In the first installment of our two-part conversation, Martha Knight, Executive Director at CCBiotech, USA, reflected on the origins of countercurrent chromatography (CCC), her early experiences with the technique, and why CCC has remained both essential and underappreciated within separation science.

Here, she looks ahead – describing the method’s distinctive strengths, paying tribute to Yoichiro Ito’s lasting influence, and exploring where countercurrent chromatography may find its most promising future applications, from proteomics and bioprocessing to environmental challenges.

For researchers who may not have hands-on experience with CCC, what do you see as its most important advantages or distinguishing strengths?

Countercurrent chromatography stands apart as a technique capable of separating virtually any type of molecule. By designing appropriate two-phase solvent systems, chromatographers can solubilize and separate a wide range of targeted components. CCC is based on partition chromatography, and the instruments developed by Yoichiro Ito beginning in the 1980s have endured because of their continued usefulness in both natural products and synthetic chemistry.

The analytical instrumentation industry offers platforms for reverse-phase, normal-phase, and supercritical fluid chromatography, but no single instrument can match the breadth of separations achievable with CCC. In many less-resourced regions, chemists continue to rely on CCC instruments and methods that have been in use for decades, not because the technique is old, but because it remains effective and adaptable. While some in the scientific community may view CCC as dated, its persistence reflects its practical value.

Looking ahead, artificial intelligence could play a role in synthesizing the extensive CCC literature to suggest effective chromatographic conditions. This would influence not only separations but also extraction strategies across applications ranging from chemical synthesis and natural products research to biotechnology, including the isolation of both small molecules and larger biomolecules.

Your recent paper (1) pays tribute to Yoichiro Ito. What do you consider his most significant contributions to the development of CCC?

Ito’s most significant contribution was the discovery of the flow-through planetary centrifuge early in his career. His original goal was to improve the separation of blood proteins beyond what could be achieved in a standard clinical centrifuge tube. He accomplished this by centrifuging a salt solution through a long coiled tube, where proteins migrated to positions corresponding to their density, with lighter components moving toward what he later defined as the “head” of the coil.

In the process, Ito carefully studied the screw direction of the coil – an Archimedean screw–like mechanism – and the directional movement of mass and density in rotating two-phase liquids. This led to the discovery of the “head” and “tail” of a rotating coil, and subsequently to the observation of partitioning between the light and heavy phases of a two-phase solvent system. His second major breakthrough was making the system flow-through by holding the inlet and outlet tubing stationary while the coil itself rotated. This was the defining innovation of the flow-through coil planetary centrifuge. Although the earliest design used planetary motion, the same principle of stationary inflow and outflow was later applied to co-axial rotating coils. Everything that followed – coiled tubing, rotating grooved plates, and other configurations – stemmed from this core concept of retaining one phase while equilibrating the other through it (2,3).

In 1985, Ito introduced a major design improvement by replacing the single-layer Teflon tubing wound on a rod with a compact, multilayer spool. This increased column volume and allowed higher rotational speeds, giving rise to high-speed CCC. These instruments proved commercially successful and were manufactured by small companies worldwide (4).

As CCC applications expanded to new classes of molecules, it became clear that some solvent systems – particularly those used for peptides and proteins – were not well retained under high-speed conditions. In my own work, I found that solvent systems containing heavier alcohols, such as n-butanol, were especially problematic. In response, Ito developed the spiral disk rotor, consisting of stacked disks with spiral grooves and narrow channels between them. Later designs incorporated multiple interwoven spirals per disk, increasing the pitch of the flow channels and significantly improving stationary-phase retention for low–interfacial tension solvent systems.

Systematic studies showed that organic-aqueous solvent pairs generally have higher interfacial tension and separate rapidly after mixing, whereas butanol-based and aqueous polymer phase systems separate more slowly. Ito addressed this by further adapting rotor and coil designs, ultimately developing the spiral tubing support rotor. This design used deep and radial channels to press Teflon tubing into multiple loops per layer, achieving solvent handling characteristics similar to the spiral disk rotor (5).

Together, these developments defined effective elution modes for all major solvent system classes – organic-aqueous, butanol-aqueous, and aqueous-aqueous polymer phases – making CCC suitable for separating small molecules as well as biomolecules such as proteins, polysaccharides, and polynucleotides. In that sense, Ito’s long-term goal of developing a countercurrent chromatography system capable of handling virtually all molecular classes was ultimately realized.

Beyond the technical innovations, what aspects of Ito’s scientific mindset or engineering philosophy do you think the community should remember?

Having known Dr. Ito through most of his years at the NIH, I saw firsthand how he developed his research from the original discovery of the planetary centrifuge and remained remarkably focused over time, while still branching into applications that were largely fruitful. He valued being part of a large research institution and made extensive use of the NIH machine shop (Mechanical Instrumentation Design and Fabrication Section, Scientific Instrumentation and Equipment Branch, NIH, Bethesda, MD). Uniquely, he was an inventor with a strong spatial intuition, and it remains impressive how much mechanical innovation he achieved without formal engineering training. Throughout, he held a quiet but firm belief that countercurrent chromatography would eventually grow and find its place.

Over the past year, after publishing a review article on Ito’s work, I found myself newly inspired when watching Ken Burns’ film on Leonardo da Vinci (6), based on Walter Isaacson’s biography (7). Da Vinci was both a master artist and a mechanical thinker, deeply engaged with the technologies of his time – designing machines, studying natural phenomena, and recording his ideas in notebooks written in mirror script. These Codices, discovered centuries later, reveal an extraordinary range of observations about mechanics and the natural world (8).

What struck me most was da Vinci’s relentless effort to create useful machines. His notebooks are filled with studies of water – its movement, power, and potential – as well as drawings of cranks, pulleys, gears, and circular geometries. Among them are depictions of the Archimedean screw, still used today for irrigation, and acknowledged by Ito as an influence on the horizontal flow-through centrifuge. Even more striking are sketches of circular channels and bucket-like forms that closely resemble Ito’s final invention, the spiral tubing support rotor with its concentric flow paths. The cross-section of the tubing inserter itself echoes some of da Vinci’s mechanical drawings.

In this sense, Ito realized a very da Vinci-like goal: creating practical, elegant mechanical devices that harness the behavior of fluids. For me, that historical connection captures both the spirit and the lasting significance of his work.

Looking ahead, where do you see the most promising opportunities/applications for CCC?

Countercurrent chromatography has potential at all scales – analytical, laboratory, and industrial. At the analytical end, specific rotor designs could be miniaturized for small-molecule separations and interfaced directly with mass spectrometry. In the life sciences, there is still an opportunity to develop compact spiral tubing support or spiral disk rotors capable of separating membrane proteins for proteomic analysis.

There are also significant opportunities for pharmaceutical and biotechnology equipment manufacturers to adapt Ito’s inventions for continuous processing. Rather than relying on batch centrifugation, products could flow directly from large cultures or containers into moving coils or rotors for isolation. For example, “foam” chromatography can concentrate or isolate a product toward the head of the system, opposite to the direction of flow. The spiral tubing support rotor, in particular, could be adapted for single-use applications in biotechnology. With 3D-printed rotors filled with fresh silicone-grade tubing, either the tubing alone or the entire rotor could be discarded after use, offering a potentially cost-effective industrial solution.

As mentioned earlier, a well-made laboratory-scale CCC instrument would also serve the research market, supporting discovery across many fields. This remains a central focus of our current work.

Although I named the company CC Biotech LLC to develop CCC for life science applications, it has become increasingly clear that some of the most compelling opportunities lie in addressing environmental challenges. Nearly 30 years ago, Ito demonstrated the separation of rare earth elements – metals whose molecular weights differ by only one unit (9). Today, these elements are critical to modern technologies, from electronics and displays to batteries for electric vehicles, yet they are largely imported and difficult to recycle.

With further development of industrial-scale CCC – using advanced spiral tubing support rotors or modified flow-through coils – there is real potential to recover rare earth elements and other critical materials from electronic waste, mining byproducts, or coal-derived residues. The energy sector is already exploring strategies such as cultivating plants or algae to concentrate metals from these sources, followed by extraction. CCC could play a key role in isolating those metals from complex extracts. While emerging technologies such as metal-organic frameworks (MOFs) may compete in this space, CCC offers a comparatively simple and cost-effective alternative.

In that context, perhaps the company name does not need to change after all.

If you were to imagine the next generation of CCC technology, what new capabilities or improvements would you most like to see?

It would be valuable to see greater engagement from industrial engineering companies – particularly those with experience in mining or rotary systems – in the further development of countercurrent chromatography. One key advance would be maintaining efficient phase equilibration at higher flow rates, effectively making CCC faster and more compatible with modern processing demands.

Adapting newer spectroscopic techniques for in-line detection would also be highly desirable. The spiral tubing support assembly, in particular, lends itself well to the incorporation of sensors and detectors for real-time monitoring and control. There is also strong potential to miniaturize CCC systems specialized for protein separations, enabling applications in proteomics.

Beyond discovery-scale work, CCC instrumentation could be developed for quality assurance, quality control, or process analytical technology (PAT) in biopharmaceutical manufacturing. More broadly, countercurrent chromatography could be integrated directly into active pharmaceutical ingredient (API) purification workflows, expanding its role in continuous and hybrid processing systems.

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