17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology - Book of Abstracts

Significance of the topic

Hyphenated separation techniques (combinations of chromatographic or electrophoretic separations with orthogonal detection and additional separation dimensions) remain central to modern analytical chemistry. They deliver the selectivity, peak capacity and structural information required across regulated and research domains: pharmaceutical and biopharmaceutical quality control, metabolomics and clinical biomarker discovery (including COVID and breathomics), environmental contaminant monitoring, food safety (MOSH/MOAH, flavors, off‑odours), and polymer/material characterization. HTC‑17 documented how advances in instrumentation, sample handling, multidimensional separations and data science together push limits on sensitivity, throughput, structural assignment and applicability to increasingly complex or volume‑limited matrices.

Goals and overview of the symposium

• Present state‑of‑the‑art developments in hyphenated techniques (GC, LC, SFC, CE) and their coupling to MS, FT‑IR, ion mobility and imaging detectors.
• Showcase methodological advances for small molecules, complex natural mixtures, macromolecules (proteins, oligonucleotides, polymers) and trace environmental analytes.
• Highlight practical innovations: sample preparation (MIP/IIP, DBS automation, TD, SPME), miniaturization (nanoLC, microfluidics, 3D‑printed sorbents), and high‑throughput GC approaches.
• Promote discussion on modeling, in‑silico method development, AI/ML for retention/time prediction and data mining for non‑target and breathomics studies.

Methodology and scientific program highlights

• Short courses: fundamentals beyond RPLC (HILIC, HIC, IEX, SEC, MM, PGC, SFC) and CE‑MS for metabolomics.
• Plenaries and keynotes focused on: breathomics and GC×GC‑TOFMS for medical applications; HILIC fundamentals and practicalities; hyphenation for large molecules; ultrafast and thermal‑gradient GC; native and hyphenated protein analyses (SEC‑MS, AF4, TIMS); and SFC‑MS interfacing.
• Parallel sessions covered multidimensional LC (online LC×LC, heart‑cutting, Total Breakthrough phenomenon), GC×GC for flavors/fragrances and VOCs, hyphenated protein characterization, sample preparation automation, environmental and food contaminant analysis, and method modelling/retention prediction.

Used instrumentation

• Comprehensive multidimensional platforms: GC×GC‑TOFMS, LC×LC‑HRMS (HILIC×RP, RP×HILIC), LC‑GC×GC‑ToFMS/FID.
• Mass spectrometry and variants: high‑resolution Orbitrap/TOF, triple quadrupole MS, APCI and APPI sources, MALDI‑IMS, native MS and TIMS (trapped ion mobility), ion‑mobility hyphenation.
• Separation hardware: UHPLC, nanoLC, capillary electrophoresis (CE) and CE‑MS interfaces, asymmetrical flow field‑flow fractionation (AF4), size‑exclusion chromatography (SEC), supercritical fluid chromatography (SFC) with make‑up solvent interfaces, micropillar array columns, superficially porous particles and novel stationary phases (polysaccharide chiral phases, porous graphitic carbon, temperature‑responsive phases).
• Sampling and sample prep: automated dried blood spot (DBS) autosamplers, thermal desorption (TD), solid phase microextraction (SPME), microwave and solvent extractions, molecularly‑ and ion‑imprinted polymers (MIP/IIP), 3D‑printed sorbents and cartridges, multicolumn countercurrent solvent gradient purification (MCSGP) for peptide purification.
• Emergent instruments and concepts: HyperChrom® hyperfast GC, Flow‑Field Thermal Gradient GC, low‑pressure (vacuum) GC, LCW (liquid core waveguide) photodegradation cell for online reaction modulation.

Main results and discussion

• Hyphenation advances for large molecules: comprehensive hyphenated designs (LC×LC, SEC‑MS, AF4–MS, native LC‑MS, TIMS) improved proteoform and aggregate profiling; affinity CE‑MS enabled proteoform‑resolved receptor binding assessments (e.g., Fc‑receptor interactions), providing functional proteoform insight rather than ensemble averages.
• HILIC progress: new practical guidance on retention mechanisms, equilibration strategies, injection solvent effects and improved retention factor estimation; HILIC demonstrated strong complementarity to RPLC for polar analytes and increased sensitivity in ESI‑MS workflows.
• Multidimensional LC innovations: demonstrations of HILIC×RP and RPLC×RPLC strategies for complex phenolics/peptides; the Total Breakthrough phenomenon was characterized and exploited to improve on‑line HILIC×RPLC peptide separations; middle‑up HILIC‑HRMS approaches enabled rapid, site‑specific N‑glycan profiling as a viable alternative to released‑glycan workflows.
• Ultrafast and thermal‑gradient GC: thermal gradient GC, flow‑field thermal gradient GC and vacuum (LPGC) strategies were reported to deliver dramatic throughput increases (up to orders of magnitude) while retaining robust chromatographic performance. Practical engineering solutions (active cooling, purged connectors, optimized injector liners) improved retention stability and robustness.
• GC×GC and volatilomics: GC×GC‑TOFMS remains a leading tool for volatilome characterization (breathomics, clinical VOCs, fragrances). Structured chromatograms, dual retention info and high mass spectral detail support better compound identification, but standardization and QC remain barriers to clinical translation.
• Sample preparation and miniaturization: tailored MIP/IIP sorbents and in‑capillary monoliths enabled selective extraction and online coupling to nanoLC; fully automated DBS extraction with LC‑MS/MS showed clinical potential for therapeutic drug monitoring; 3D‑printed SPE devices and sorbents provide rapid prototyping and custom formats for microextraction workflows.
• Analytical purification and production scale: MCSGP (multicolumn continuous chromatography) demonstrated major gains in yield/productivity and solvent‑use for peptide purification compared to batch methods, addressing the yield‑versus‑purity tradeoffs of center‑cut separations.
• Methods for problematic matrices and contaminants: integrated LC‑GC×GC‑ToFMS platforms were proposed for MOSH/MOAH analysis and for detailed MOAH subfraction and toxicity assessment; TD‑GC×GC workflows correlated VOCs from LDPE materials to human sensory panels for odor assessment.
• Modeling and data science: thermodynamic and retention‑time modeling for GC×GC under vacuum outlet conditions and for loop dispersion in 2D‑LC contributed to rational method development. Graph neural networks and other ML approaches outperformed classical descriptor‑based models for retention prediction, facilitating candidate filtering in non‑targeted workflows. Advances in untargeted data processing for GC×GC and metabolomics were also presented.

Benefits and practical applications

• Faster analytical pipelines (ultrafast GC, short and ultra‑short LC columns, automated extraction) reduce time‑to‑result in QC and high‑throughput screening.
• Higher structural resolution (multidimensional separations, ion mobility, high‑resolution MS, MALDI imaging) enables more confident identification of isomers, proteoforms and complex mixtures important for safety, efficacy and forensic questions.
• Miniaturized and automated sampling (DBS, microextraction, nanoLC‑CE) enable analysis of volume‑limited biological samples and support clinical/field deployments.
• Selective sorbents (MIP/IIP) and MCSGP continuous purification enhance selectivity and downstream processing in both analytics and manufacturing.
• Model‑driven method translation and ML‑assisted retention prediction speed method setup and reduce trial‑and‑error in multidimensional method development.

Future trends and potential applications

• Deeper integration of orthogonal separations (LC×LC×IMS×MS) with real‑time data reduction and AI‑driven interpretation to cope with extremely complex matrices (e.g., polymers, plant metabolomes, environmental UCMs).
• Wider adoption of ion mobility (TIMS, trapped mobility) and native MS workflows for routine proteoform and complex‑assembly characterization in biopharma and structural biology.
• Standardization efforts for clinical volatilomics and breathomics to enable robust, multi‑site biomarker validation and regulatory acceptance.
• Increased automation from sample collection (home DBS), online sample prep, to hyphenated analysis and automated reporting for routine TDM, QC and environmental monitoring.
• Green and high‑throughput chromatography: expansion of SFC and unified‑CO2 mobile phase methods for sustainable, fast separations; further maturation of solvent‑saving continuous purification techniques (MCSGP).
• Growing role for in‑silico method development, mechanistic modeling and ML (including graph neural nets) to predict retention, optimize multidimensional workflows, and prioritize candidate identifications in non‑targeted studies.
• Rapid prototyping (3D printing) and custom sorbent manufacturing will broaden bespoke extraction formats and lab‑scale consumables for niche applications.

Conclusion

The HTC‑17 program underlined that the field of hyphenated separation science is maturing along multiple complementary axes: instrumental innovation (faster, higher‑resolution separations, better hyphenation hardware), advanced sample handling (selective miniaturized sorbents, automated DBS workflows), and data‑centric method development (modeling and ML). Together these elements substantially expand the analytical reach for complex and clinically relevant samples, for high‑throughput industry workflows, and for in‑depth characterization in research. Remaining priorities are standardization (especially for clinical breathomics and complex food matrices), robust interfaces for pairing SFC and MS, and improved turnkey software for processing the massive data from multidimensional separations. Ongoing cross‑disciplinary work (engineering, surface chemistry, informatics, statistics) will be essential to convert demonstrated advances into routine, validated laboratory practice.

References

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