Since the 1960s, when the first high-performance liquid chromatography (HPLC) column was packed, scientists and manufacturers have steadily pursued smaller particles to improve separation performance. This is because the resolving power of a chromatographic column increases as particle size decreases. Smaller particles allow analytes to move more rapidly between the mobile and stationary phases, enhancing column efficiency.
This evolution began with the original 45–60 µm pellicular particles: non-porous glass beads coated with a thin, porous layer, developed by Csaba Horváth in 1968 (1). By 2004, Waters Corporation introduced fully porous 1.7 µm particles alongside new ultra-high-pressure liquid chromatography (UHPLC) systems (2). Although finer particles exist today, further particle miniaturization raises significant engineering challenges, including pump design, heat generation, extra-column dispersion, and column packing under extremely high pressures (5,000 bar). As of 2025, commercial columns packed with particles smaller than 1.3 µm are not available.
In parallel, over the past two decades, many manufacturers have succeeded in producing highly uniform (monodisperse) 2 µm particles. Unlike conventional particles, which vary in size by 20–30 percent, monodisperse particles have nearly identical diameters, with a relative standard deviation close to zero.
This has raised an important question in the LC community: does uniformity in particle size result in superior column efficiency and resolution compared to traditional polydisperse particles?
Looks can be deceiving
Viewing scanning electron microscopy (SEM) images of monodisperse particles, those with nearly identical sizes, immediately evokes a sense of order and perfection. Their uniform appearance stands in contrast to the visual randomness of polydisperse particles, which vary significantly in size. As humans, we are naturally drawn to symmetry, order, and structure, and we tend to trust what looks neat and well-organized.
This visual appeal – I believe – has contributed to a widespread belief in the chromatography field: that monodisperse particles must deliver superior column efficiency in liquid chromatography for the same average particle size. However, this assumption, though appealing, remains more myth than proven fact. The idea that particle size uniformity translates directly into higher chromatographic efficiency is a hypothesis that still awaits rigorous validation.
Several companies – including Advanced Materials Technologies and Phenomenex for core-shell or superficially porous particles (SPPs), and Sigma-Aldrich, Fortis Technologies, ThermoFisher, Hitachi, and Nanomicro Technologies for fully porous particles (FPPs) – have developed and marketed monodisperse particle technologies, each with distinct materials and formats. Each has highlighted on their respective websites the intrinsic advantages of monodisperse particles over conventional polydisperse ones in terms of column efficiency. Remarkably, it is not possible to find proof of such claims based on the rigorous measurement of reduced van Deemter curves (normalized to the average particle diameter) over a wide range of flow rates compared to the same van Deemter curves of benchmark columns packed with polydisperse particles. Nevertheless, over the past decade, this message of superior performance has gained traction and has been shared widely at major international analytical chemistry conferences such as Pittcon, the Eastern Analytical Symposium, AAPS National Biotechnology conference, and the HPLC Symposium, among others. They even appear in peer-reviewed journals, such as LCGC International (3).
In this latter example, the method described omits essential data needed for a fair comparison, specifically, the complete reduced van Deemter plots over a wide range of flow rates for both monodisperse and standard reference polydisperse particles. These plots are crucial for assessing column efficiency across a range of flow rates and comparing the minimum reduced plate height (RPH) – the column length divided by the column efficiency or plate number. It’s important to emphasize that conventional polydisperse particles, when properly packed, can even achieve RPHs as low as 1.7. The ultimate limit is set to be around 0.9 for FPPs and 0.7 for SPPs (4). Therefore, any minimum RPH observed for monodisperse particles that exceeds 1.5 for SPPs and 1.7 for FPPs cannot be considered evidence of superior intrinsic performance. Without such comparative benchmarks, claims of enhanced efficiency remain speculative.
Over the past two decades, academic researchers have explored two main approaches to test whether monodisperse particles truly enhance the performance of HPLC columns.
The first approach involves digitally reconstructing the actual structure of randomly packed columns using the exact experimental particle size distribution measured by the well-adopted Coulter counter technique. This means simulating how analytes move through a digitalized column where every particle’s position is preserved as in the real column. The results have been striking. Reducing the relative standard deviation (RSD) of particle size distribution from 25.3 percent (typical for conventional fully porous particles) to just 3.4 percent (as seen in superficially porous particles) had virtually no measurable impact on column performance (4). These findings strongly suggest that the width of the particle size distribution has little to no intrinsic impact on column efficiency. It has also been demonstrated, based on structural reconstructions of capillary columns, that the superior column efficiency of monodisperse superficially porous particles (SPPs) over polydisperse fully porous particles (FPPs) is not due to the narrower particle size distribution (PSD). Rather, it stems from a more uniform interparticle porosity profile across the column, from the wall to the central bulk region, which is achieved through a more effective packing procedure (5). However, despite being based on realistic simulations of analyte transport through actual randomly packed structures, one could still argue that simulations, no matter how detailed, are not a substitute for direct experimental proof.
This brings us to the second approach: physically mixing particles of different sizes in controlled ratios to vary the RSD of the size distribution. Indeed, this has been done and once again, the outcome was clear: increasing the RSD from 16 percent to 41 percent did not produce any measurable decrease in column efficiency (6). Additionally, more recently, we tested this further by sieving conventional <2 µm UHPLC particles (specifically, 1.7 µm BEH-C18) to narrow their size distribution. The ratio between the largest and smallest particles (the so-called D90/D10 ratio corresponding to the 10 percent and 90 percent points of the cumulative size distribution) was reduced from 1.42 to 1.25. Yet, when we measured column efficiency using reduced (normalized to the average particle size) van Deemter plots over a large range of flow rates, no improvement in column performance was observed.
These findings align with earlier academic studies conducted on larger particles [7–10], reinforcing a consistent conclusion: regardless of how tightly particle sizes are controlled, analytical columns remain randomly packed, and axial dispersion, the spreading of analytes along the column, remains largely unchanged. In essence, the inherent disorder of the packed bed structure outweighs any projected advantage of particle size uniformity. The past literature has unambiguously demonstrated that column performance is therefore primarily governed by the selection and optimization of individual particle morphology and the packing procedure. These factors determine the uniformity of the random bed structure across the entire column diameter, rather than the narrowness of the particle size distribution.
A worrying trend
It worries me that we have not yet seen a broad, critical scientific debate in the chromatography community or calls for further exploration and validation. I fear this is part of an emerging trend – one that risks becoming dominant when the checks and balances essential to a technically complex field like liquid chromatography begin to erode. This coincides with a broader shift in academic training, particularly in the United States, where universities are producing fewer specialists in the fundamental science of liquid chromatography. As a result, opportunities for critical discussion and scientific debate at major conferences have diminished, leaving many claims about new technologies, such as monodisperse particles, largely unchallenged.
To counter this trend, we must maintain a high standard of education in both the theoretical and practical foundations of the discipline. Strong academic training fosters critical thinking among users, enabling them to evaluate new technologies with a discerning eye. When users are well-informed, they are more likely to challenge manufacturers' claims and demand evidence-based innovation. This dynamic not only strengthens the scientific community but also drives meaningful progress in analytical science.
Finally, to ensure the integrity of scientific communication, it is equally important that conferences and journals, especially those with strong reputations, critically evaluate submitted work before publication or presentation. When unverified claims are inadvertently relayed, it can mislead the community and dilute the standards of evidence-based research. A more robust review process, supported by experienced scientists with deep expertise in the field, would help safeguard against such oversights. This approach reinforces the credibility of the platforms and ensures that new ideas are grounded in rigorous scientific validation.
The path forward
This discussion concerning the development and evaluation of new liquid chromatography columns, particularly those incorporating monodisperse particles, highlights the importance of maintaining rigorous scientific standards.
If there’s one key lesson for readers and reviewers when assessing particle performance, it’s that they should make sure to verify three essential elements:
Accurate and transparent measurements of reduced plate heights across a broad range of flow rates
Meaningful benchmark comparisons against well-established LC column performance standards, as observed in 2025
Precise assessment of the uniformity of the random bed structure across the whole column diameter.
These criteria are vital to ensure that performance claims are not only scientifically valid but also practically relevant for solving real-world analytical challenges.
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References
- C Horváth, B Preiss, S Lipsky, Anal. Chem, 39, 1422 (1967).
- J Mazzeo, U Neue, M Kele, R Plumb, Anal. Chem, 77, 460A–467A (2005).
- K Butchart, M Woodruff, LCGC North America, 41, 446–452 (2023).
- A Daneyko, A Holtzel, S Khirevich, U Tallarek, Anal. Chem, 83, 3903–3910 (2011).
- S Bruns, D Stockel, B Smarsly, U Tallarek, J. Chromatogr. A, 1268, 53–63 (2012).
- F Gritti, T Farkas, J Heng, G Guiochon, J. Chromatogr. A, 1218, 8209–8221 (2011).
- I Halasz, M Naefe, Anal. Chem, 44, 76 (1972).
- J Done, J Knox, J. Chromatogr. Sci, 10, 606 (1972).
- R Endele, I Halasz, K Unger, J. Chromatogr, 99, 377 (1974).
- C Dewaele, M Versele, J. Chromatogr, 260, 13 (1983).
