Innovative column technology for liquid chromatographyThomas Pfeiffer, Ph.D., AlphaCrom OHG, Karlstrasse 38, 89129 Langenau/Germany
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Fast method development and upscaling shown by the example of a root extrakt.
Rotor volume 200 ml, flow rate: 10 ml/min, detection UV 254 nm, solvent system: hexane-ethylacetate-methanol-water
| Fig. 1||Fig. 2 |
|Fig. 3||Fig. 4|
The analytical HPLC chromatogram (Fig.1) shows the normal complexity of such a root extract. The original content of 6% was enriched to 25% by one extraction step. The enriched extract still contents many impurities (Fig.2). By selection of an appropriate solvent system 5 g of the filtered raw extract will be injected. The target compound (peak 3 in Fig. 3) is quantitatively purified. The HPLC chromatogram of the target fraction demonstrates this impressively (Fig. 4). This fast chromatography can only be realized without a solid phase to avoid matrix effects of the sample. This method has been upscaled to production scale successfully.
Separation of 8 peptides with pH-zone-refining
Solvent System: Methyl-tert.-butyl-ether/acetonitril/water, rotor volume: 200 mL, stationary phase: organic + 44 mM TFA, mobile phase: aqueous + 15 mM NH3, detection: 254 nm, Sample injection: 800 mg (100 mg of each peptide) in 20 mL of stationary phase
| Fig. 5|
The method of pH-zone-refining is perfectly tailored for the separation of polar compounds. Because of the exchange of mobile phase with stationary phase over the time of the run, a pH change takes place over the entire length of the rotor. This pH-gradient enhances the separation performance by better sample solubility and selectivity. There will be no irreversible adsorption leading to a 100% sample recovery.
Aqueous phase system for the separation of proteins
Rotor volume: 200 ml, flow rate: 5 ml/min, 20 mg per proteine, Phase 1: 12.5% PEG8000-25% in distilled water. Phase 2: K2HPO4 + KH2PO4 (pH 6.5) in distilled water.
| Fig. 6|
The CPC technology is ideal for separating macromolecules and/or proteins. The PEG/salt (phosphat) buffer shows excellent results regarding resolution of compounds in an aqueous 2-phase system. Low differences of the biomolecules in polarity and the especially designed system of rotor ducts lead to highly efficient separations.
Chiral separation with and w/o chiral selector
Separation of four DNB-amino acid racemates with N-dodecanoyl-L-proline-3,5-dimethyl-anilin as chiral selector. Solvent system: hexane-ethylacetate-methanol-10 mM HCl.
| Fig. 7|
History and CPC technology
Liquid 2-phase systems have always been a well known technology in chemistry and pharmacy for the separation of a compound mixture. The first devices for continuous separation were built according to the technology developed by Dr. Craig (Fig.8) and can still be found today in some industrial companies. Technical realization for the laboratory took place when Prof. Ito, USA, invented a device for continuous partition chromatography. In this technology a teflon tube, winded around a central axis to form a coil, was rotating in a planetary motion. Companies like PharmaTec, USA, Prof. Sutherland in England as well as a manufacturer from China, guided this technology into industrial application. But it took until the end of last century to build a device for the durable use in the industry. It began when the manufacturing of pressure stable rotors made out of stainless steel were invented by the French company KromatonTechnologies. The first device has been the FCPC-Fast Centrifugal Partition Chromatograph (Fig.9). In it's rotor (Fig.10 ) are more then 1000 little ducts, interconnected in series by small channels.The rotor consists of many round metal discs which are sealed from each other by thin teflon sheets. This technology is focussed on separations of complex mixtures of compounds from milligram to kilogram.
In the rotor ducts the liquid stationary phase will be penetrated by the liquid mobile phase. The separation of the two liquid phases in the ducts takes place by centrifugal forces. After equilibration of the two liquid phases, the sample can be injected into the rotor. Now the separation process takes place in the two non immiscible phases by partition of several compounds of the sample according to their partition coefficient KD. The compounds move with different velocity to the end of the rotor and will be fractionized.
|Fig. 9||Fig. 10|
A general system set up is built from a HPLC pump, sample injection, CPC rotor as the column, detector and fraction collector.
Because of the achieved technical perfection of the CPC technology it enables for a general industrial use. In comparison to the solid phase HPLC columns CPC is not limited by the miscibility of the sample and allows to run much larger sample amounts with same flow rates. The possibility to fractionize the liquid mobile and the liquid stationary phase leads to a 100% sample recovery. These abilities enable the operator to separate very different samples fast, easy and cost saving. The CPC technology in combination with HPLC devices speeds up the complete chromatographic process up to the purified product while the upscaling procedure from analytical to production scale is linear because the separational results are the same, independent of the rotor volume size and the direct correlation of sample amount and rotor volume. Due to the missing solid phase there is no need to upscale the flowrates. The total costs for prep separations in comparison to solid phase HPLC can be reduced up to a factor of 10.
1. A.Berthod, in Comprehensive Analytical Chemistry, Volume 28, Countercurrent Chromatography, Elsevier Science , 2002
2. J.-M. Menet and Didier Thiébaut, Countercurrent Chromatography, Chromatographic Science Series Volume 82, Marcel Dekker, New York, Basel, 1999
3. Y.Ma, Y.Ito and A. Foucault, J. Chromatogr. A, 704 (1995) 75
4. I.A.Sutherland, L.Brown, S. Forbes, G. Games, D. Hawes, K.Hostettmann, E.H. McKerrell, A. Marston, D.Wheatley and P.Wood, J. Liq. Chromatogr. Relat. Technol. 21(3):279 (1998)
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