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Centrifugal Partition Chromatography (CPC)

 

Our laboratory is outfitted with several Kromaton CPC units (Russell Robatell, France). The flagship R&D instrument, model FCPC A through its proprietary rotor design offers best in class loading capacity, stationary phase retention, and improved resolution, which results with high throughput operation.

What is CPC?

Centrifugal Partition Chromatography (CPC) is a technique based on general principles of liquid-liquid partitioning, and is often referred to as countercurrent chromatography (CCC). In CPC, the chromatographic separation occurs as a “mobile phase” elutes the sample through a “stationary phase” that is immobilized in the CPC’s rotor by centrifugal force. The mobile and stationary phases relate to the two phases of bi-phasic solvent systems.

The separation potential of the CPC is a result of the rotor design. The rotor comprises hundreds of microcells connected in series. These microcells emulate liquid-liquid partitioning similar to that performed in separatory funnels, however repeated hundreds of times through the flow path, extrapolating the otherwise small variance in partition coeffecients into substantial separations.

The development of suitable solvent systems is the most involved and critical element in CPC. The solvent systems are composed of complex mixtures of solvents which will result in the production of two phases, a hydrophobic and a hydrophilic phase. The solvents and ratios are determined by a combination of experience and experimentation to produce favorable solubility and partition coeffients for the components of the sample. The optimal range of partition coefficients is between 0.2 and 5. Partition coefficients that are too high will result in early elution of the components and overlapping, while partitioning coefficients that are too low will lead to components being retained excessively long. By manipulating the ratios of the solvents, the partition coefficients can be altered, not only effecting the elution but the selectivity of the components.

The CPC mode described is only one of many, and already offers such a wide selectivity. CPC contains many other modes of operation, offering a breadth of approaches that can lead to very interesting and useful selectivities. This versatility makes CPC one of the most powerful instruments in separation sciences.

CPC Flow Diagram for Ascending Phase Operation

What is the significance of CPC for your projects?

Solid phases based on silica dominate the current chromatographic market, and although these columns are invaluable in HPLC analysis there are many issues in applying them to a preparative scale. One of the major detractors is that the free silanols found in silica-based solid phases contribute to the degradation of labile components, reducing yields and destroying priceless material. Additionally, to find good separation, very often many expensive preparative columns must be tested. Alternately, CPC relies on a liquid stationary phases that providing the most gentle environment, protecting labile components. Furthermore, as the system relies on bi-phasic CPC systems, the selectivity is highly customizable through manipulation of the composition of the phases. The CPC can be easily switched between normal and reverse phase modes. Perhaps the main factor responsible for the preparative advantage of the CPC is the large volume of stationary phase, which is thousands times larger than the volume of stationary phase in reverse phase operations. This allows dissolution of larger quantities of poorly soluble substances, which often is prohibitive in RP HPLC. In our hands the CPC purification often produces higher purity than preparative HPLC.

Other benefits of CPC

Direct Method Scale-Up

A CPC method designed on an analytical scale CPC can be linearly scaled to 10-liter pilot-scale CPC without a loss of resolution. Additionally, as the process is scaled up the sample loading capacity may increase by a factor higher than the increase of the rotor volume.

Large Loading Capacity

CPC offers a higher loading capacity than many solid phases. A unit with a 1-liter rotor can handle up to tens of grams per run.

Flexibility Through non-Conventional Methods

In addition to optimization of selectivity through manipulation of solvent system, the CPC can be used in other known modes of operation, such as pH-Zone refining, which relies on the pKa of acids and bases to separate the components. At Planta Analytica we have discovered novel, proprietary modes that provide outstanding benefits.

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