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I have had enough conversations with experts in the field of high performance liquid chromatography (HPLC) stationary-phase supports to know that there is more to the increased efficiency provided by the use of superficially porous particles (SPP) compared to fully porous particles (FPP) than simply mass transfer effects. Yet, I would argue that this is still one of the biggest misconceptions propagated by some members of the chromatography community.
I have had enough conversations with experts in the field of high performance liquid chromatography (HPLC) stationary-phase supports to know that there is more to the increased efficiency provided by the use of superficially porous particles (SPP) compared to fully porous particles (FPP) than simply mass transfer effects. Yet, I would argue that this is still one of the biggest misconceptions propagated by some members of the chromatography community. It is very tempting to assume that increased efficiency is achieved simply because the intra-particle diffusion path is significantly reduced in SPP. The fact of the matter is that for small molecules, diffusion is sufficiently fast that the mass transfer effects are much smaller than other kinetic contributions, which lead to increased efficiency.
Last week, we were fortunate to have Dr. Fabrice Gritti join us at U.T. Arlington for a Friday seminar. Dr. Gritti is a research scientist at the University of Tennessee – Knoxville, and for the past decade has been the right-hand man for luminary Prof. George Guiochon, who passed away late last year. Dr. Gritti is widely renowned as an expert on chromatographic theory, a fact that was recognized by his recent receipt of the 2013 Chromatography Society Jubilee Medal, awarded at HPLC 2013 in Amsterdam, The Netherlands. The first part of his seminar provided a retrospective of his work assessing and designing separation systems since his graduate school days. The second part focused on his work on fundamental thermodynamic and kinetic aspects of separation theory, and the use of advanced measurements to gain new insights into these processes. It was an extremely educational talk. I appreciated the detail placed on chromatographic theory beyond what is usually covered in the modern textbook, but especially his coverage of efficiency related to the difference between SPP and FPP. In fact, it was his work on the latter that was specifically cited for his Jubilee Medal award. I appreciated Dr. Gritti’s ability to effectively convey quite complex topics with practical examples, which made them more understandable for the audience. For me, I gained a new appreciation for the nuances differentiating modern stationary-phase supports, and I thought this a good venue to distill some of what I learned.
The fundamentals of separation science in the context of Dr. Gritti’s work were segregated into the thermodynamics of adsorption events and the kinetics associated with band-broadening processes. Measurements related to thermodynamics have been heavily focused on adsorption and retention mechanisms as they pertain to preparative-scale HPLC and production optimization. These measurements have included the use of frontal analysis and the inverse method to determine different adsorption isotherms for analytes under various chromatographic conditions. The determination of the type of adsorption isotherm (for example, Langmuirian, anti-Langmuirian, and so forth) provides valuable insight into the analyte-dependent retention mechanism to maximize performance of the separation system, especially under high analyte loads. Some concrete examples included optimizing the purification of C60 versus C70 fullerenes (1), selecting the best buffer for purification of a pharmaceutical active ingredient (2), and elucidating bi- and triphasic adsorption conditions for phenol in the presence of methanol versus acetonitrile mobile-phase conditions (3).
From the kinetics side, and aspects of band broadening, the discussion quickly led into fundamental concepts established through the well-known van Deemter equation, with some added nuances. Following the seminar, I was pointed to an exceptional article by Gritti and Guiochon (4), which discusses facts and myths associated with the performance of SPP. From the discussion in the seminar, and reading the article, it became clear that a more in-depth knowledge of the factors contributing to band broadening must be appreciated. In fact, the details can be much more complex than presented in even these venues, but I was seeking a very basic understanding that I could successfully convey to others - I wanted to understand the elevator pitch first, before delving into the more intricate complexities.
As is well known according to van Deemter theory, the height equivalent to a theoretical plate (HETP), a measure of chromatographic efficiency, is determined by contributions from eddy dispersion, longitudinal diffusion, mass transfer processes, and linear velocity of the mobile phase. The primary misconception about why SPP are more efficient than FPP is that the reduced diffusion path in the SPP lessens contributions from mass transfer processes. Closer analysis, such as that performed by Gritti and Guiochon, indicated that this is not the primary factor when small molecule analysis is considered. Small molecules have high enough diffusion coefficients so that the difference in mass transfer found between SPP and FPP is minimal. In fact, overall, mass transfer contributions to HETP in this case are very small compared to contributions from longitudinal diffusion and eddy dispersion. Their experiments confirmed that the primary reason for lower HETP in SPP was reduced eddy dispersion. Longitudinal diffusion is also reduced, but not to the degree to which eddy dispersion is under practical operational flow rates. Furthermore, when eddy dispersion is split into short-range and long-range processes, the researchers found that a significant reduction in short-range eddy dispersion, specifically trans-particle and inter-particle eddy dispersion, was most effected. I still need to do some more reading to appreciate the fundamental differences between short- and long-range eddy dispersion.
It is believed that along the cross-sectional diameter of the column, packing with SPP is more uniform than that achieved with FPP. This packing uniformity leads to a reduction in wall effects for columns packed with SPP. Yet, SPP are considered to have a rougher surface than FPP, which limits slip of particles during packing. For FPP, it may be that an uneven distribution of particle diameters closer to the wall compared to in the center of the column is created based on the difference in pressures experienced near versus away from the wall during packing, combined with the ability of the FPP to slip past each other. The roughness of the SPP may resist the slip that creates an unequal distribution of particle sizes across the cross-section of the column; one interpretation is that the SPP columns are not packed quite as tightly as smooth FPP packings, but overall, the packing of SPP may be more uniform. The wall effects, or the fact that the packing material is necessarily contained within a tube and endcapped with frits, are another undeniable issue. It was interesting to hear that it would be theoretically possible to achieve a surprising 800,000 plates per meter in HPLC packings if a bed with no boundaries could be created. The lag in flow created by the interaction of mobile phase and analytes with the wall of the column creates a major reduction in efficiency. Yet, this reduction is not as severe with the use of SPP compared to that for FPP.
Csaba Horvath invented SPP for macromolecule separations in the 1960s (5,6). The reduction in mass transfer was his goal. Given that the diffusion for large molecules like proteins is much slower than for small molecules, this was a reasonable strategy. Because SPP have become more popular in recent years, primarily for small molecule separations, it has been necessary to revisit the fundamental reasons why they provide improved chromatographic performance. In the recent seminar by Gritti, I gained a better understanding and appreciation for these reasons. As I delve into the supporting literature, I realize that it is going to take some studying to really describe the underlying theory in detail. This is one of the things that I love about chromatography. I liken it to a statement I heard about learning the English language - with a little practice, one can become a functional practitioner, but to become an expert, it can take a lifetime. Gritti closed his seminar by summarizing his motivations. If one can understand the thermodynamics associated with retention, then they can design new materials to optimize selectivity. If one can understand the kinetics associated with mass transfer, diffusion, and dispersion, then they can design new architectures to optimize efficiency. In this manner, it seems that there is much more work that could be done to improve chromatography in the years to come - but one just needs to home in on and appreciate the detailed fundamentals associated with the process.
(1) F. Gritti and G. Guiochon, J. Chromatogr. A1053, 59–69 (2004).
(2) F. Gritti and G. Guiochon, Anal. Chem.76, 7310–7322 (2004).
(3) F. Gritti and G. Guiochon, Anal. Chem.77, 4257–4272 (2005).
(4) F. Gritti and G. Guiochon, LCGC North Am.30(7), 586–595 (2012). (http://www.chromatographyonline.com/facts-and-legends-columns-packed-sub-3-m-core-shell-particles?id=&pageID=1&sk=&date=).
(5) C.G. Horváth, B.A. Preiss, and S.R. Lipsky, Anal. Chem.39, 1422–1428 (1967).
(6) C.G. Horváth and S.R. Lipsky, Anal. Chem.41, 1227–1234 (1969).
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