The history of HPLC reminds us that although core principles are bound by physics, innovation often thrives in system hyphenation and application expansion – as we’re seeing with cell and gene therapies and vaccines – Fabrice Gritti argues
In 2015, we gathered together a group of experts to ask: have we reached the limits of liquid chromatography? Our experts returned a resounding, “no!” – as they did two years later in the follow-up piece: LC on the Edge. However, ten years on – and with HPLC 2025 just around the corner – we feel the time right to reach out gurus old and new, take stock of the field’s progress over the past decade, and revisit our provocative question…
When you look back over the past 10 years, has HPLC innovation lived up to expectations?
Yes, I believe HPLC innovation has lived up to expectations – though not through breakthroughs in resolution, selectivity, or throughput, which have remained relatively stable (notably, UHPLC celebrated its 20th anniversary last year). Instead, the last decade has seen HPLC evolve in response to the analytical challenges posed by complex biomolecules such as monoclonal antibodies (mAbs), mRNA, adeno-associated viruses (AAVs), and lipid nanoparticles (LNPs). This growing demand has catalyzed significant advances in column and system technologies.
The most impactful development of the past decade has been the emergence of fully bio-inert systems and columns, designed to overcome issues like sample loss and resolution degradation caused by metal-analyte interactions. Manufacturers have introduced metal-free hardware, new surface chemistries, and specialized columns – such as robust SEC columns with ultra-wide pores (up to 2000 Å for LNPs) and slalom chromatography columns tailored for large DNA/RNA molecules. These solutions have dramatically improved the analysis of sensitive compounds and accelerated innovation in biopharmaceutical research.
Hyphenation has also advanced notably. Beyond the essential use of mass spectrometry, non-optical detectors – including refractometers, light-scattering, and zeta potential detectors—have emerged as valuable tools for deeper molecular insight, particularly in size and charge characterization. Complemented by increasingly user-friendly software interfaces, multi-detector systems are now more accessible and effective, broadening the scope and precision of biopharmaceutical applications.
Are there any developments from the past couple of years that have stood out?
Yes, and although I may be slightly biased, a standout development in recent years is the revival of “slalom chromatography,” a technique originally discovered in 1988 for separating large DNA and RNA molecules. It was initially set aside due to unclear retention mechanisms, poor-quality particles, and limited practical use. Recently, I’ve been directly involved in reintroducing this method using innovative SEC particles and fully bio-inert UHPLC systems. These advancements now enable effective characterization of large plasmid DNA and the separation of mRNA vaccines from dsRNA impurities – critical needs in cell and gene therapy research. When combined with complementary techniques like light-scattering, charge-detection mass spectrometry, and mass photometry, slalom chromatography will likely emerge as a valuable tool for analyzing very large nucleic acids (>2 MDa). I believe it has strong potential to shape the future of biopharmaceutical analysis.
So, has HPLC peaked?
Although certain aspects of HPLC, like particle size reduction and packed column performance, may have reached practical limits due to physical and chemical constraints, the technique has not peaked. About 15–20 years ago, discussions at international conferences predicted a lower limit around 1.5 µm for the particle size, due to challenges with pressure, heat dissipation, and system dispersion. Those limitations still hold today, making further gains in speed and performance from smaller particles unlikely. Similarly, major breakthroughs in column selectivity are rare, aside from some promises in mixed-mode HPLC. However, many other areas – such as bio-inert column and system design, advanced detection methods, automation, hyphenation, data handling and processing – continue to evolve rapidly.
The history of HPLC reminds us that while core principles are bound by physics, innovation often thrives in novel surface chemistry, system hyphenation and integration and application expansion.
What are some of the hottest trends in HPLC today?
In addition to the development of improved columns and systems designed to meet the needs of application chemists working on the characterization of complex biological systems discussed above, which I would say is the hottest trend, another major trend is the rising importance of artificial intelligence in chromatography, particularly for system diagnostics and predicting compound retention based on molecular structure in untargeted metabolomics, proteomics, and lipidomics. Although still in its early stages, this approach holds significant promise due to the vast amount of data being generated in these fields.
Is AI having an impact on the HPLC field today?
Currently, AI has a limited impact on the HPLC field, as it remains primarily at the research stage rather than being a fully adopted and validated tool within the broader HPLC community or by regulatory bodies. That said, AI-supported HPLC is highly attractive and holds great promise. It has the potential to significantly accelerate method development and data handling, reduce failures in large-scale chemical processes through automated process analytical technologies, and improve the environmental footprint of preparative HPLC.
Yet, trusting AI predictions in the HPLC field is a risky business. Nevertheless, these efforts are expected to continue and expand in the coming years, especially as this area of research continues to receive strong support from grant funding agencies.
Where do you expect HPLC to be in 5 years’ time?
In five years, the resolution, speed, and selectivity of HPLC columns and instruments are unlikely to change dramatically. However, I believe that many of the analytical challenges posed by the rapidly evolving biopharmaceutical industry, particularly with emerging modalities, will be addressed through the development of well-designed, specialized HPLC columns (such as SEC, affinity, and field flow fraction) and dedicated systems.
More importantly, it's worth noting that chromatographic sciences are no longer being taught as thoroughly as they should be in many countries, including the United States. As a result, the fundamental knowledge of chromatography among entry-level professionals is declining. This trend suggests that, within five years, HPLC systems will become increasingly user-friendly and more heavily controlled by AI algorithms, helping to reduce manual input and enhance the speed and efficiency of analysis.
Gazing further into your crystal ball… In the decade(s) to come, what might the next “HPLC gamechanger” look like?
Looking into the decades ahead, I believe the next HPLC game-changer will be the integration of artificial intelligence across nearly every stage of the workflow – from sample preparation and method development to data handling and processing. Process development for large-scale bioreactors will also benefit significantly from AI and hybrid modeling approaches (e.g., digital twins), helping to reduce both costs and carbon footprint.
Moreover, I believe that generative design – combining fundamental principles of physics and chemistry with the vast amounts of data generated today – will drive the discovery of new 3D column structures, enhancing both speed and resolution. These innovations could become accessible to HPLC users once 3D printers capable of producing at 1-micron resolution across large build volumes (~ 1 cm3) are widely available.
Overall, are you optimistic about the future of HPLC?
Absolutely. HPLC will always remain a force to be reckoned with. It is one of the most sensitive analytical techniques available, capable of detecting subtle differences in free energy, approximately 25 J/mol using recycling chromatography (for selectivity α = 1.01), while the weakest dispersive intermolecular interactions in nature are around 50 J/mol.
That said, HPLC and multi-dimensional HPLC are not a panacea for sample characterization. Rather, they will continue to play a vital role by complementing and being hyphenated with other analytical techniques. In a world where both the amount and complexity of chemical systems to be analyzed are constantly increasing, HPLC will remain an essential tool for the success of analytical scientists.
Fabrice Gritti is Principal Consulting Scientist, Waters Corporation, USA
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