CPC vs Legacy Chromatography
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Chromatography is a process that was designed and developed in 1903 by Russian botanist Mikhail Tswett to separate components of a mixture. After 120 years and several technological milestones later, chromatography is used to test and analyze various compounds and contaminants in foods, beverages, and even antibody analysis for vaccine creation. The separation occurs as the material is moved by the mobile phase through a column filled by the stationary phase.
Traditional chromatography uses a stationary phase often with silica gel that creates many challenges especially in the areas of performance, consistency, waste management and scaling. Inappropriate packing of the column results in “air pockets” impacting overall performance. Even a properly packed column’s efficiency decreases run after run as the stationary phase adsorbs particles. When a column becomes saturated, it no longer provides the required retention. At this point, the stationary phase must either be regenerated and cleaned in the best-case scenario, or, in the worst case, be safely disposed of without creating environmental hazards. These challenges make it very difficult to scale legacy columns. If the diameter and/or the length of the column are increased there can be more air pockets. A very tall column also requires a tall building. Industrial-scale separation and purification can require a column that is so large it would not be feasible.
In contrast, centrifugal partition chromatography (CPC) as it is commonly referred to, produces consistent results as it can utilize a non-degrading liquid stationary phase, which fills out the column perfectly. CPC is a liquid-liquid separation technique that scientists started utilizing in laboratories more than 40 years ago. It is the next stage in the evolution of chromatography as it is highly adaptable and easily scalable.
In CPC, the chromatography “column” consists of cells interconnected by metal tubing arranged in a series on a rotor. CPC column capacity can be scaled by increasing the number and size of the cells. CPC offers high precision as every run is performed with a refreshed stationary phase, adaptability as the solvent system can be easily switched, and it is cost effective as both stationary and mobile solvents can be reused extensively.
In CPC, separation occurs between two immiscible liquid phases. The stationary phase is immobilized inside the rotor by a strong centrifugal force. The mobile phase, containing the sample to be purified, is fed under pressure into the rotor and pumped through the stationary phase in the form of tiny droplets, or percolation. This is a simple and reproducible mechanism that leverages different compound partition coefficients.
Given the wide range of benefits and increasing industry demand, more and more leading universities are incorporating the science of CPC into their programs. The CPC chromatogram provides a great starting point to understand the chemistry as it demonstrates the separation of compounds of interest based on their unique partition coefficients achieved through a centrifugal partition chromatography system. The partition coefficient is the ratio of concentrations of a compound in a mixture of two immiscible solvents at equilibrium. This ratio is therefore a comparison of the solubilities of the solute in these two liquid phases.
CPC is a true alternative to conventional chromatography with many advantages, such as:
- Since CPC does not have a traditional solid stationary phase, total recovery can be expected as there is no irreversible adsorption on the stationary phase.
- Since CPC only utilizes solvents, the theoretical combinations are almost infinite, which provides a lot of options to fine tune a system for the individual separation task.
- It is quite versatile and lends itself well to experimenting with the chemistry of the process. The system has an extremely broad polarity range.
- The liquid-liquid separation allows constant separation performance, as refreshed solvents are used in each run.
- The CPC method also has incredible flexibility. The type of input material can be also changed from batch to batch. Customers can easily change their compound of interest if the volatility of the market calls for it.
- High load-ability with an outstanding stationary phase ratio.
- The CPC technology is also considered to be forgiving. Suppose that the system was not set up properly. The financial impact caused would be negligible.
- Solvents used in CPC can be recycled and reused, an important and sustainable effort.
- Scalability is another advantage that could refer to the method itself, meaning that once the system is working on a lab scale, it could be easily adapted almost seamlessly for large-scale applications. Scalability could also refer to the throughput.
CPC comes with some advantages but also requires a different thought process during the purification step. CPC is not something new from the theory side, however having an instrument that is truly operating at an industrial scale is new as the design and creation of such an instrument came with several technical challenges that had to be resolved.
Some scientists thought that CPC could only be achieved at the laboratory scale. The inventors at RotaChrom Technologies wanted to prove that it could be feasible on a larger scale and started designing a new system 15 years ago. RotaChrom has managed to develop a complete industrial-scale CPC solution with integrated solvent recovery. Unlike traditional, legacy chromatography, where a stationary phase and their properties (available surface, pore size, etc.) define the retention and separation performance mainly, CPC uses only liquid phases. This means that one solvent is immobilized via gravitational force and the other mobile phase is forced through in the form of tiny droplets. As a result of this main difference, the mass transfer and the separation are different, so it is not surprising that method development and capabilities to solve a separation issue have to be different as well.
While traditional chromatography increases the resolution by pushing the properties of the solid stationary phase, such as particle size, to obtain a higher number of plates, CPC utilizes selectivity to obtain the necessary peak to peak separation.
CPC has the advantage over traditional chromatography to compensate for a lower plate number, often with extreme selectivity numbers. Furthermore, CPC is also not as susceptible to samples with significant content of ballast materials, such as fermentation broth or chlorophyll, and can be loaded with much higher volume and concentration. This, and the fact that the stationary phase is refreshed between every run, allows for CPC to operate not only with fine distillates but also with heavier crude input materials. This does not deprecate the purification performance over time, which does occur in traditional chromatography.
With CPC, there is also an almost infinite combination of solvents. The challenge is to find the perfect combination that enables enough selectivity for the given separation problem. Apart from the fact that this creates an extremely flexible array of tools for the CPC separation, the method development results in a very cost-effective setup, where the final method on the machine utilizes the very same solvent system.
The infinite combination of solvents also provides a high flexibility, so changing between product lines or compounds of interest is easy. The same instrument can be used for targeted and non-targeted purification problems, as opposed to a single and well-defined compound.
Ownership cost structure and comparing costs
When selecting a purification technology, it is highly recommended to evaluate the total cost of ownership together with other considerations. Here are a few key points:
One of the most important considerations is the purification objective and the load capacity, as this will basically define the level of investment that will be required to address the task ahead. Finding a purification solution on an industrial scale is not the simplest task, especially in an industry that is undergoing many changes and the target keeps moving.
The second most important factor is consumables, including energy, solvents, and stationary phase costs. Legacy technologies utilizing solid-stationary phases, mainly C18 Silica, have a larger cost burden due to either disposal fees or regeneration/cleaning fees. In all-liquid chromatography technologies, the mobile phase is a solvent, which can be extensively reused with solvent recovery equipment.
Andras Gaspar, PhD, is RotaChrom’s chief product officer. He studied bioengineering at the Budapest University of Technology and Economics and went on to earn his PhD in chemistry at the Technical University of Munich. After more than a decade of engineering work, he joined RotaChrom Technologies in 2020.