<p><strong>🎤Dr. Leigh Terry (University of Alabama)</strong></p><p><strong>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.</strong></p><p>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.</p><h4><strong>In this episode, we cover:</strong></h4><ul><li>Why PFAS are called “forever chemicals” (and why that label is evolving)</li><li>The limitations of targeted<strong> LC-MS/MS methods</strong></li><li>What the fluorine mass balance problem reveals about PFAS destruction</li><li>How combustion ion chromatography (<strong>CIC</strong>) 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><strong>Key Insight:</strong></h4><p>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.</p><h3><strong>Video Transcription</strong></h3><p>In this episode of <i>Concentrating on Chromatography</i>, 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.</p><h4><strong>Understanding PFAS and Their Significance</strong></h4><h5><strong>The University of Alabama Water Quality Research Center</strong></h5><p>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.</p><p>The laboratory specializes in:</p><ul><li>Drinking water analysis</li><li>Organic contaminants</li><li>Emerging contaminants</li><li>PFAS monitoring</li><li>Water quality research</li></ul><p>Their work supports both research initiatives and practical solutions for water utilities and environmental agencies.</p><h4><strong>Why Focus on PFAS?</strong></h4><p>Although PFAS compounds have existed since the 1940s, public awareness has increased dramatically only in recent years.</p><p>According to Dr. Terry, several factors contributed to the rapid growth of PFAS research:</p><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><p>PFAS are not new contaminants. Rather, society is only beginning to understand their prevalence in the environment and their potential impacts on human health.</p><h4><strong>The Development of Standardized PFAS Methods</strong></h4><p>One major advancement in PFAS analysis has been the development of standardized EPA methods.</p><p>These methods allow:</p><ul><li>Consistent testing procedures</li><li>Comparable results between laboratories</li><li>Regulatory monitoring</li><li>Reliable data interpretation</li></ul><p>Dr. Terry notes that many of these standardized approaches began appearing around 2018–2019, providing a common framework for analytical laboratories and researchers.</p><h4><strong>How Large Is the PFAS Universe?</strong></h4><p>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.</p><p>PFOA and PFOS are often referred to as:</p><ul><li>Legacy PFAS compounds</li><li>Original PFAS contaminants</li><li>Early commercial PFAS chemicals</li></ul><p>The challenge is that even minor modifications to molecular structure create entirely new PFAS compounds.</p><p>Researchers estimate:</p><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><p>Scientific debate continues regarding classification and definition, making precise counting difficult.</p><h4><strong>Why PFAS Are Called "Forever Chemicals"</strong></h4><p>Dr. Terry notes that the phrase "forever chemicals" has become widely used, although she suggests the terminology could benefit from rebranding.</p><p>PFAS persistence results from two key chemical properties.</p><h5><strong>1. Amphiphilic Structure</strong></h5><p>PFAS molecules contain:</p><ul><li>A water-loving (hydrophilic) head group</li><li>A water-repelling (hydrophobic) fluorinated tail</li></ul><p>This unusual combination allows PFAS to partition between:</p><ul><li>Water</li><li>Solids</li><li>Air-water interfaces</li><li>Foam</li></ul><p>Unlike many organic compounds, PFAS can interact effectively with multiple environmental phases.</p><h5><strong>2. Extremely Strong Carbon–Fluorine Bonds</strong></h5><p>The carbon–fluorine bond is among the strongest bonds in organic chemistry.</p><p>As Dr. Terry explains:</p><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><p>These properties make PFAS extremely persistent in both environmental systems and biological organisms.</p><h4><strong>Toxicity, Regulations, and Environmental Behavior</strong></h4><h5><strong>Understanding PFAS Toxicity</strong></h5><p>One of the biggest challenges in PFAS regulation is the incomplete understanding of toxicity.</p><p>Dr. Terry explains that EPA regulations generally consider three factors:</p><ul><li>Presence in drinking water sources</li><li>Toxicity to humans</li><li>Availability of practical treatment options</li></ul><p>While some PFAS compounds are known to be harmful, many others remain insufficiently studied. Furthermore, PFAS often occur as mixtures rather than individual compounds.</p><p>This creates important scientific questions:</p><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><p>These uncertainties continue to drive active research.</p><h4><strong>What Happens to PFAS in Wastewater Treatment Plants?</strong></h4><p>PFAS do not behave like many conventional contaminants.</p><p>Rather than remaining fully dissolved in water, they often accumulate at interfaces such as:</p><ul><li>Water-solid interfaces</li><li>Water-air interfaces</li><li>Foam layers</li><li>Biological solids</li></ul><p>As a result:</p><ul><li>Some PFAS accumulate in biosolids</li><li>Some partition into foam</li><li>Some remain in treated effluent</li></ul><p>Their distribution depends on wastewater composition and treatment processes.</p><h4><strong>Regulatory Monitoring and Targeted LC-MS Analysis</strong></h4><p>Current EPA regulations focus on targeted analysis of specific PFAS compounds.</p><p>Examples include:</p><ul><li>PFOA</li><li>PFOS</li></ul><p>Drinking water regulations currently establish maximum contaminant levels (MCLs) for these compounds at extremely low concentrations.</p><p>Advantages of targeted LC-MS analysis include:</p><ul><li>Clearly defined analytes</li><li>Established regulatory limits</li><li>High analytical sensitivity</li><li>Reliable quantitation</li></ul><p>However, limitations remain:</p><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><p>As Dr. Terry notes, targeted analysis provides only part of the overall picture.</p><h4><strong>The Fluorine Mass Balance Problem</strong></h4><p>One of the most important concepts discussed in the interview is fluorine mass balance.</p><p>When PFAS degrade, fluorinated fragments may remain in the environment.</p><p>This creates a critical question:</p><p>Are treatment technologies truly destroying PFAS, or merely transforming them into shorter-chain PFAS compounds?</p><p>To answer this, researchers perform fluorine mass balance calculations using:</p><ul><li>Targeted PFAS analysis</li><li>Fluoride measurements</li><li>Total organic fluorine measurements</li></ul><p>A successful destruction process should ultimately convert PFAS into fluoride ions rather than simply producing smaller PFAS molecules.</p><h4><strong>Advanced Analysis, Treatment Technologies, and Future Directions</strong></h4><h5><strong>Combining Targeted and Bulk Fluorine Measurements</strong></h5><p>Dr. Terry's group complements targeted LC-MS analysis with measurements of total organic fluorine.</p><p>Approaches include:</p><ul><li>Absorbable Organic Fluorine (AOF)</li><li>Extractable Organic Fluorine (EOF)</li><li>Combustion Ion Chromatography (CIC)</li></ul><p>In these methods:</p><ul><li>Organic fluorine is captured</li><li>Samples are combusted</li><li>Released fluoride is quantified</li></ul><p>This provides insight into total fluorinated content, although contributions may originate from sources beyond PFAS, including pharmaceuticals and consumer products.</p><h4><strong>Surprising Findings from Combined Analysis</strong></h4><p>One important finding from wastewater studies was that targeted PFAS measurements accounted for only a small fraction of total organic fluorine.</p><p>Dr. Terry reports that:</p><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><p>This suggests that environmental fluorine chemistry is considerably more complex than currently represented by routine monitoring programs.</p><h4><strong>Preventing PFAS Contamination in the Laboratory</strong></h4><p>PFAS analysis requires exceptionally strict quality control.</p><p>Dr. Terry's laboratory routinely:</p><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><p>These practices help prevent contamination from laboratory materials and consumables, a common challenge in ultra-trace PFAS analysis.</p><h4><strong>Current PFAS Treatment Technologies</strong></h4><p>Three technologies currently dominate PFAS removal in drinking water treatment:</p><ul><li>Granular Activated Carbon (GAC)</li><li>Ion Exchange Resins</li><li>Reverse Osmosis (RO)</li></ul><p>Selection depends on:</p><ul><li>PFAS concentration</li><li>Water quality</li><li>Local operating conditions</li><li>Utility infrastructure</li></ul><p>Although many emerging technologies are being investigated, these remain the most widely implemented solutions.</p><h4><strong>Membrane Filtration and Pretreatment</strong></h4><p>Dr. Terry collaborates with colleagues studying membrane-based PFAS treatment.</p><p>Her own work focuses on pretreatment through biological filtration.</p><p>The strategy involves:</p><ul><li>Removing natural organic matter</li><li>Reducing membrane fouling</li><li>Improving membrane performance</li><li>Extending operational efficiency</li></ul><p>This integrated approach demonstrates how multiple treatment technologies can work together to improve PFAS removal.</p><h4><strong>Regional Monitoring Projects</strong></h4><p>The University of Alabama team participates in EPA-supported monitoring efforts along the Gulf Coast.</p><p>Their observations indicate:</p><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><p>Examples include:</p><ul><li>Industrial facilities</li><li>Manufacturing operations</li><li>Military installations</li><li>Carpet manufacturing facilities</li></ul><p>Source identification remains a critical component of environmental monitoring programs.</p><h4><strong>Future Directions for PFAS Monitoring</strong></h4><p>Looking ahead, Dr. Terry sees several opportunities for improvement.</p><p>Desired advances include:</p><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><p>Current PFAS testing requires substantial expertise, specialized instrumentation, and rigorous quality control, making widespread implementation challenging.</p><h4><strong>Dr. Terry's Perspective on Long-Term Solutions</strong></h4><p>When asked what major PFAS challenge she would most like to solve, Dr. Terry offered a broader perspective.</p><p>Rather than focusing solely on remediation, she emphasized prevention.</p><p>Her view is that society should:</p><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><p>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.</p><h4><strong>Final Thoughts</strong></h4><p>Dr. Terry encourages consumers to:</p><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><p>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.</p><h4><strong>Conclusion</strong></h4><p>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.</p><p>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.</p><blockquote><p><strong>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.</strong></p></blockquote><blockquote><h4><strong>Concentrating on Chromatography Podcast</strong></h4><p>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!</p><p>You can find <strong>Concentrating on Chromatography Podcast </strong>in podcast apps:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL"><strong>Spotify</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185"><strong>Apple podcast</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography"><strong>podscan</strong></a></li></ul><p>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos"><strong>YouTube channel</strong></a></p></blockquote>

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

<p><strong>This study evaluates an immobilized enzyme reactor (IMER) containing peptide N-glycosidase from </strong><i><strong>Rudaea cellulosilytica</strong></i><strong> (PNGase Rc) for rapid online deglycosylation of intact glycoproteins. Human glycoproteins and therapeutic antibodies were used as model substrates to assess reactor performance.</strong></p><p>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.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577"><strong>Online Deglycosylation of Monomeric Intact Proteins Using the PNGase Rc Immobilized-Enzyme Reactor</strong></a></h4><p>Katarína Molnárová, Petr Novák, Jana Nováková, and Petr Pompach*</p><p>ACS Omega 2026, 11, 17, 25724–25729</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.6c00577">https://doi.org/10.1021/acsomega.6c00577</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>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 <i>N</i>-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></p><p>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-<i>N</i>-glycosidase F (PNGase F) is most used for glycan removal. It cleaves the linkage between the Asn residue and the <i>N</i>-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-<i>co</i>-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 <i>N</i>-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-<i>S</i>-transferase–glutathione affinity coupling was employed to release <i>N</i>-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></p><p>Gramlich et al. showed the potential of PNGase Rc for <i>N</i>-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 <i>tris</i>(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></p><p>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, <i>anti</i>-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.</p><h4><strong>Experimental Section</strong></h4><h5><strong>Instrumentation and Experimental Conditions</strong></h5><p>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"><strong>Agilent 1260 Infinity III isocratic pump</strong></a><strong> </strong>(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"><strong>Agilent 1290 Infinity II HPLC</strong></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"><strong>maXis Q-TOF mass spectrometer</strong></a> (Bruker Daltonics, Bremen, Germany) was operated in the survey MS mode in a mass range of <i>m</i>/z 50–3000. The acquired data were analyzed and processed by <strong>DataAnalysis 4.4</strong> (Bruker Daltonics) and deconvoluted by UniDec <a href="javascript:void(0);">(17)</a> with the following parameters: data processing range, <i>m</i>/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.</p><p><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"></p><h4><strong>Results and Discussion</strong></h4><p>The performance of the PNGase Rc IMER for the removal of complex <i>N</i>-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 <i>N</i>-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 <i>m</i>/<i>z</i> 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 <i>m</i>/<i>z</i> 9190 and 15,940. When the PNGase Rc IMER was integrated into the flow system, a signal at <i>m</i>/<i>z</i> 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).</p><p><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"></p><p>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 <i>N</i>-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 <i>N</i>-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.</p><p>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.</p><p>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>).</p><p><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"></p><h4><strong>Conclusions</strong></h4><p>Here, we demonstrate the potential of the PNGase Rc IMER for the online removal of <i>N</i>-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.</p></div>

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

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

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Waters ACQUITY Advanced Polymer Chromatography (APC) System

The ACQUITY APC System Increase sample throughput and laboratory operations by obtaining accurate and repeatable sample separation up to 20 time faster than before.

<div><div><h4><strong>The highest performance for polymers and oligomers</strong></h4></div></div><div><div><p><strong>Today’s polymer scientists operate within an increasingly dynamic market space, one with a growing demand for high-performing materials, bio-material innovations, and intense competition. But the long run times of traditional chromatography polymer characterization has made keeping up with this demand a challenge for many labs.</strong></p><p>Not anymore. With the <strong>next era in polymer separation</strong>, the <strong>ACQUITY Advanced Polymer Chromatography (APC) System</strong>, you can achieve the <strong>highest resolution size-based separations</strong> for <strong>chromatographic polymer and oligomer characterization</strong>, all <strong>faster than ever</strong>. With the <strong>ACQUITY APC System</strong>, you can <strong>improve data consistency and quality</strong> with <strong>fast, daily calibration</strong>, and <strong>automate your method development</strong> with <strong>advanced system technologies</strong> – so you can <strong>innovate faster</strong> while <strong>improving lab operations.</strong></p><div><div><div><div><h3><strong>Features and Benefits</strong></h3><ul><li><strong>Accelerate your insights</strong> - Reduce laboratory turnaround times for polymer samples by getting answers to your development labs, your manufacturing operations teams, and your customers 5 to 20 times faster than traditional SEC/GPC with the ultra-low system dispersion of the ACQUITY APC System paired with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1950"><strong>ACQUITY APC Columns</strong></a>.</li><li><strong>Get the accurate data you need for informed decisions</strong> - Gain more insight into your polymer samples through compatibility with a wide range of specific detection techniques (refractive index, UV/Vis, PDA, ELS) and third-party advanced detectors like multi-angle light scattering and viscometry. Get accurate molecular weight (MW) data every day with the precise flow delivery of the isocratic solvent manager.</li><li><strong>Optimize method development, even in harsh conditions</strong> - Optimize method development via rapid and automated solvent switching and aggressive solvent compatibility, which supports the harsh separation conditions associated with polymer analyses.</li><li><strong>Improve lab efficiency with one system</strong> - Increase analytical scope and laboratory efficiency by leveraging one system to perform multiple applications, including basic liquid chromatography (LC), gradient, isocratic, reverse-phase, and gel permeation chromatography (GPC).</li><li><strong>Simplify your column management</strong> - Simplify and optimize column bank calibration through standards that are matched to column bank MW ranges. Versatile column management allows you to automatically select up to two banks of <strong>ACQUITY APC</strong> columns and up to two banks of traditional GPC columns - all housed in a stable, temperature-controlled environment.</li></ul></div></div><div><div><h4><strong>Recommended Use</strong></h4><p>Highest-resolution, size-based separations for chromatographic polymer and oligomer characterization.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_ACQUITY_APC_System_nakonfigurovany_pro_automaticke_prepinani_jak_APC_tak_GPC_kolon_e198c774f4/Waters-ACQUITY-APC-System-nakonfigurovany-pro-automaticke-prepinani-jak-APC-tak-GPC-kolon.jpg" alt="Waters ACQUITY Advanced Polymer Chromatography (APC) System." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_ACQUITY_APC_System_nakonfigurovany_pro_automaticke_prepinani_jak_APC_tak_GPC_kolon_e198c774f4/thumbnail_Waters-ACQUITY-APC-System-nakonfigurovany-pro-automaticke-prepinani-jak-APC-tak-GPC-kolon.jpg 213w," sizes="100vw" width="969" height="710"></p><div><div><h3><strong>Accelerate innovation with enhanced insight</strong></h3><p>Harnessing the benefits of advanced polymer chromatography with the ACQUITY APC System means obtaining accurate and repeatable polymer and oligomer molecular weight information anywhere between<strong> 5 to 20 times faster than was possible</strong> with traditional<strong> GPC/SEC technology</strong> - so you can innovate faster while improving laboratory operations.</p><p><strong>With the ACQUITY APC System, you can:</strong></p><ul><li><strong>Reduce laboratory turnaround times for polymer samples&nbsp;</strong>by getting answers to your development labs, your manufacturing operations teams, and your customers - faster.</li><li><strong>Drive innovation</strong>&nbsp;with faster results and more information so that your entire operation can respond more rapidly by decreasing development cycle times and accelerating time to market.</li><li><strong>Streamline process monitoring</strong>&nbsp;and achieve better batch control with the agility to make timely decisions “on-the-fly” about process and synthesis optimization<strong>.</strong></li><li><strong>Significantly reduce the analysis cost per sample</strong>&nbsp;by reducing solvent consumption and waste disposal volumes.</li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_ca5154865a.jpg" alt="Waters ACQUITY Advanced Polymer Chromatography (APC) System: Accelerate your innovation with enhanced insight into polymer chemical structure with the ACQUITY APC System." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_ca5154865a.jpg 245w," sizes="100vw" width="1024" height="600"></p><div><div><h3><strong>Optimize method development with solvent switching and compatibility</strong></h3><p>Using the ACQUITY APC System with <strong>Polymer Quaternary Solvent Manager</strong> (p-QSM) <strong>technology </strong>provides chemists and polymer scientists with the ultimate flexibility to analyze the most complex polymer blends and additives using standard polymer chromatography, gradient polymer elution chromatography (GPEC), and reverse-phase LC with a single system.</p><p>Additional system features allow automated solvent selection of up to six different solvents. Automated column bank switching capabilities can also be paired with the rigid and solvent-resilient particles of the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1950"><strong>ACQUITY APC Columns</strong></a> to achieve the world’s first truly automated solution for method development of size-based polymer separations. This supports separation of polymers in hours, instead of days.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_2_b95f96f0a2.jpg" alt="Waters ACQUITY Advanced Polymer Chromatography (APC) System: Repeatable analysis of poly (methyl methacrylate co ethylacrylate) using the same two running ACQUITY APC 4.6 x 150 mm Columns (450 Å and 125 Å) in series after automated switching of the solvents from THF to toluene to DMF and back to THF." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_2_b95f96f0a2.jpg 245w," sizes="100vw" width="1024" height="609"></p></div></div><div><div><h3><strong>Use advanced detection for even more insights</strong></h3><p>Get the valuable decision-making information you need from a single analysis by equipping the ACQUITY APC System with advanced detectors. Combining the ACQUITY APC System with advanced detection solutions significantly increases information attainable from <strong>size-exclusion chromatography</strong> (SEC) analysis through addition of <strong>refractive index</strong> (RI), <strong>ultraviolet </strong>(UV),<strong> photodiode array </strong>(PDA), <strong>light scattering</strong> (LS), and <strong>differential viscometry</strong> (IV). Advanced detection options from <strong>Wyatt Technology</strong> are seamlessly integrated with ACQUITY APC System, enabling you to fully characterize samples to better inform the structure-property relationship of novel complex polymers.</p></div></div><div><div><h3><strong>Keep pace with your research with dedicated consumables</strong></h3><p>The ACQUITY APC System is even more powerful with <a target="_blank" rel="noopener noreferrer" href="https://www.waters.com/nextgen/global/products/columns/acquity-apc-columns.html"><strong>ACQUITY APC Columns</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://www.waters.com/nextgen/global/products/standards-and-reagents/acquity-advanced-polymer-chromatography-calibration-standards-ki.html"><strong>ACQUITY APC Calibration Standards Kits</strong></a>. The Ethylene Bridged Hybrid (BEH) Technology in ACQUITY APC Columns creates particles that ensure extreme column performance and long column lifetime under harsh operating conditions – ideal for general purpose, reversed-phase chromatography, and for method development due to extreme pH stability and applicability to the broadest range of compound classes.</p><p>With run times of less than 10 minutes, a bank of ACQUITY APC Columns can be <strong>calibrated in less than 30 minutes</strong> using ACQUITY APC Calibration Standards Kits.&nbsp;These standard kits have been matched to the MW range of ACQUITY APC Columns, generating a 10-point calibration for any bank combination in a simple dilute-and-shoot format. With daily column bank calibration, you can greatly improve data consistency to increase confidence in batch-to-batch measurements. Select the best column and calibration standards for a given application with our convenient <a target="_blank" rel="noopener noreferrer" href="https://www.waters.com/waters/promotionDetail.htm?id=134823417">selector tool</a>.&nbsp;</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_4_056bb6d284.jpg" alt="Waters ACQUITY Advanced Polymer Chromatography (APC) System: Ethylene Bridged Hybrid (BEH) Technology helps you keep up with the pace of your research with state-of-the-art reversed-phase and HILIC HPLC columns." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_4_056bb6d284.jpg 245w," sizes="100vw" width="1024" height="620"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_5_7a814e249b.jpg" alt="Waters ACQUITY Advanced Polymer Chromatography (APC) System: Improve data and consistency with ACQUITY APC Calibration Standards." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_ACQUITY_Advanced_Polymer_Chromatography_APC_System_5_7a814e249b.jpg 245w," sizes="100vw" width="1024" height="591"></p></div></div></div></div></div></div></div></div></div></div>

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