Navigating The Path To Modern Slalom Chromatography: An Interview with Fabrice Gritti

Can you define slalom chromatography (SC)?
The definition of SC is historical. The term "slalom chromatography" was first introduced, somewhat arbitrarily, as a new mode and mechanism of retention/separation in liquid chromatography in a 1989 publication: Size-Dependent Chromatographic Separation of Double-Stranded DNA Which Is Not Based on Gel Permeation Mode (Analytical Biochemistry, 178, 336–341). (1). This study was authored by Jun Hirabayashi and Ken-ichi Kasai, two Japanese researchers from the Department of Biological Chemistry, Faculty of Pharmaceutical Sciences in Sagamiko, Japan.

Fabrice Gritti, consultant scientist at Waters Corporation, spoke to LCGC International about the history of slalom chromatography (SC) and why he decided that the technique was worth re-investigating.

In 1988, they, along with a group of American researchers—Barry Boyes, Douglas Walker, and Patrick McGeer—independently discovered that double-stranded DNA (dsDNA) fragments could be separated using standard size-exclusion chromatography (SEC) columns. The Japanese group used 5 µm and 9 µm SEC particles (1) while the American group used 4 µm SEC particles (2). Both particles have an average mesopore size of 250 Angstrom and were used to separate large proteins such as mAb. Notably, the elution order of dsDNA fragments was found to be the reverse of the conventional exclusion mechanism typically observed in SEC or hydrodynamic chromatography (HDC) principles. Nobody really understood and could explain why this was happening.

It is fascinating to note that the term "slalom chromatography" was purely speculative in 1989 immediately after the facts were reported. The authors imagined, without direct observation, that long dsDNA biopolymers would behave like a skier, "slaloming" or frequently turning around the packed particles during their chromatographic migration along the gel permeation column. This explanation was proposed to account for why long dsDNA fragments were more retarded than shorter ones.

Thirty-seven years later, we have discovered that this “slalom” concept of the retention mechanism and separation of dsDNA macromolecules was somewhat inaccurate and misleading. Nevertheless, the term “slalom chromatography” has endured and will continue to be used. We will later provide further insights into the actual retention mechanism, which is grounded in sound physical chemistry. In fact, SC is an out-of-equilibrium retention mode of chromatography, as evidenced by direct observations of dsDNA (labeled with YOYO-1, a fluorescent dye) migrating with the flow of the mobile phase between packed chromatographic particles (3) or indirect measurement of retention factors under various experimental conditions of dynamic parameters such as flow rate, dynamic viscosity. Slalom would better be replaced by peristalsis mode of motion.

How and why did your company become interested in developing slalom chromatography?
This is a rather long story that started about 15 years ago, following discussions and suggestions from the late Edward Bouvier, a former student of the high performance liquid chromatography (HPLC) pioneer Csaba Horváth. At that time, Ed suggested exploring SC as an alternative chromatographic separation mode for handling very large molecules like DNA because the largest pore size available was only 450 Å, which could not accommodate such large analytes.

The main challenges at that time were: (i) No one within or outside Waters Corporation had a clear fundamental understanding of how SC worked, (ii) SEC particles at the time contained non-specific adsorption sites, and (iii) column hardware lacked the protective coatings now available for analyzing carboxylate-rich (multi-acid) or phosphate-rich compounds, such as oligonucleotides and DNA.

A breakthrough occurred approximately two years ago, in 2023, when Matt Lauber, Pen Chen, and Kevin Wyndham from Waters' development group transferred SC to the Waters' research teams in which I was involved. This allowed us to advance our understanding of SC and prepare the most suitable SC columns and methods for biochemists and biologists working in cell and gene therapies.

I was immediately and deeply interested in this problem because it inherently relied on the physicochemical properties of packed beds (specifically, the average shear rate generated by the mobile phase under laminar flow conditions) and the characteristics of individual DNA or RNA chains, such as their persistence length, the DNA elongation vs. shear force relationship, and the relaxation kinetics.

To address this, I conducted a comprehensive series of experiments, systematically varying one critical parameter at a time, including particle size, linear velocity, mobile phase viscosity, and the size of linear dsDNAs. After gathering and analyzing these empirical results, it became possible to construct a robust theory of retention in SC, which operates as an out-of-equilibrium retention mechanism. Three publications have been published on SC during the year 2024 (4–6) and one review paper on the history and applications of SC has been published in 2025 (7).

Eventually, we submitted a SC patent application targeting methods to analyze and characterize complex DNA and RNA mixtures using modern and inert ultrahigh-pressure liquid chromatography (UHPLC) column technologies. All the teams, from the physical chemists to the particle chemists, and the application chemists, were then very proud to have revived this promising but abandoned mode of separation in LC.

What makes SC particularly useful for separating and studying DNA and RNA molecules and compared other chromatographic techniques?
Slalom chromatography is particularly useful for the analysis and characterization of large DNA and RNA biopolymers, ranging in size from 500 Å to approximately 0.5 μm. These molecules are increasingly central to the development of cell and gene therapies. SC offers several advantages, combining: (i) the simplicity of a UHPLC method, (ii) the speed and high throughput typical of UHPLC techniques, (iii) the high-resolution power and specificity of UHPLC as a separation tool, and iv) the lower detection limits and reproducibility inherent to UHPLC systems and columns. SC thus represents a promising alternative to conventional analytical techniques, many of which are reaching their limits when analyzing such large molecules.

For example, traditional separation modes such as ion-pair reversed-phase liquid chromatography (IP-RPLC), which involves gradient changes in organic solvent and ion-pairing agent(s), or ion-exchange (IEX) and hydrophobic interaction chromatography (HIC), which rely on varying salt concentrations, are limited by the pore sizes of their packing particles—typically no larger than 500 Å. SEC, using ultrawide mesopores up to 3000 Å, can accommodate some of these large DNA and RNA molecules, but its resolution power is inadequate. HDC with narrow internal diameter (i.d). (~5 μm) open capillary columns offers a viable alternative but requires extremely long analysis times (several hours) and specialized detection methods, such as laser-induced fluorescence (LIF), due to the short optical path length for UV–vis detection. Similarly, asymmetric flow field-flow fractionation (AF4) can handle such large molecules but lacks sufficient resolution for complex samples.

Currently, the most used techniques are agarose gel electrophoresis (AGE), which accommodates dsDNA up to 25 kbp, and pulsed-field gel electrophoresis (PFGE), which can analyze dsDNA up to 10 Mbp. However, these methods require very long analysis times, typically 1–2 hours for AGE and up to 24 hours for PFGE.

The appeal of SC lies in its ability to separate double-stranded DNA (dsDNA) ranging from 2 to 25 kbp with a peak capacity of approximately 20 in less than 2 min, achieving a limit of detection as low as 1 ng. No other available separation technique matches SC’s combination of resolution power, speed, and sensitivity.

How does the separation mechanism of SC differ from other chromatographic techniques?
The primary distinction between the separation mechanism of SC and other chromatographic modes lies in its out-of-equilibrium retention mechanism with respect to the conformation of DNA and RNA (ribonucleic acid) biopolymers because they migrate along the SC column. This characteristic imparts unique selectivity properties to SC. In contrast, other chromatographic methods, such as SEC, HDC, and AF4, are equilibrium separation modes, where DNA and RNA molecules are eluted in their equilibrium random coil conformations.

To better understand the retention mechanism in SC, it is important to consider the dynamics within the interparticle spaces of a packed column. A wide range of shear flow rates is generated in these spaces, with the maximum shear rate occurring near the particle surface and the shear rate being nearly zero at the center of the flow channels. This shear gradient causes dsDNA or dsRNA biopolymers to stretch and elongate near the particle surface, resulting in their retardation. Conversely, when these biopolymers migrate to the central regions of the flow channels, they tend to relax back into their random coil conformations, where they are transported by convection through the packed bed.

Longer dsDNA molecules spend more time near the particle surface than shorter ones. This is because the likelihood of an extended polymer chain being close to the particle surface increases with the length of the molecule. Qualitatively, this describes the fundamental separation mechanism of SC.

Moreover, quantitative models of SC retention are now available to predict the retention factor of any linear dsDNA based on user-selected experimental parameters, including particle size, column length, pressure drop, temperature, aqueous mobile phase composition, and salt content.

What recent improvements in technology have made SC more effective for DNA and RNA analysis?
One significant advancement in SC technology compared to the LC systems and columns of the late 1990s is the easy integration of sub-3-μm particles. This improvement coincides with the 20th anniversary of UHPLC, introduced by Waters Corporation in 2004. The use of small 1.7-μm UHPLC particles has enabled unprecedented baseline resolution for 2 to 6 kbp dsDNA, achieving a peak capacity close to 10—something that was not feasible with the larger 5-μm particles previously available.

A second important improvement is the development of SC particles free from non-specific adsorption sites, along with the fabrication of metal (stainless steel) column hardware coated with advanced materials that prevent irreversible interactions between DNA/RNA analytes and the column surface. This eliminates analyte loss during analysis. As a result, users can now employ simple, fully aqueous buffer solutions (around physiological pH 7.4) without the need for specific mobile phase additives such as organic solvents, high-ionic-strength salts, or metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA).

These inert bioparticles and specialized column hardware were not available in the late 1980s and 1990s, making these recent innovations critical for the advancement of SC technology for the characterization of biological molecules.

What are the limitations of other techniques used to analyze DNA and RNA and what benefits does SC offer over these methods?
The primary limitations of conventional: Ion-pair reversed-phase liquid chromatography (IP-RPLC), ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) and separation modes stem from the small pore sizes of their particles, which cannot accommodate very large (>500 Å) DNA and RNA macromolecules, resulting in limited selectivity. The same limitation applies to SEC, even though recent ultrawide mesopores can reach sizes as large as 2000 Å. Most large DNA and RNA molecules are essentially excluded from the pore volume of the latest SEC particles.

HDC is a viable technique for separating such large nucleic acid polymers, but it requires nanoflow systems, long analysis times, and complex detection methods such as LIF. Similarly, AGE and PFGE are the current reference techniques for characterizing large DNAs, but their long analysis times and lack of compatibility with high-throughput workflows make them less practical. AF4 suffers from insufficient resolving power, while analytical centrifugation techniques are too complex to interpret and are unable to resolve mixtures of complex DNA samples effectively.

In a recent report (6) entitled Ultra-High Pressure Slalom Chromatography: Application to the Characterization of Large DNA and RNA Samples Relevant in Cell and Gene Therapy, Journal of Chromatography A, 1738 (2024) 465487), we demonstrated several proofs of concept for SC, addressing the four critical attributes biochemists and biologists in the field of cell and gene therapy require.

• Ease of use: The SC technique is as straightforward to apply as classical UHPLC methods.

• High selectivity: SC methods offer exceptional selectivity based on the size of linear dsDNA or dsRNA, achieving baseline resolution for size differences as small as 0.3 to 0.5 kbp.

• Speed: SC methods are extremely fast, completing analyses in just a few minutes.

• Sensitivity: SC enables the detection of as little as 1 ng of DNA material, with nearly a 3-log linear dynamic range using optical detection at 260 nm.

What are the critical parameters to optimize when developing SC methods for analyzing complex biomodalities like DNA/RNA, and how do these parameters affect separation efficiency and reproducibility?
Interestingly, there is a simple rule in SC to maximize resolution for a targeted DNA size. In a recent report (5) we demonstrated that the highest resolution is achieved when the average extended length of the DNA chain is approximately half the particle size. Under these conditions, both HDC and SC contribute to the DNA retention mechanism.

For example, small DNAs (2 to 9 kbp) are best separated using 1.7-μm SC particles in 15-cm columns at a pressure drop of 10,000 psi and room temperature. At the same pressure and temperature, 2.5-μm particles are optimal for analyzing DNAs ranging from 4 to 12 kbp in 30-cm columns. For DNAs between 6 and 16 kbp, 3.5-μm particles packed in 60-cm columns provide the best separation.

Even with a fixed particle size and column length, SC remains a highly adaptable technique. The average extension length of DNA and RNA macromolecules can be easily adjusted by modifying the linear velocity or eluent viscosity, both of which directly influence the shear flow rate and the extension of the biopolymer. For negatively charged SC particles, the ionic strength of the mobile phase also affects the shear flow rate as DNA molecules can be excluded from regions near the particle surface.

Models of retention have been developed to predict and optimize the resolution of an SC column, considering the combined effects of electrostatic repulsion (ER), HDC, and SC on DNA retention.

To maintain high SC efficiency, it is important to ensure that the extended length of DNA does not exceed approximately twice the particle size. Exceeding this limit can result in excessively long DNA relaxation times relative to the characteristic convection time of UHPLC, leading to increased peak widths and potential DNA mass losses. The repeatability of retention times in SC is excellent (< 1%) and comparable to that of standard UHPLC SEC methods used for analyzing large compounds.

How does SC help identify impurities in RNA used for making mRNA therapeutics?
Remarkably, SC capitalizes on differences in the entropic elasticity of polymer chains. dsDNA and dsRNA are highly extensible due to their rigid double-helix structures. Their persistence length (the shortest distance along the polymer chain beyond which the correlation between the tangent direction is lost) is approximately 500 Å. Consequently, a force of only 1 pN is required to stretch a 48.5 kbp λ-DNA to 80% of its contour length, with overstretching of the double-helix structure occurring at 60 pN.

In contrast, single-stranded DNA (ssDNA) and messenger RNA (mRNA) are extremely flexible and compact. Their persistence length is only about 10–15 Å, and they require a significantly higher force of 40 pN to achieve 80% extension of their contour length. In practical terms, mRNA remains in a random-dense coil conformation in an SC column, eluting according to an exclusion HDC retention mechanism.

It is important to note that mRNA produced via the in vitro transcription (IVT) enzymatic reaction contains a variety of impurities, including uncapped mRNA, mRNA with incomplete or absent polyA tails, and dsRNA contaminants. Unlike mRNA, dsRNA impurities adopt an extended chain conformation and are significantly retained in SC columns. This provides excellent selectivity between mRNA and dsRNA, allowing the isolation and quantification of dsRNA impurities down to as low as 0.1% in IVT-produced mRNA samples.

How does SC ensure accurate results when measuring the size or structure of DNA and RNA molecules?
The accurate measurement of the length or number of base pairs in an unknown linear dsDNA or dsRNA is straightforward using linear dsDNA standards, for example, 1 kbp DNA ladder, λ-DNA BstEII digest, λ-DNA HindIII digest, and λ-DNA Monocut Mix. A combined HDC/SC retention model that we recently published (7) is calibrated to the experimental retention factors measured for DNA sizes ranging from 0.5 to 48.5 kbp. Using this model, the size of an unknown DNA can be determined unambiguously based on its measured retention factor and the best-fit model predictions.

The accuracy of these measurements is as follows:

±0.3 kbp for DNA sizes up to 6 kbp,

±0.4 kbp for sizes up to 10 kbp,

±1 kbp for sizes up to 20 kbp, and

±2–3 kbp for sizes up to 50 kbp.

How could SC help improve the development of new medicines, especially in gene and cell therapy?
SC has significant impact at various stages of developing new treatments in cell and gene therapies. It enables rapid characterization of plasmid DNA, including gene length determination and distinguishing between circular and linearized DNA forms. As previously discussed, SC also facilitates the measurement of dsRNA impurities, ensuring the safety of mRNA treatments. Moreover, it can differentiate between various dsRNA structures, such as strictly linear dsRNA, partially hybridized dsRNA, and hairpin loop structures.

Most importantly, SC is a fast and efficient technique that supports the accelerated development of new mRNA vaccines, from discovery to clinical trials and preparative manufacturing stages.

Could SC possibly be used in other application areas?
Absolutely. SC is applicable to any large macromolecules that may differ in their persistence length. Thus far, we have primarily investigated the applications of SC for biopolymers with persistence lengths on the order of 500 Å. However, it is expected that SC will also find applications in the polymer field, particularly for semi-flexible or rigid chains influenced by factors such as the nature of the monomers and co-monomers, the degree of branching, and more. For example, we are currently exploring the use of SC for characterizing synthetic polyethylene (PE) polymer chains with varying degrees of branching. Such rapid characterization of PE could help companies such as Dow Chemical design monomers more effectively for achieving targeted bulk properties in polymer materials.

References

(1) Hirabayashi, J.; Kasai, K.-I. Slalom Chromatography: A New Size-Dependent Separation Method for DNA. Nucleic Acid Res. Symp. Ser. 1988, 20, 67–68.

(2) Boyes, B. E.; Walker, D. G.; McGeer, P. L. Separation of Large DNA Restriction Fragments on a Size-Exclusion Column by a Nonideal Mechanism. Anal. Biochem. 1988, 170 (1), 127–134. https://doi.org/10.1016/0003-2697(88)90099-1.

(3) Ström, O. E.; Beech, J. P.; Tegenfeldt, J. O. High-Throughput Separation of Long DNA in Deterministic Lateral Displacement Arrays. Micromachines 2022, 13 (10), 1754. https://doi.org/10.3390/mi13101754.

(4)Gritti, F.; Wyndham, K. Retention Mechanism in Combined Hydrodynamic and Slalom Chromatography for Analyzing Large Nucleic Acid Biopolymers Relevant to Cell and Gene Therapies. J. Chromatogr. A 2024, 1730, 465075. https://doi.org/10.1016/j.chroma.2024.465075.

(5)Gritti, F. Theoretical Predictions to Facilitate the Method Development in Slalom Chromatography for the Separation of Large DNA Molecules. J. Chromatogr. A 2024, 1736, 465379. https://doi.org/10.1016/j.chroma.2024.465379.

(6)Gritti, F. Ultra-High Pressure Slalom Chromatography: Application to the Characterization of Large DNA and RNA Samples Relevant in Cell and Gene Therapy. J. Chromatogr. A 2024, 1738, 465487. https://doi.org/10.1016/j.chroma.2024.465487.

(7) Gritti, F. Retention Mechanism in Slalom Chromatography: Perspectives on the Characterization of Large DNA and RNA Biopolymers in Cell and Gene Therapy. J. Chromatogr. A 2025, 1743, 465691. https://doi.org/10.1016/j.chroma.2025.465691.

Biography
Fabrice G. Gritti received a Ph.D. in Chemistry and Physics of Condensed Matter from the University of Bordeaux I (France) in 2001. He then worked as a research scientist at the University of Tennessee (Knoxville, TN) from 2002 to 2014 in the research group of the late Prof. Georges Guiochon. He joined Waters Corporation in 2015 where he is currently a consulting scientist.

Dr. Gritti’s main research interests involve liquid/solid adsorption thermodynamics and mass transfer in heterogeneous media used in the field of separation science. During the last 25 years, he has provided fundamental insights in preparative liquid chromatography and on the retention mechanisms in liquid chromatography (LC), refined the detailed theory of band broadening in modern LC columns, and contributed to improve column and instrument technologies in both LC and supercritical fluid chromatography for various relevant applications.

Dr. Gritti has been invited to give about thirty seminars on diverse topics of chromatographic sciences worldwide. He has delivered over a hundred invited keynote lectures and published over three hundred peer-reviewed articles. Dr. Gritti was the recipient of the 2013 Chromatographic Society Jubilee Medal, the 2019 JFK Huber Lecture Award, the 2022 Eastern Analytical Symposium Award for Outstanding Achievements in Separation Science, the 2023 Csaba Horvath Memorial Award for propagation of separation sciences throughout the world and co-operation in the development of chromatography in Hungary, and the 2024 A.J.P. Martin Medal from the UK-based Chromatographic Society.