Countercurrent chromatography (CCC) sits slightly outside the mainstream of separation science, yet its influence runs deep. Based on liquid-liquid partitioning rather than solid stationary phases, the technique has enabled separations that are difficult – or impossible – by other means.
In this two-part interview, Martha Knight, Executive Director at CCBiotech, USA, traces CCC’s evolution from its early days as countercurrent distribution to the modern planetary centrifuge systems pioneered by Yoichiro Ito. Here, she reflects on her first encounters with the technique at Rockefeller University, its role throughout her career in peptide synthesis and natural products research, and why CCC remains underappreciated despite its unique capabilities.
How did you first encounter countercurrent chromatography, and what drew you to the technique?
Shortly after graduating from college, I started in 1968 as a lab technician for John Morrow Stewart in Rockefeller University’s Biochemistry Department, working alongside R. Bruce Merrifield. It was an exciting time, when much of Merrifield’s revolutionary work on solid-phase peptide synthesis – later recognized with the 1984 Nobel Prize in Chemistry – was taking shape.
Busily humming in the lab was one of the first amino acid analyzers, invented by Sanford Moore and William Stein, who shared the 1972 Nobel Prize. Moore would often stop by: tall, white-haired (said to have once turned blue after a mishap with ninhydrin), and invariably dressed in a dark suit and tie while everyone else wore lab coats. For his distinguished demeanor, we referred to him as “the Parson.”
Moore’s work on peptide and protein structure relied on rigorous compositional analysis and high-quality separations, which made countercurrent distribution (CCD) a routine and essential tool in the same Rockefeller labs where these analytical advances were unfolding.
A technician worked full-time tending the amino acid analyzer, while my own duties included checking peptide purity and purifying products on the countercurrent distribution (CCD) instrument in the hallway. This was a large, glass-based system of 100 interconnected tubes that typically ran overnight to complete a single separation.
CCD had been invented in the late 1930s by Lyman Craig, a senior Rockefeller scientist, with instruments built by Otto Post from the institute’s workshop. Craig occasionally stopped by the lab; on one visit he quietly watched me setting up a run – and offered a half-smile.
I later moved with Dr. Stewart to the University of Colorado School of Medicine, where we established a peptide research lab and continued to rely on CCD, now scaled up to a 200-tube system, to purify cardiovascular peptides such as angiotensin and bradykinin analogs. I completed my MSc there before taking a fellowship that brought me to the University of Buenos Aires. The department, chaired by Alejandro Paladini – himself a former Craig postdoc – ran an even larger CCD system, which I used to purify newly discovered peptides such as thyrotropin-releasing hormone.
During my PhD work at the NIH, I was introduced to Yoichiro Ito’s compact coil planetary centrifuge countercurrent chromatograph. Compared with traditional CCD, this system was a revelation: small, efficient, and capable of true preparative separations. Ito readily helped purify my samples, allowing me to move forward with studies on enkephalin metabolism.
From that point on, countercurrent chromatography became central to my work. After completing my PhD, I adopted the new generation of CCC instruments at NIH, publishing both biological studies and separation methods. In truth, I hadn’t chosen CCD early on – it was simply the only preparative option available – but I was naturally drawn to Ito’s approach as countercurrent chromatography evolved into a far more versatile and powerful technique.
Over the course of your career, how has CCC figured into your work, and what kinds of separation challenges has it helped you to address?
In 1984, I founded Peptide Technologies Corp. to provide custom peptide synthesis, analytical chemistry services, and research supported by the US government’s SBIR program. The company launched before preparative HPLC was widely available, so purification relied on two CCC instruments of the horizontal flow-through coil design originally developed at the NIH by Yoichiro Ito.
As preparative reverse-phase HPLC matured, we eventually adopted it – using one-inch ID columns – because eluents could be monitored in real time and purified fractions lyophilized directly. However, CCC retained important advantages, particularly higher sample loading and the ability to handle compounds with low water solubility. This proved critical for challenging targets, including hydrophobic peptide fragments of the HIV gp120 coat protein for a National Institute of Allergy and Infectious Diseases vaccine program. Peptides that could not be recovered from C18 columns could often be produced in useful quantities by CCC, while acidic peptides were handled using tailored two-phase solvent systems containing basic components.
Through a series of grants, we applied CCC to a wide range of molecular classes. These included the isolation of water-soluble flavonoids from the African medicinal plant Sutherlandia frutescens, where closely eluting compounds in reverse-phase HPLC were cleanly resolved by optimizing two-phase solvent systems. We also used CCC to isolate additional natural products, such as sutherlandins and glycosylated derivatives, using alternative solvent combinations. In more exploratory work, we achieved the separation of chiral carbon nanotube species using polyethylene glycol-dextran aqueous systems originally developed for protein and biopolymer separations.
How would you describe the state of countercurrent chromatography now?
Countercurrent chromatography is generally more widely appreciated in Europe, Asia, and tropical regions than in the US, where adoption has been slower. Much of the current activity comes from entrepreneurial chemists and engineers applying the technique in the food and natural products sectors, with more recent growth in the cannabis industry.
Several companies now produce large-volume centrifugal partition chromatographs (CPCs) designed for higher solvent flow rates. These systems use co-axial, non-planetary rotating drums with peripheral separation cells and rotating inlet and outlet seals. Yoichiro Ito was involved in early CPC design efforts in Japan, but he viewed them as closer in principle to his early droplet CCC systems and argued that they lacked the mixing efficiency – and therefore the resolution – of continuous, planetary coil-based designs. Engineers, however, favored CPCs for their mechanical simplicity and scalability.
In China, countercurrent chromatography has developed along two parallel paths. Large-scale horizontal flow-through systems – descended from the earliest CCC designs – are used to process kilogram-scale samples at relatively modest flow rates. At the same time, compact high-speed CCC instruments have been produced for many years by established manufacturers in Shanghai, serving both academic and industrial research laboratories.
What recent developments are shaping the way CCC is applied in modern analytical and preparative settings?
Among large instrument companies, development of truly large-scale countercurrent chromatography systems remains limited, with most efforts focused on niche applications in natural-product-related industries. In contrast, much of the innovation in application development is happening in academia, where new solvent systems are being designed for both small and large molecules across the life sciences and nanotechnology.
Advances in solvent chemistry – including ionic liquids, deep eutectic solvents (DES), naturally occurring DES, and more sustainable, environmentally sourced components – are increasingly being incorporated into CCC workflows. Organic two-phase solvent systems continue to be used for poorly water-soluble organic and inorganic compounds, while polyethylene glycol (PEG)-based aqueous two-phase systems are well suited to large biomolecules.
At the same time, the growth of multi-angle light scattering (MALS) and other hybrid spectroscopic detection techniques is driving demand for complementary high-resolution separation methods for proteins and other large molecules. CCC can meet this need through PEG-salt aqueous two-phase systems, provided they are compatible with the detector. In addition, another Ito-developed technique – the centrifugal precipitation chromatograph (Rotify), which separates proteins based on their distinct precipitation points – offers potential for MALS-based analytical quality control. CCC instruments can therefore be interfaced with detection modules already offered by major instrument manufacturers, extending their relevance in modern analytical workflows.
Do you feel CCC receives the attention it deserves within separation science?
Countercurrent chromatography still does not receive the attention it deserves within separation science. The analytical instrumentation industry rarely promotes CCC or CPC at major conferences, and online visibility remains limited. There is, however, clear demand in the research community for well-engineered, laboratory-scale CCC instruments. When these become more widely available, adoption is likely to grow, and larger, diversified companies will take greater notice.
Some of the barriers are rooted in lingering misconceptions. Earlier CCC instruments were noisy, mechanically complex, and limited by rotor designs that struggled to maintain high stationary-phase retention across a broad range of solvent systems. Modern bench-top planetary centrifuges have addressed many of these issues, but further improvements – such as better temperature control for aqueous two-phase systems and more robust, quieter construction – would help broaden acceptance.
There is also a tendency to overestimate the mechanical demands of the technique. Planetary centrifugation does not require the extreme g-forces associated with ultracentrifugation for proteins or viruses. In fact, analytical-scale CCC studies have shown that relatively modest rotational speeds can achieve efficiencies comparable to HPLC. This opens the door to smaller, quieter instruments operating under fully non-denaturing conditions, particularly for protein separations.
Despite limited industrial promotion, CCC maintains a strong research presence. Since 2000, an international CCC community has met biennially across different continents – most recently at CCC2024 in Rio de Janeiro – with proceedings published as special issues of Journal of Chromatography A. More recently, the International CCC & CPC Network based at the University of Reims has launched online seminars to engage researchers at all career stages. While major analytical meetings still lack dedicated CCC sessions, interest remains strong in Europe and Asia, and CCC research continues to appear regularly across separation science journals.
Ultimately, CCC offers a compelling economic and technical proposition: operating costs are largely limited to solvents, with no columns to replace. As instrument design continues to improve and misconceptions fade, the technique is well positioned to gain broader recognition within the separation science community.
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