🎤Dr. Leigh Terry (University of Alabama)In this episode of Concentrating on Chromatography, we sit down with Dr. Leigh Terry from the University of Alabama to explore one of the biggest challenges in environmental analysis today: understanding—and accurately measuring—PFAS in water systems.While most regulatory methods focus on a small number of compounds like PFOA and PFOS, there may be thousands of PFAS compounds in the environment—and much of that burden goes undetected.<h4>In this episode, we cover:</h4><ul><li>Why PFAS are called “forever chemicals” (and why that label is evolving)</li><li>The limitations of targeted LC-MS/MS methods</li><li>What the fluorine mass balance problem reveals about PFAS destruction</li><li>How combustion ion chromatography (CIC) helps measure total organofluorine</li><li>Why “99.99% removal” doesn’t always mean what you think</li><li>Where PFAS actually end up in wastewater treatment systems</li><li>The most effective technologies for PFAS removal today</li><li>Why drinking water may only account for ~20% of total PFAS exposure</li></ul><h4>Key Insight:</h4>Even when analyzing dozens of PFAS compounds, researchers may only be capturing a small fraction of total organic fluorine—raising major questions about how we monitor and regulate these contaminants.<h3>Video Transcription</h3>In this episode of Concentrating on Chromatography, Dr. Leigh Terry discusses PFAS contamination in drinking water, the analytical challenges associated with measuring these compounds, current treatment technologies, and future directions for monitoring and regulation. The conversation highlights how analytical chemistry, environmental engineering, and public health intersect in addressing one of today's most discussed classes of emerging contaminants.<h4>Understanding PFAS and Their Significance</h4><h5>The University of Alabama Water Quality Research Center</h5>Dr. Leigh Terry is part of the University of Alabama's Center for Water Quality Research. The center operates an analytical laboratory focused on drinking water quality and the detection of contaminants at extremely low concentrations.The laboratory specializes in:<ul><li>Drinking water analysis</li><li>Organic contaminants</li><li>Emerging contaminants</li><li>PFAS monitoring</li><li>Water quality research</li></ul>Their work supports both research initiatives and practical solutions for water utilities and environmental agencies.<h4>Why Focus on PFAS?</h4>Although PFAS compounds have existed since the 1940s, public awareness has increased dramatically only in recent years.According to Dr. Terry, several factors contributed to the rapid growth of PFAS research:<ul><li>Increased media attention</li><li>New regulatory initiatives</li><li>Growing evidence of human exposure</li><li>Expanding research opportunities</li><li>Greater understanding of potential health risks</li></ul>PFAS are not new contaminants. Rather, society is only beginning to understand their prevalence in the environment and their potential impacts on human health.<h4>The Development of Standardized PFAS Methods</h4>One major advancement in PFAS analysis has been the development of standardized EPA methods.These methods allow:<ul><li>Consistent testing procedures</li><li>Comparable results between laboratories</li><li>Regulatory monitoring</li><li>Reliable data interpretation</li></ul>Dr. Terry notes that many of these standardized approaches began appearing around 2018–2019, providing a common framework for analytical laboratories and researchers.<h4>How Large Is the PFAS Universe?</h4>Many people are familiar with PFOA and PFOS, two of the most heavily studied PFAS compounds. However, these represent only a small fraction of the broader PFAS family.PFOA and PFOS are often referred to as:<ul><li>Legacy PFAS compounds</li><li>Original PFAS contaminants</li><li>Early commercial PFAS chemicals</li></ul>The challenge is that even minor modifications to molecular structure create entirely new PFAS compounds.Researchers estimate:<ul><li>Thousands of PFAS compounds exist</li><li>Some estimates exceed 10,000 compounds</li><li>Exact numbers depend on how PFAS are defined</li></ul>Scientific debate continues regarding classification and definition, making precise counting difficult.<h4>Why PFAS Are Called "Forever Chemicals"</h4>Dr. Terry notes that the phrase "forever chemicals" has become widely used, although she suggests the terminology could benefit from rebranding.PFAS persistence results from two key chemical properties.<h5>1. Amphiphilic Structure</h5>PFAS molecules contain:<ul><li>A water-loving (hydrophilic) head group</li><li>A water-repelling (hydrophobic) fluorinated tail</li></ul>This unusual combination allows PFAS to partition between:<ul><li>Water</li><li>Solids</li><li>Air-water interfaces</li><li>Foam</li></ul>Unlike many organic compounds, PFAS can interact effectively with multiple environmental phases.<h5>2. Extremely Strong Carbon–Fluorine Bonds</h5>The carbon–fluorine bond is among the strongest bonds in organic chemistry.As Dr. Terry explains:<ul><li>Large amounts of energy are required to break these bonds</li><li>Natural degradation processes are often ineffective</li><li>Destruction can require highly aggressive treatment conditions</li></ul>These properties make PFAS extremely persistent in both environmental systems and biological organisms.<h4>Toxicity, Regulations, and Environmental Behavior</h4><h5>Understanding PFAS Toxicity</h5>One of the biggest challenges in PFAS regulation is the incomplete understanding of toxicity.Dr. Terry explains that EPA regulations generally consider three factors:<ul><li>Presence in drinking water sources</li><li>Toxicity to humans</li><li>Availability of practical treatment options</li></ul>While some PFAS compounds are known to be harmful, many others remain insufficiently studied. Furthermore, PFAS often occur as mixtures rather than individual compounds.This creates important scientific questions:<ul><li>What is the toxicity of individual PFAS?</li><li>What is the toxicity of PFAS mixtures?</li><li>How should cumulative exposure be evaluated?</li></ul>These uncertainties continue to drive active research.<h4>What Happens to PFAS in Wastewater Treatment Plants?</h4>PFAS do not behave like many conventional contaminants.Rather than remaining fully dissolved in water, they often accumulate at interfaces such as:<ul><li>Water-solid interfaces</li><li>Water-air interfaces</li><li>Foam layers</li><li>Biological solids</li></ul>As a result:<ul><li>Some PFAS accumulate in biosolids</li><li>Some partition into foam</li><li>Some remain in treated effluent</li></ul>Their distribution depends on wastewater composition and treatment processes.<h4>Regulatory Monitoring and Targeted LC-MS Analysis</h4>Current EPA regulations focus on targeted analysis of specific PFAS compounds.Examples include:<ul><li>PFOA</li><li>PFOS</li></ul>Drinking water regulations currently establish maximum contaminant levels (MCLs) for these compounds at extremely low concentrations.Advantages of targeted LC-MS analysis include:<ul><li>Clearly defined analytes</li><li>Established regulatory limits</li><li>High analytical sensitivity</li><li>Reliable quantitation</li></ul>However, limitations remain:<ul><li>Only a small subset of PFAS is measured</li><li>Many PFAS compounds remain undetected</li><li>Total PFAS burden may be underestimated</li></ul>As Dr. Terry notes, targeted analysis provides only part of the overall picture.<h4>The Fluorine Mass Balance Problem</h4>One of the most important concepts discussed in the interview is fluorine mass balance.When PFAS degrade, fluorinated fragments may remain in the environment.This creates a critical question:Are treatment technologies truly destroying PFAS, or merely transforming them into shorter-chain PFAS compounds?To answer this, researchers perform fluorine mass balance calculations using:<ul><li>Targeted PFAS analysis</li><li>Fluoride measurements</li><li>Total organic fluorine measurements</li></ul>A successful destruction process should ultimately convert PFAS into fluoride ions rather than simply producing smaller PFAS molecules.<h4>Advanced Analysis, Treatment Technologies, and Future Directions</h4><h5>Combining Targeted and Bulk Fluorine Measurements</h5>Dr. Terry's group complements targeted LC-MS analysis with measurements of total organic fluorine.Approaches include:<ul><li>Absorbable Organic Fluorine (AOF)</li><li>Extractable Organic Fluorine (EOF)</li><li>Combustion Ion Chromatography (CIC)</li></ul>In these methods:<ul><li>Organic fluorine is captured</li><li>Samples are combusted</li><li>Released fluoride is quantified</li></ul>This provides insight into total fluorinated content, although contributions may originate from sources beyond PFAS, including pharmaceuticals and consumer products.<h4>Surprising Findings from Combined Analysis</h4>One important finding from wastewater studies was that targeted PFAS measurements accounted for only a small fraction of total organic fluorine.Dr. Terry reports that:<ul><li>Approximately 4% of measured AOF could be explained by targeted PFAS compounds</li><li>The majority of organic fluorine remained unidentified</li></ul>This suggests that environmental fluorine chemistry is considerably more complex than currently represented by routine monitoring programs.<h4>Preventing PFAS Contamination in the Laboratory</h4>PFAS analysis requires exceptionally strict quality control.Dr. Terry's laboratory routinely:<ul><li>Tests every new batch of SPE cartridges</li><li>Tests carbon tubes before use</li><li>Uses DI water blanks</li><li>Verifies consumables labeled as PFAS-free</li><li>Collects field blanks</li><li>Monitors analytical blanks throughout workflows</li></ul>These practices help prevent contamination from laboratory materials and consumables, a common challenge in ultra-trace PFAS analysis.<h4>Current PFAS Treatment Technologies</h4>Three technologies currently dominate PFAS removal in drinking water treatment:<ul><li>Granular Activated Carbon (GAC)</li><li>Ion Exchange Resins</li><li>Reverse Osmosis (RO)</li></ul>Selection depends on:<ul><li>PFAS concentration</li><li>Water quality</li><li>Local operating conditions</li><li>Utility infrastructure</li></ul>Although many emerging technologies are being investigated, these remain the most widely implemented solutions.<h4>Membrane Filtration and Pretreatment</h4>Dr. Terry collaborates with colleagues studying membrane-based PFAS treatment.Her own work focuses on pretreatment through biological filtration.The strategy involves:<ul><li>Removing natural organic matter</li><li>Reducing membrane fouling</li><li>Improving membrane performance</li><li>Extending operational efficiency</li></ul>This integrated approach demonstrates how multiple treatment technologies can work together to improve PFAS removal.<h4>Regional Monitoring Projects</h4>The University of Alabama team participates in EPA-supported monitoring efforts along the Gulf Coast.Their observations indicate:<ul><li>PFAS rarely occur alone</li><li>Multiple PFAS compounds frequently co-occur</li><li>Contamination often originates from identifiable point sources</li></ul>Examples include:<ul><li>Industrial facilities</li><li>Manufacturing operations</li><li>Military installations</li><li>Carpet manufacturing facilities</li></ul>Source identification remains a critical component of environmental monitoring programs.<h4>Future Directions for PFAS Monitoring</h4>Looking ahead, Dr. Terry sees several opportunities for improvement.Desired advances include:<ul><li>In situ monitoring technologies</li><li>Portable detection systems</li><li>Reduced analytical complexity</li><li>Lower instrument costs</li><li>Less expensive consumables</li><li>Faster analysis workflows</li></ul>Current PFAS testing requires substantial expertise, specialized instrumentation, and rigorous quality control, making widespread implementation challenging.<h4>Dr. Terry's Perspective on Long-Term Solutions</h4>When asked what major PFAS challenge she would most like to solve, Dr. Terry offered a broader perspective.Rather than focusing solely on remediation, she emphasized prevention.Her view is that society should:<ul><li>Better evaluate chemical toxicity before commercialization</li><li>Assess risks before large-scale deployment</li><li>Reduce introduction of harmful compounds into the environment</li><li>Support utilities tasked with contaminant removal</li></ul>She notes that water utilities often bear the responsibility of removing contaminants they did not create, highlighting the need for broader societal and regulatory support.<h4>Final Thoughts</h4>Dr. Terry encourages consumers to:<ul><li>Learn where their drinking water originates</li><li>Read annual water quality reports</li><li>Stay informed about local water quality</li><li>Consider appropriate home filtration where needed</li></ul>At the same time, she emphasizes confidence in municipal water systems and the importance of continued investment in water quality research, monitoring, and treatment technologies.<h4>Conclusion</h4>PFAS remain one of the most complex challenges in environmental chemistry and water treatment. Through advanced analytical methods such as LC-MS/MS, combustion ion chromatography, fluorine mass balance approaches, and large-scale monitoring programs, researchers like Dr. Leigh Terry are helping improve our understanding of these persistent contaminants.The interview highlights that solving the PFAS challenge will require a combination of better analytical tools, improved treatment technologies, stronger regulatory frameworks, and a greater emphasis on prevention before contaminants enter the environment.<blockquote>This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.</blockquote><blockquote><h4>Concentrating on Chromatography Podcast</h4>Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!You can find Concentrating on Chromatography Podcast in podcast apps:<ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL">Spotify</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185">Apple podcast</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography">podscan</a></li></ul>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos">YouTube channel</a></blockquote>

PFAS in Water: Why We’re Missing 96% of the Problem

This study evaluates an immobilized enzyme reactor (IMER) containing peptide N-glycosidase from Rudaea cellulosilytica (PNGase Rc) for rapid online deglycosylation of intact glycoproteins. Human glycoproteins and therapeutic antibodies were used as model substrates to assess reactor performance.The PNGase Rc IMER efficiently removed complex sialylated and core-fucosylated glycans, simplifying protein characterization and glycosylation-site analysis. The approach offers a fast and effective solution for bioanalytical workflows, biopharmaceutical quality control, and stability testing of therapeutic proteins.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577">Online Deglycosylation of Monomeric Intact Proteins Using the PNGase Rc Immobilized-Enzyme Reactor</a></h4>Katarína Molnárová, Petr Novák, Jana Nováková, and Petr Pompach*ACS Omega 2026, 11, 17, 25724–25729<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.6c00577">https://doi.org/10.1021/acsomega.6c00577</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.<div>Protein glycosylation, in which monosaccharide units are attached to a protein backbone through enzymatic pathways, is involved in many important biochemical processes and is considered one of the most important post-translational modifications. <a href="javascript:void(0);">(1)</a> It affects not only the stability and secretion of proteins, cell–cell interactions, and receptor–ligand recognition mechanisms but also the biological activity, plasma half-life, immunogenicity, pharmacodynamics, and pharmacokinetics of biotherapeutics. <a href="javascript:void(0);">(2−5)</a> Therapeutical monoclonal antibodies (mAbs) are among the best known pharmaceuticals that are based on glycoproteins. <a href="javascript:void(0);">(6)</a> Over 90% of the mAbs are derived from immunoglobulin G (IgG), in which N-glycosylation occurs at Asn297 of the Fc region and is responsible for modulating immune responses and interactions with other proteins. <a href="javascript:void(0);">(7,8)</a> In cancer treatment, mAbs are used to increase the response of the immune system against the tumor cell, thus inhibiting the cancerous cell growth and helping with the destruction and elimination. <a href="javascript:void(0);">(9)</a> Among the most commonly used mAbs are trastuzumab, bevacizumab, cetuximab, or margetuximab, which are used as an additional treatment for breast, colorectal, prostate, or lung cancer. <a href="javascript:void(0);">(10)</a>Mass spectrometry (MS)-based methods have become a method of choice in the analysis of mAbs that can provide information on sequence, post-translational modifications, and glycosylation profiles. <a href="javascript:void(0);">(11)</a> The enzymatic removal of the attached glycans from the protein backbone can reduce the complexity of the MS analysis, which can lead to a better understanding of the protein structure and modifications. In glycoproteomics, peptide-N-glycosidase F (PNGase F) is most used for glycan removal. It cleaves the linkage between the Asn residue and the N-acetylglucosamine (GlcNAc). <a href="javascript:void(0);">(12)</a> Jmeian et al. developed a monolithic PNGase F enzyme reactor fabricated in fused silica using a glycidyl methacrylate-co-ethylene dimethacrylate polymer and integrated it into an LC–MS workflow. The authors demonstrated the applicability of this system for the characterization of released neutral and acidic N-glycans by LC–MS analysis. <a href="javascript:void(0);">(13)</a> In another study, oriented immobilization of PNGase F onto a methacrylate-based monolithic support via glutathione-S-transferase–glutathione affinity coupling was employed to release N-glycans from denatured ribonuclease B and fetuin, as well as from native IgG. The released glycans and the corresponding deglycosylated proteins were subsequently analyzed by MALDI-MS. <a href="javascript:void(0);">(14)</a>Gramlich et al. showed the potential of PNGase Rc for N-glycan hydrolysis from both tryptic peptides and native glycoproteins at a low pH. The favorable properties of PNGase Rc, including activity at acidic pH and compatibility with reducing agents such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and denaturants like urea, make this enzyme well suited for online intact protein deglycosylation workflows. <a href="javascript:void(0);">(15,16)</a>In this report, we demonstrate the potential of the PNGase Rc IMER for rapid online deglycosylation of reduced monomeric glycoproteins, including human haptoglobin (Hp), human transferrin, anti-hHD6, and the therapeutic monoclonal antibodies trastuzumab and bevacizumab. The column retained its activity over more than 150 consecutive measurements, highlighting its robustness, suitability for repeated use in bioanalytical workflows, and applicability to pharmaceutical research and development.<h4>Experimental Section</h4><h5>Instrumentation and Experimental Conditions</h5>Automated sample handling and online deglycosylation were carried out using the HDX workstation (AffiPro, Czech Republic). The scheme of the system is shown on <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig1">Figure 1</a>. Samples of 50 mM ammonium bicarbonate buffer (pH 8.2) and 40 mM TCEP were placed in a sample tray at 4 °C. Ten microliters of the protein sample at a concentration of 0.03 mg/mL (0.3 μg per injection) were added to 40 μL of 50 mM ammonium bicarbonate and then transferred to 50 μL of 40 mM TCEP, pH 2.3. The disulfide bond reduction was carried at 40 °C for 5 min. The automated sample handling system injected 100 μL of the reaction mixture into the sample loop and switched the injection valve. Online deglycosylation was performed at room temperature using a PNGase Rc IMER (2.1 × 20 mm, AffiPro, Czech Republic) at a flow rate of 50 μL/min, followed by protein desalting on an OPTI-TRAP (0.5 × 2 mm) trap column (Optimize Technologies Inc., Oregon City, Oregon, USA) heated to 55 °C for 10 min with 0.2% formic acid delivered by an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+Infinity+III+isocratic+pump">Agilent 1260 Infinity III isocratic pump</a> (Agilent Technologies, Santa Clara, California, USA). By switching the trap valve, the protein was eluted by an acetonitrile gradient [(min)/% B] 0/10-9/10-16/70-16.5/90-17/90-18/10-23/10] delivered by the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC">Agilent 1290 Infinity II HPLC</a> system (Agilent Technologies, Santa Clara, California, USA) at a flow rate of 20 μL/min. The mobile phase A consisted of 5% acetonitrile with 1% propan-2-ol and 0.1% formic acid, and the mobile phase B consisted of 95% acetonitrile with 1% propan-2-ol and 0.1% formic acid. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=8&amp;search=maXis+Q-TOF">maXis Q-TOF mass spectrometer</a> (Bruker Daltonics, Bremen, Germany) was operated in the survey MS mode in a mass range of m/z 50–3000. The acquired data were analyzed and processed by DataAnalysis 4.4 (Bruker Daltonics) and deconvoluted by UniDec <a href="javascript:void(0);">(17)</a> with the following parameters: data processing range, m/z 500–3000; charge range, 1–80; allowed mass range, 1,000–80,000; sampling rate, 10 Da; peak detection range, 500 Da; and peak detection threshold, 0.1.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_17_25724_25729_Figure_1_Schematic_of_the_automated_sample_handling_system_for_online_protein_deglycosylation_and_desalting_d4ee48b627.jpg" alt="ACS Omega 2026, 11, 17, 25724–25729: Figure 1. Schematic of the automated sample handling system for online protein deglycosylation and desalting. Protein samples, along with 50 mM ammonium bicarbonate and 40 mM TCEP stock solutions were placed in a sample tray chilled to 4 °C, while a second tray heated to 40 °C was used for disulfide bond reduction with TCEP buffer." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_17_25724_25729_Figure_1_Schematic_of_the_automated_sample_handling_system_for_online_protein_deglycosylation_and_desalting_d4ee48b627.jpg 245w," sizes="100vw" width="1287" height="386"><h4>Results and Discussion</h4>The performance of the PNGase Rc IMER for the removal of complex N-glycans from intact proteins was tested using heavily glycosylated human Hp with four glycosylation sites (Asn184, Asn207, Asn211, and Asn241) <a href="javascript:void(0);">(18)</a> located at the β-chain and occupied by complex N-glycans terminated with sialic acid. Haptoglobin phenotype 2-1, used in this study, forms multimers consisting of β-chain and disulfide linked α1 and α2 chains. <a href="javascript:void(0);">(19)</a> The detection of glycosylated Hp β-chain requires reduction of the disulfide bonds by incubating the protein with a reducing reagent. TCEP is a common reducing reagent used for fast reduction of disulfide bonds at various pH levels. Forty degrees Celsius, the maximum temperature of the PAL tray, and 5 min of incubation were sufficient for complete reduction of Hp disulfide bonds. Individual signals corresponding to the glycosylated β-chain were observed in the m/z range of 35,800–37,400 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig2">Figure 2</a>a). The limited resolution of the mass spectrometer did not allow assigning the different glycoforms of the haptoglobin β-chain, which is occupied by biantennary, triantennary, or tetraantennary glycans with various amounts of sialic acid and fucose. <a href="javascript:void(0);">(20,21)</a> The nonglycosylated α1 and α2 chains were observed at m/z 9190 and 15,940. When the PNGase Rc IMER was integrated into the flow system, a signal at m/z 27,270 was detected, demonstrating complete deglycosylation of the haptoglobin β-chain (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig2">Figure 2</a>b).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_17_25724_25729_Figure_2_A_Mass_spectrum_of_human_Hp_without_the_PN_Gase_Rc_IMER_implemented_in_the_system_0ecca84ed4.jpg" alt="ACS Omega 2026, 11, 17, 25724–25729: Figure 2. (A) Mass spectrum of human Hp without the PNGase Rc IMER implemented in the system. The base peak signals of the spectrum correspond to both α1 and α2 chains. The signal of the β chain is spread across its different glycoforms. (B) Mass spectrum of human Hp with the PNGase Rc IMER implemented in the system. The signal of the deglycosylated β chain is higher compared to its glycosylated form and observed only at m/z 27,270. This indicates complete removal of glycans from the Hp β chain." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_17_25724_25729_Figure_2_A_Mass_spectrum_of_human_Hp_without_the_PN_Gase_Rc_IMER_implemented_in_the_system_0ecca84ed4.jpg 245w," sizes="100vw" width="2015" height="913">In a previous study by Krenkova et al., the PNGase F IMER was used for deglycosylation of IgG, RNase B, and bovine fetuin with follow-up mass spectrometry detection of both released glycans and intact proteins using MALDI-TOF MS. <a href="javascript:void(0);">(14)</a> The authors demonstrated an effective method for rapid protein deglycosylation, which, however, requires additional sample handling prior to mass spectrometry measurement. The other study by Jmeian et al., which focused on released N-glycans, implemented a PNGase F IMER with online LC–MS detection. <a href="javascript:void(0);">(13)</a> The authors show the effectiveness of the method by identifying many different glycans released from a single protein or from a complex plasma mixture. Our approach is not directly intended for released N-glycan analysis; instead, it demonstrates the ability to measure intact proteins after rapid online deglycosylation using PNGase Rc at low pH. Samples prepared immediately prior to mass spectrometry analysis using the automated sample-handling platform can reduce variability associated with autosampler residence time, as some proteins may undergo degradation or precipitation during storage in the autosampler.Although all glycoproteins in this study were effectively deglycosylated, the method’s efficiency depends on the characteristics of the individual glycoprotein. Therefore, optimizing method parameters─including the isocratic flow rate, glycoprotein incubation time with TCEP, and/or the addition of denaturing reagent─may improve the overall efficiency of the deglycosylation process.The PNGase Rc IMER stability over a series of injections was tested by using the trastuzumab antibody. The measurements were carried out with the enzyme column kept at the laboratory temperature. As shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig5">Figure 5</a>, the results indicate that the column performance remained stable over the course of the 150 measurements, and no glycosylated form of the trastuzumab antibody heavy chain was detected. The activity of PNGase Rc in the presence of 5% acetonitrile and 5% propan-2-ol was evaluated using a bottom-up approach with human 2–1 haptoglobin, which was online-digested by a coimmobilized nepenthesin-2/pepsin IMER. In the absence of an organic solvent, the peptide IEKVVLHPNYSQVD remained fully deglycosylated throughout 30 injections. In contrast, in the presence of organic solvent, a marked loss of enzymatic activity was observed after only a few injections (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.6c00577/suppl_file/ao6c00577_si_001.pdf">Figure 2</a>).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_17_25724_25729_Figure_5_Monitoring_the_stability_and_performance_of_PN_Gase_Rc_IMER_4a9b5a9c53.jpg" alt="ACS Omega 2026, 11, 17, 25724–25729: Figure 5. Monitoring the stability and performance of PNGase Rc IMER. Trastuzumab heavy chain was completely deglycosylated after 150 injections." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_17_25724_25729_Figure_5_Monitoring_the_stability_and_performance_of_PN_Gase_Rc_IMER_4a9b5a9c53.jpg 170w," sizes="100vw" width="996" height="918"><h4>Conclusions</h4>Here, we demonstrate the potential of the PNGase Rc IMER for the online removal of N-glycans from intact monomeric glycoproteins reduced with TCEP. The column showed high sensitivity toward Hp, which has multiple glycosylation sites with various glycan structures, confirming its suitability for the profiling of complex glycoproteins. Bevacizumab and trastuzumab exhibited similar glycosylation profiles, with core-fucosylated biantennary glycans being the most abundant. Online deglycosylation with the PNGase Rc column resulted in a complete removal of the glycans from the heavy chains of both antibodies, proving that the column can be used for the rapid online deglycosylation of intact therapeutic mAbs. Moreover, the column remained stable over 150 measurements without any loss of activity, even when it was maintained at laboratory temperature. The significantly reduced reaction time, together with full automation and the absence of manual handling steps, substantially improves analytical throughput compared to conventional off-line deglycosylation workflows. These findings highlight the potential of the PNGase Rc IMER in the development of glycoprotein-based biopharmaceuticals, where detailed structural characterization of the protein part is critical for quality control, monitoring batch-to-batch variability, and stability testing.</div>

Online Deglycosylation of Monomeric Intact Proteins Using the PNGase Rc Immobilized-Enzyme Reactor

In the week of June 29, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.<blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinars">👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</a></h4>👉 Search and filter in the largest global database with over 5 500 GC, LC, MS and spectroscopy webinars.</blockquote><h5>1. Thermo Fisher Scientific: Insights from an IC specialist: To be suppressed or not suppressed?</h5><ul><li>Tu, 30.6.2026 09:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6310">Registration</a></li></ul>Join Thermo Fisher’s Ask the Experts session to learn how suppressor technology impacts ion chromatography performance, sensitivity, reliability, maintenance, and troubleshooting.<h5>2. SCIEX: Advancing Proteomics with ZenoTOF: From Single-Cell Analysis to Plasma Biomarker Discovery</h5><ul><li>Tu, 30.6.2026 11:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6311">Registration</a></li></ul>Discover how ZenoTOF high-resolution mass spectrometry is accelerating single-cell proteomics and plasma biomarker discovery through enhanced sensitivity, throughput, and automation.<h5>3. Agilent Technologies: Measuring Diverse Samples with UV/VIS Spectrometers and Integrating Spheres</h5><ul><li>Tu, 30.6.2026 20:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6052">Registration</a></li></ul>This webinar will discuss diffuse transmission and reflection UV/VIS measurements, including how to perform measurements with Agilent’s Cary 60 UV/VIS Spectrometer.<h5>4. Waters Corporation: Informatics series #33 Tips, Tricks and Best Practices</h5><ul><li>Th, 2.7.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6315">Registration</a></li></ul>This interactive session revisits essential features, shortcuts, and practical techniques in Empower software that can streamline everyday laboratory work and reduce unnecessary effort.<h5>5. Waters Corporation: Unlocking Therapeutic Peptide Characterisation (GLP-1RAs) with Cyclic IMS</h5><ul><li>Th, 2.7.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6313">Registration</a></li></ul>Discover how cyclic ion mobility spectrometry (cyclic IMS) improves characterization of GLP-1 analogues and therapeutic peptides, supporting development, quality control, and regulatory submissions.<h5>6. Agilent Technologies: Spectral Flow Cytometry Masterclass - Session 1</h5><ul><li>Fr, 3.7.2026 07:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6316">Registration</a></li></ul>Learn how full-spectrum flow cytometry enables high-dimensional cell analysis. Explore spectral flow technology, experimental design, and applications in immunology, oncology, and vaccine research.

Webinars LabRulezLCMS Week 27/2026

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Webinars

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Everything from the world of analytical chemistry in one place. We connect people in solving their problems. At Labrulez you will find all the necessary information easily, quickly and clearly. Stop searching and start finding.

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Organomation, founded in 1959, designs and manufactures high-quality nitrogen evaporators and extraction systems. Known for innovation and durability, their lab instruments are used globally for efficient sample preparation with strong customer support.

Organomation

Separation Science

Agilent Technologies

Phenomenex

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Waters Corporation

<ul>
<li>Wait what?! Who knew!? There are so many things that a mass spec can do.
</li>
<li>I&#39;ve used it for years, yes this may be true.
</li>
<li>I&#39;ve processed some samples, more than a few.
</li>
<li>But between me and you, I had no idea all the things it could do!
</li>
<li>From sample prep to separation, you just won&#39;t believe…
</li>
<li>All of the tips and tricks we have up our sleeve.
</li>
<li>So, take a few minutes, about 15 should do.
</li>
<li>And learn all the things your mass spec can do.
</li>
</ul>
Thinking of developing a new method utilizing MS? Selecting the best analytical method begins with a decision on how to best separate the analytes of interest. This choice will affect sample preparation and sample introduction, so it is important to get it right early. In this webinar we will review analyte structural properties that make the compound more amenable to either liquid chromatography or to gas chromatography.
<blockquote>
Presenter: Karen Yannell (LC/MS Application Engineer, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Karen E. Yannell is a LC/MS Application Engineer at Agilent Technologies. She is an expert in small molecule high throughput analysis by triple quadrupole (TQ) and quadrupole time-of-flight (QTOF) mass spectrometry for applied markets. Her recent work has focused on developing routine Q-TOF workflows for both expert and non-expert users alike. Previously, Karen worked on forensic, metabolomics, and biomarker discovery applications with ambient ionization sources. She received her PhD in Analytical Chemistry from Purdue University where studied under the advisement of Prof. R. Graham Cooks. Outside of the laboratory, she enjoys cooking unique dishes and hiking along the beach with her dog.
</blockquote>
<blockquote>
Presenter: David Weil, PhD (Senior Applications Scientist, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
David Weil is a senior applications scientist in Agilent located in Wood Dale with over 30 years of experience using mass spectrometry. Prior to joining Agilent in 2004, he worked for 14 years at 3M in the Corporate Research Analytical Laboratories (St.Paul, MN) and 4 years at Nicolet/Extrel doing ultra-high resolution mass spectrometry. David received his Ph.D. in Physical Chemistry from the University of Minnesota and Post Doc with Dr Charles Wilkins at University of California-Riverside developing laser desorption FTMS and ion-molecule reactions. David has three grown sons and live with his wife Naomi just blocks from Wrigley Field and Lake Michigan.
</blockquote>
<blockquote>
Presenter: Matt Curtis, PhD (GC/MS Applications Scientist, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Matt joined Agilent in 2013 as a GC/MS applications scientist in the Santa Clara, CA Center of Excellence. His primary responsibility is focused on providing instrument demos for customer interested in each technology Agilent offers; the GC/MS single quadrupole, triple quadrupole and the quadrupole Time-of-Flight. The variety of different samples submitted to the demo lab provide a unique knowledge of optimizing parameters for different applications to provide the best quality data from the instrument.
</blockquote>

Agilent Technologies: Selecting your Separation: Are my Compounds Better for GC or LC

Selecting your Separation: Are my Compounds Better for GC or LC

We will review analyte structural properties that make the compound more amenable to either liquid chromatography or to gas chromatography.

Separation selection: Are my Compounds Better for GC or LC

Having good peak shape can improve resolution and produce accurate and reliable quantitation results. Problems with peak shape, like fronting, tailing, shoulders, and splits, are all indicative of chromatography issues.
We will discuss factors affecting peak shape, such as the column, sample, mobile phase, and more, and offer remedies for improving peak shape.
<blockquote>
Presenter: Golnar Javadi (Application Engineer, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Golnar Javadi is an applications engineer providing online applications assistance and technical support for LC columns and supplies, Sample Preparation products, and Spectroscopy supplies.
</blockquote>
<blockquote>
Before joining Varian and then Agilent, Golnar worked in the pharmaceutical and nutraceutical industries on method development and quality control. She also taught college level chemistry courses for a few years.
</blockquote>
<blockquote>
Golnar then joined Varian as a technical support specialist, providing customers with method development assistance and technical support on Sample Preparation (SPE, SLE, and Filtration), HPLC, GC, ToxiLab and DAT. After the Varian-Agilent merger, Golnar joined Agilent Technologies as an Applications Engineer. She provides technical support to customers on Sample Preparation products, Liquid Chromatography columns, and Spectroscopy supplies.
</blockquote>

Peak Shape and Why It Matters: How to Get Good Peak Shape

We will discuss factors affecting peak shape, such as the column, sample, mobile phase, and more, and offer remedies for improving peak shape.

Three Webinars and a Roundtable with Experts from Merck KGaA, Darmstadt, Germany, Metrohm &amp; Mettler Toledo
A Week Dedicated to Karl Fischer Titration!
Karl Fischer titration is still the most used, most accurate, and fastest method to determine water content in all kinds of sample matrices. This titration week covers different aspects that need to be considered when determining the water content in products, raw materials, and other substances. Experts from the life science business of Merck KGaA, Darmstadt, Germany, Metrohm AG, and Mettler Toledo will present and discuss different steps of the Karl Fischer titration process and how to improve it. Learnings include:
<ul>
<li>Karl Fischer titration process and tips for how to improve it</li>
<li>Karl Fischer oven method and its advantages</li>
<li>Reference materials to use for titer determination, instrument check, and result verification</li>
<li>Parameters needed to validate a Karl Fischer titration application</li>
<li>News and future aspects of Karl Fischer titration</li>
</ul>
<h3>The Karl Fischer Oven Method – Step by Step</h3>
arl Fischer (KF) titration is a standard method for the water content determination in all types of substances. However, for various reasons, some substances are not suitable for a direct Karl Fischer titration. These reasons include that they undergo side reaction with KF reagents; they contaminate the titration cell and/or the electrodes; the substances are poorly soluble in alcohol; they release their water only slowly; or the water is released at high temperatures only. These problems can be avoided by using the oven method. This makes the KF oven method the perfect solution for the analysis of difficult samples. By attending this presentation, you will learn:
<ul>
<li>The principle of the oven method</li>
<li>How to properly prepare your titration system with an oven</li>
<li>How to prepare your sample</li>
<li>How to select the optimal oven temperature</li>
</ul>
After viewing this webinar, you will know how to perform accurate sample measurements using the oven method.
<blockquote>
Presenter: Michael Margreth (Application Specialist - Karl Fischer Titration and Water Determination, Metrohm)
</blockquote>

The Karl Fischer Oven Method – Step by Step

After viewing this webinar, you will know how to perform accurate sample measurements using the oven method.

Purification is critical to many scientific workflows. Compounds of interest are rarely found to be the right purity in their native state. Join Waters and Virscidian for this 6-part webinar series to learn the basics of purification and application of purification principles for more advanced workflows.
<h3>PART 2 | Basics of Supercritical Fluid Chromatography Purification</h3>
Supercritical Fluid Chromatography (SFC) is an effective and green technique to isolate a compound(s) of interest from a mixture. SFC utilizes liquid CO2 to quickly isolate compounds while minimizing the efforts required to dry the collected fraction. In this module, we will discuss factors involved in SFC purification. 
Learning Objectives: 
<ul>
<li>Understand how to use SFC purification</li>
<li>Make informed decisions on method optimization</li>
<li>Understand impact of decisions made during purification on larger workflow</li>
</ul>
<blockquote>
Presenter: Andrew Leightner (Market Development Manager – Americas Field Marketing, Waters Corporation)
</blockquote>
<blockquote>
Andy Leightner has over 10 years of experience serving the pharma market from contract research organizations. He specialized in method development and validation leveraging LC, GC, and mass spectrometry to meet complex client needs. He supports pharmaceutical and biopharmaceutical discovery and development in the Americas for Waters Corporation. Andy received a bachelors degree in biology from the University of Wisconsin-Madison.
</blockquote>

Waters Corporation: Basics of Supercritical Fluid Chromatography Purification

Basics of Supercritical Fluid Chromatography Purification

We will discuss factors involved in SFC purification.

<h5>Key learning objectives:</h5>
<ul>
<li>What is chiral chromatography, chiral screening, why do it, what are the benefits...</li>
<li>What the chiral screening process looks like</li>
<li>Scope, deliverables, and timelines for chiral screening</li>
<li>How to request chiral screening services</li>
</ul>
<blockquote>
Presenter: Zandra Baja (Applications Scientist)
</blockquote>
<blockquote>
Zandra is an Applications Scientist at PhenoLogix specializing in chiral screening, USP and PhEur applications, and environmental GC-MS applications. Zandra obtained her bachelor&#39;s degree in chemistry from California State University of Long Beach. Prior to joining Phenomenex in 2021, she spent 3 years working as a chemist in the petroleum industry and 8 years as an analytical chemist working with gas chromatography in an environmental air testing laboratory. Zandra has also spent several years in quality assurance management where she was responsible for maintaining ISO/IEC 17025 and TNI Standard laboratory accreditations. 
</blockquote>

Phenomenex: Chiral Chromatography Screening 101

Chiral Chromatography Screening 101

What is chiral chromatography, chiral screening, why do it, what are the benefits.... What the chiral screening process looks like....

How about a 20 minute coffee break along with a bite of Empower? Here you go!
What you can learn: 
<ul>
<li>What&#39;s the difference between Archive and Backup and why do you need both</li>
<li>The cheapest, the fastest, or the most definitive data management strategy is meaningless if the data is untrustworthy</li>
<li>How including a complementary archive strategy to your existing backup will not only improve efficiency, but can reduce costs and regulatory risk</li>
<li>How both approaches are viable and the business value each one can offer</li>
</ul>
Register and prepare a coffee!
<blockquote>
Presenter: Ralf Schröder (Informatics Product Marketing Europe·Waters GesmbH)
</blockquote>

Waters Corporation: Waters Informatics series #5: Archive vs Backup (Po2)

Waters Informatics series #5: Archive vs Backup (Po2)

What's the difference between Archive and Backup and why do you need both?

The calculation of signal to noise ratio is a critical parameter during method development, method validation and routine monitoring the performance of a chromatographic method. Both Limit of Detection (LOD or LDL) and Limit of Quantitation (LOQ or LQL) are based on signal to noise ratio. Empower can calculate signal to noise ratio in many ways. While we designed this functionality in Empower to comply with pharmacopeia requirements, there are alternative methods for this calculation.
Key points of discussion will be:
<ul>
<li>Calculating Signal to Noise ratio as per USP/EP/JP Requirements</li>
<li>Calculating Signal to Noise ratio using other options within the System Suitability option</li>
<li>Calculating Signal to Noise ratio without the System Suitability Option</li>
</ul>
Please join us for a live discussion. As always, don’t forget to bring your Empower questions for this exclusive opportunity to ask our experts anything about Empower Software.
<blockquote>
Presenter: Neil Lander (Principal Product Manager, Waters Corporation)
</blockquote>
<blockquote>
Neil has worked for Waters Corporation since 1994 and has held a variety of positions including Technical Support Specialist, Technical Trainer, Manager Customer Education and currently, Principal Marketing Manager focusing on Informatics solutions. He provides internal support for Empower Chromatography Data System and is focused on developing laboratory software solutions that help organizations achieve their scientific and operational objectives. Prior to joining Waters, he was an analytical chemist where he gained experience in both the cosmetics and specialty chemical industries.
</blockquote>

Waters Corporation: Empower Tips: Ask Me Anything About Calculating Signal to Noise Ratio

Empower Tips: Ask Me Anything About Calculating Signal to Noise Ratio

The calculation of signal to noise ratio is a critical parameter during method development, validation and monitoring the performance.

Empower Tips: Calculating Signal to Noise Ratio

While making a UV-Vis measurement is simple, making an accurate measurement requires an understanding of instrument parameters and their effects on the measurement. Parameters include Baseline correction, spectral bandwidth, data interval, and many additional settings.
This presentation will provide an insight into these parameters and more.
<blockquote>
Presenter: Mark Fisher, PhD (Application Engineer, Molecular Spectroscopy, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Mark, an Application Scientist for Agilent Technologies, has over 35 years of experience in molecular spectroscopy. He has held roles in Agilent&#39;s UV-Vis-NIR, fluorescence, and atomic absorption product portfolios. In addition to identifying industry trends and new business needs, Mark&#39;s responsibilities include application development, pre and post-sales support, as well as acting as a customer advocate for new product and technology opportunities. Mark received his doctorate in analytical chemistry from the University of Nebraska-Lincoln.
</blockquote>
<blockquote>
Presenter: Scott Melis, PhD (Application Scientist, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Scott Melis has a PHD in Physics from Georgetown University in Washington, DC and started with Agilent in 2021 as a Molecular Spectroscopy Application Scientist. In his graduate work, Scott studied nanoparticle formation and growth for a variety of applications. Projects that he worked with include developing processes to deposit and characterize semiconducting molecular nano-cocrystals for use in optoelectronic devices as well as controlling polymer nanoparticle size through rapid mixing conditions.
</blockquote>

Agilent Technologies: Understanding UV-Vis Instrumental Parameters

Understanding UV-Vis Instrumental Parameters

This presentation will provide an insight into baseline correction, spectral bandwidth, data interval, and many additional settings

In this session, explore the operation and latest developments of automated SPME for VOCs and SVOCs by headspace, direct immersion, and derivatization techniques.
Furthermore, discover how SPME has key applications for food, water, environmental and forensic samples for use in laboratories for GC-MS and LC-MS and direct MS analysis.
<blockquote>
Presenter: Hans-Joachim Huebschmann (Consultant, VP Sample Preparation, CTC Analytics)
</blockquote>

SelectScience: Solid phase microextraction – How does it work for my samples?

Solid phase microextraction – How does it work for my samples?

Explore the operation and latest developments of automated SPME for VOCs and SVOCs.

Solid phase microextraction–How does it work for my samples?

Key Learning Points:
<ul>
<li>Understand how to obtain good resolution by utilizing selectivity
</li>
<li>Learn the basics of different stationary phase particle characteristics 
</li>
<li>Discover how to choose different stationary phases based on compound classes
</li>
<li>Find how mobile phase can be used to change selectivity of columns
</li>
</ul>
Who Should Attend:
<ul>
<li>Any LC users who are looking for are looking for a basic overview of Reverse Phase Method Development by looking at different small molecule compound classes</li>
</ul>
<blockquote>
Presenter: Genevieve Hodson (Senior Application Scientist, Phenomenex)
</blockquote>
<blockquote>
Genevieve graduated from the University of Texas with a BS in Chemistry located in her home town of Austin Texas. She spent 2 years of her undergraduate performing research in an Organic Chemistry Lab synthesizing intermediates. Out of school she spent 4 years as an Analytical Chemist in the QC and R&amp;D Department of Cerilliant (a Sigma Millipore Company). There she worked with many analytical instruments but became an expert at LC by performing purity analysis, customer specific methods, validations, qc testing and stability studies on a wide range of small molecules including pharmaceuticals, pesticides, explosives and illegal substances. From there she moved to Los Angeles California where she worked at Johnson and Johnson and then a small dietary supplement company. After Genevieve spent a year living abroad in Israel raising her first son, she moved back to LA where she got hired as a Technical Specialist at Phenomenex. At Phenomenex she enjoys continuing to learn about chromatography every day through helping customer with all their chromatography needs!
</blockquote>

Phenomenex: Understanding Reverse Phase Selectivity for Different Compound Classes

LCMS webinars focusing on Academy/Principles - page 21

Understanding Reverse Phase Selectivity for Different Compound Classes