienced a week of catching up with long-time friends in a variety of fields, discussing potential new collaborations, and of course getting a refresher course on recent technical advances in analytical chemistry broadly, and separation science specifically. Without exception, each day there was at least one talk I attended that addressed a topic that fits squarely in the domain of “LC Troubleshooting.” In this installment, I briefly discuss three of these topics, in each case providing a little background, a quick summary of what was presented at Pittcon, and relevant references for those who are interested in learning more about the topic.

Improving Sensitivity for Oligonucleotide Analysis by Mass Spectrometry

On Sunday, March 2, Bingchuan Wei of Genentech gave a presentation entitled “Unveiling the Structural Complexity of Guide RNA, A Critical Reagent used in CRISPR Gene Therapy.” Although the bulk of the talk was focused on experiments aimed at understanding the higher order structure of guide RNAs (gRNAs), there was a short portion of the presentation that I found especially interesting from the point of view of method development and troubleshooting for LC separations of RNAs in general. Several reports in the literature have indicated the challenges associated with detection of oligonucleotides (ONs) by mass spectrometry (MS) that arise from the formation of adducts upon association of the ONs with alkali metals ions such as sodium and potassium ions. One example of this is shown in a 2022 paper by Wei and co-workers where they compared the spectral quality for gRNAs in terms of the overall signal-to-noise ratio, and the quality of deconvoluted mass spectra used to identify ONs based on accurate mass (1). Their results were consistent with previous work by Birdsall and associates., which was focused on smaller ONs (2). Figure 1 shows a comparison of mass spectra obtained under “normal” LC-MS conditions (Figure 1a) or conditions where steps have been taken to reduce adduct formation (Figure 1b). In Figure 1b, we see that the spectra are much cleaner, with better signal-to-noise ratio, which improves sensitivity and increases confidence in deconvoluted mass spectra obtained from these data. In this work, Wei and coworkers discussed the following instrument and sample considerations, and demonstrated that a dramatic improvement in spectral quality can be realized if the following practices are followed:

• Use plastic containers for both mobile phase solvents (replacing the more commonly-used glass bottles) and sample vials. This eliminates the potential for alkali metal ions leaching from the glass into the solvents or samples.
• Use MS-grade solvents and reagents (for example, solvent additives) that are less likely to contain alkali metal ions at concentrations that will lead to significant adduct formation.
• Use freshly purified water that is not exposed to glass before using it in the LC system. Again, this reduces contamination of the water with alkali metal ions that leach from glass surfaces into the water.
• Flush the LC system with 0.1% formic acid in water overnight prior to use to remove alkali metal ions from the flow path.

FIGURE 1: Comparison between (a) ON mass spectra obtained under “normal” LC–MS conditions, and (b) spectra obtained after steps have been taken to reduce adduct formation. This figure was generously provided by Bingchuan Wei and Lance Cadang.

During his Pittcon presentation, Wei indicated these guidelines are generally helpful, but that maintaining a “metal-free” flow path over a period of days can be difficult, due in part to contamination of mobile phase solvents by metal ions from ambient laboratory air. In previous work (3), Wei’s Genentech colleagues had shown that small-pore reversed-phase columns can be used to separate low molecular weight sample constituents (including alkali metal ions) from gRNAs by a size-exclusion (SEC) mechanism prior to MS detection. This is a very simple, but effective, approach to reduce the level of metal adduction of ONs during MS analysis, providing a way to get much cleaner spectra. Finally, Wei showed that the SEC approach can be used in the second dimension of a two-dimensional liquid chromatography (2D-LC) system to provide simple, but effective, cleanup of ON materials eluting from a first dimension separation, such as ion-pairing reversed-phase chromatography. This is a clever approach that seems poised to find broad applicability for the analysis of ONs by MS.

Common Analytical Targets for Analysis by Chromatography Coupled with MS Adsorb to Microplastics

On Wednesday, March 5, Emanuela Gionfriddo from the State University of New York (SUNY) at Buffalo gave a presentation entitled “Solid-Phase Microextraction Reveals Microplastic-Mediated Transfer of Semi-Volatile Pesticides.” One of the take-home messages of her talk was that solid-phase microextraction (SPME) provides a convenient means to study the adsorption of molecules to microplastics (defined as plastic-derived microparticles ranging in size from 1 µm to 5 mm) in situ. Adsorption to microplastics is known to be affected by a large number variables, including temperature, ionic strength, and pH (3,4). Traditional approaches to quantitative analysis of pesticides and other organic compounds of environmental significance, such as filtration and extraction, affect these parameters. This, in turn, may unintentionally affect the distribution of the compounds under study between the aqueous environment and the microplastic particles, thus calling into question the results obtained from these approaches. During her talk, Gionfriddo explained that when carried out in the “negligible depletion” mode, the SPME approach provides a unique, effective means of studying adsorption equilibria without altering the equilibria through steps that change the composition of the system. Only trace amounts of the target analytes are extracted onto the SPME sorbent, leaving the rest of the system unaffected. Moreover, multiple SPME extractions can be made from the same system, adding to the analytical power of the approach.

We have dabbled in environmental work in my own laboratory over the years. As I listened to this talk, I could not help but think about all of the prior work in my laboratory, and others, using approaches that first filter particulates out of river, lake, and wastewater, followed by extraction of the remaining liquid using solid-phase (SPE) extraction and similar techniques. The emerging work on microplastics is making it clear that past studies where the particulates were simply discarded have missed a significant fraction of the mass of many organic compounds in the environment. It is going to be very interesting to follow the work of Gionfriddo’s group and others as they work to learn about the interaction of organic compounds and microplastics in the environment.

Advice on Troubleshooting and Experimental Design from a Legend in Separation Science

On Tuesday, March 4, LCGC International hosted a session organized to honor this years’ recipients of the LCGC Emerging Leader in Chromatography Award (Katelynn Perrault Uptmor of William and Mary in Williamsburg, Virginia) and the LCGC Lifetime Achievement Award (Christopher Pohl). I found it fascinating to hear Pohl tell several stories about his professional development from the early days working as a chemist at a fertilizer manufacturer to the later stages of his career in senior leadership at Dionex and Thermo Scientific. A holder of 112 issued patents, he has made tremendous contributions to the field of ion chromatography, and analytical chemistry more generally.

Pohl closed his presentation with a few bullet points that I think can be described as advice to younger scientists distilled from a lifetime of achievement in separation science. Two of them that I think are relevant to anyone engaged in a troubleshooting exercise are as follows:

Change one thing at a time: This is advice both John Dolan and I have articulated in past “LC Troubleshooting” installments (5), but it is so important that I don’t think it’s possible to have too many reminders of the principle. In the context of a troubleshooting exercise, the idea is that we should only change one thing at a time, see what happens, and then decide what to do next. For example, a wavy ultraviolet (UV) detector baseline could be caused by an air bubble in the flow cell, or a sticky check valve in the pump. An “all-in” approach to resolving this problem could involve both flushing the flow cell with isopropanol to remove air bubbles, and changing from machine-mixed mobile phase (pure solvents from pump heads A and B are combined to produce the mobile phase) to pre-mixed mobile phase pumped from just one channel of the pump. If we do this, we’re changing two things in the system at once. Now, suppose the problem goes away; the question is “Which of the two changes resolved the problem?” We will never know. While this is not necessarily a problem in the moment, if we fail to isolate the actual problem and its solution this time, we will not know which problem to pursue first in the future when we see the same symptoms. Furthermore, the all-in approach is often overly expensive, where it is likely that we will replace parts that are in fact perfectly functional. It is very tempting in the moment when we are trying to get a failing instrument back online to say; I’ll come back later and figure out what the actual problem was. But the reality of these situations is that “later” rarely ever happens. We simply move on and lose the opportunity to learn things from the troubleshooting exercise that could help us in the future.

Plan your experiments carefully (this includes troubleshooting experiments): Pohl’s point with this piece of advice was that it is important to plan experiments carefully to avoid preventable mistakes or oversight. If a new experiment fails, it is highly likely that you will not repeat that experiment again, instead moving on to try other ideas or potential solutions to problems. In other words, the idea tested in that experiment—which may, in fact, be a very good one—probably won’t get a second chance if it fails the first time. I really appreciated this point, because it certainly is consistent with my own scientific experience. We are often interested in making sure that a successful, or useful experiment is reproducible, but we rarely hear people express interest in making sure that an experiment that has failed one time also fails reproducibly. This advice is another way of saying what I have heard from one of my scientific mentors for a long time—“If you don’t have time to do the experiment right the first time, when will you have time to do it right?”

Summary

In this installment, I have discussed some troubleshooting tips and advice gleaned from presentations at the recent Pittcon 2025 conference held in Boston. If we pay close attention to these conference talks, there is actually a lot of troubleshooting knowledge lurking in them. Specifically, at this Pittcon I was able to pick up advice on reducing metal adducts in the analysis of oligonucleotides by mass spectrometry, learned about adsorption of commonly-studied organic compounds to microplastics in the environment, and took away troubleshooting advice distilled from a lifetime of work by separation science legend Christopher Pohl. I learned a lot from this Pittcon, in addition to the opportunity to catch up with friends in the field.

Acknowledgments

I want to thank Emanuela Gionfriddo and Bingchuan Wei for reviewing this article and providing helpful feedback.

References

(1) Wei, B.; Wang, J.; Cadang, L.; et al. Development of an Ion Pairing Reversed-Phase Liquid Chromatography-Mass Spectrometry Method for Characterization of Clustered Regularly Interspaced Short Palindromic Repeats Guide Ribonucleic Acid. J. Chromatogr. A 2022, 1665, 462839. DOI: 10.1016/j.chroma.2022.462839

(2) Crittenden, C. M.; Lanzillotti, M. B.; Chen, B. Top-Down Mass Spectrometry of Synthetic Single Guide Ribonucleic Acids Enabled by Facile Sample Clean-Up. Anal. Chem. 2023, 95 (6), 3180–3186. DOI: 10.1021/acs.analchem.2c03030

(3) Concha-Graña, E.; Moscoso-Pérez, C. M.; López-Mahía, P.; Muniategui-Lorenzo, S. Adsorption of Pesticides and Personal Care Products on Pristine and Weathered Microplastics in the Marine Environment. Comparison between BioBased and Conventional Plastics. Sci. Total Environ. 2022, 848, 157703. DOI: 10.1016/j.scitotenv.2022.157703

(4) Yu, F.; Yang, C.; Zhu, Z.; Bai, X.; Ma, J. Adsorption Behavior of Organic Pollutants and Metals on Micro/Nanoplastics in the Aquatic Environment. Sci. Total Environ. 2019, 694, 133643. DOI: 10.1016/j.scitotenv.2019.133643

(5) Stoll, D., R. Some Essential Principles of Effective Troubleshooting. LCGC North Am. 2020, 38 (10), 505–509.

About the Author

Dwight R. Stoll is the editor of “LC Troubleshooting.” Stoll is a professor and the co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. He has authored or coauthored more than 75 peer-reviewed publications and four book chapters in separation science and more than 100 conference presentations. He is also a member of LCGC’s editorial advisory board.