Peter Hewitson

Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal medicine

This paper describes intermittent counter-current extraction, a novel method of using a conventional twin column counter-current chromatograph to either split a sample into two groups of compounds or extract and enrich a target compound from a crude extract. The first method is demonstrated by splitting a model mixture of four compounds into two groups. The second method is demonstrated by the extraction and enrichment of a high value target compound, triptolide, from a Chinese herbal medicine crude extract of Tripterygium wilfordii Hook. f., where it is found at low concentration (2%). This was achieved by retaining and enriching the target compound within the column while washing away all other components of the crude material. The success of the first method allowed the second method to be carried out without the need for costly preliminary experiments with the high value sample. 188mg of triptolide at greater than 98% purity was separated from 9.2g of crude extract, using 10l of solvent in a 3-h separation.

New 18-l process-scale counter-current chromatography centrifuge

A new Dynamic Extractions Maxi-counter-current chromatography (CCC) centrifuge with a column volume of 18-l has been installed in the Advanced Bioprocessing Centre at Brunel. This instrument has four times the capacity of the 4.6-l Maxi-CCC centrifuge which has been operating robustly for 3 years. Tests using the model sample system benzyl alcohol and p-cresol with a heptane:ethyl acetate:methanol:water (HEMWat) phase system (1.4:0.1:0.5:1.0) show that resolution is almost double with this new high capacity device. Commissioning tests with a mixture of caffeine, K(D)=0.21; ferulic acid, K(D)=0.82; umbelliferone, K(D)=1.2 and vanillin, K(D)=1.49 using a HEMWat phase system of 1:1.5:1:1.5 on the 9-l column show that resolutions equivalent to analytical instruments will be possible using the full 18-l capacity. They also show that predictable scale-up from simple test tube tests is feasible with knowledge of the stationary phase retention for the planned process scale run.

Scale-up of counter-current chromatography: Demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale

Predictable scale-up from test tube derived distribution ratios and analytical-scale sample loading optimisation is demonstrated using a model sample system of benzyl alcohol and p-cresol in a heptane:ethyl acetate:methanol:water phase system with the new 18 L Maxi counter-current chromatography centrifuge. The versatility of having a liquid stationary phase with its high loading capacity and flexible operating modes is demonstrated at two different scales by separating and concentrating target compounds using a mixture of caffeine, vanillin, naringenin and carvone using a quasi-continuous technique called intermittent counter-current extraction.

Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide

Reversed phase HPLC (RP-HPLC) and high performance countercurrent chromatography (HPCCC) were compared for the pilot scale purification of two semi-synthetic spinosyns, spinetoram-J and spinetoram-L, the major components of the commercial insecticide spinetoram. Two, independently performed, 1 kg, purification campaigns were compared. Each method resulted in the isolation of both components at a purity of >97% and yields for spinetoram-J and spinetoram-L of >93% and ≥ 63% of theoretical, respectively. The HPCCC process produced a 2-fold higher throughput and consumed approximately 70% less solvent than preparative scale RP-HPLC, the volume of product containing fractions from HPCCC amounted to 7% of that produced by HPLC and so required much less post-run processing.

Evaluation of dual flow counter-current chromatography and intermittent counter-current extraction.

The aim of this research is to compare two continuous extraction technologies, intermittent counter-current extraction (ICcE) and dual flow counter-current chromatography (DFCCC), in terms of loading and throughput using the GUESSmix, and show the advantages and disadvantages of the two methods. A model sample containing caffeine, vanillin, naringenin and carvone, with a total load of 11.2 g, was employed with a hexane-ethyl acetate-methanol-water (2:3:2:3) phase system to evaluate an ICcE method on a preparative (912 ml coil volume) DE-Midi instrument. While DFCCC was carried out on a specially designed preparative (561 ml coil volume) bobbin installed in a similar Midi instrument case. While similar throughputs of 7.8 g/h and 6.9 g/h were achieved for the ICcE and DFCCC methods respectively, ICcE was demonstrated to have a number of advantages over DFCCC.

Intermittent counter-current extraction—Effect of the key operating parameters on selectivity and throughput

Intermittent counter-current extraction (ICcE) has proved itself as a method for splitting compounds into streams and/or concentrating compounds in the column. In this paper a model mixture sample based on a modified GUESSmix (containing salicin, caffeine, aspirin, coumarin, salicylic acid, carvone, ionone and biphenyl) was separated into two eluant streams across a range of HEMWat phase system polarities from the polar system 11 through to non-polar system 23. ICcE could provide throughput of over 1 kg/day with this model sample, at the preparative scale, Changing the time cycle to adjust where the sample mixture is split into two streams was demonstrated. It is established that for the continuous running of ICcE, on a conventional twin bobbin counter-current chromatograph instrument, it is necessary to adjust the dead volumes of the flying leads to maintain similar phase retention in each column so the instrument does not become hydrodynamically and mechanically unbalanced due to the difference in densities between the upper and lower phases.

Scale-up of protein purifications using aqueous two-phase systems: Comparing multilayer toroidal coil chromatography with centrifugal partition chromatography

Two different laboratory scale liquid-liquid extraction processes using aqueous two-phase systems (ATPS) are compared: centrifugal partition chromatography (CPC) and multilayer toroidal coil chromatography (MTCC). Both use the same phase system, 12.5% (w/w) PEG-1000:12.5% (w/w) K(2)HPO(4), the same flow rate of 10 mL/min and a similar mean acceleration field of between 220 × g and 240 × g. The main performance difference between the two processes is that there is a continuous loss of stationary phase with CPC, while for MTCC there is not - even when sample loading is increased. Comparable separation efficiency is demonstrated using a mixture of lysozyme and myoglobin. A throughput of 0.14 g/h is possible with CPC despite having to refill the system with stationary phase before each injection. A higher throughput of 0.67 g/h is demonstrated with MTCC mainly due to its ability to tolerate serial sample injections which significantly reduces its cycle time. While CPC has already demonstrated that it can be scaled to pilot scale, MTCC has still to achieve this goal.

Steady-state and non-steady state operation of counter-current chromatography devices☆

Different variants of separation processes based on steady-state (continuous sample loading) and non-steady state (batch) operating modes of CCC columns have been analyzed and compared. The analysis is carried out on the basis of the modified equilibrium cell model, which takes into account both mechanisms of band broadening - interphase mass transfer and axial mixing. A full theoretical treatment of the intermittent counter-current chromatography with short sample loading time is performed. Analytical expressions are presented allowing the simulation of the intermittent counter-current chromatography separations for various experimental conditions. Chromatographic and extraction separations have been compared and advantages and disadvantages of the two methods have been evaluated. Further technical development of the CCC machines to implement counter-current extraction separations is considered.

Centrifugal partition chromatography in a biorefinery context: separation of monosaccharides from hydrolysed sugar beet pulp.

A critical step in the bioprocessing of sustainable biomass feedstocks, such as sugar beet pulp (SBP), is the isolation of the component sugars from the hydrolysed polysaccharides. This facilitates their subsequent conversion into higher value chemicals and pharmaceutical intermediates. Separation methodologies such as centrifugal partition chromatography (CPC) offer an alternative to traditional resin-based chromatographic techniques for multicomponent sugar separations. Highly polar two-phase systems containing ethanol and aqueous ammonium sulphate are examined here for the separation of monosaccharides present in hydrolysed SBP pectin: l-rhamnose, l-arabinose, d-galactose and d-galacturonic acid. Dimethyl sulfoxide (DMSO) was selected as an effective phase system modifier improving monosaccharide separation. The best phase system identified was ethanol:DMSO:aqueous ammonium sulphate (300gL(-1)) (0.8:0.1:1.8, v:v:v) which enabled separation of the SBP monosaccharides by CPC (200mL column) in ascending mode (upper phase as mobile phase) with a mobile phase flow rate of 8mLmin(-1). A mixture containing all four monosaccharides (1.08g total sugars) in the proportions found in hydrolysed SBP was separated into three main fractions; a pure l-rhamnose fraction (>90%), a mixed l-arabinose/d-galactose fraction and a pure d-galacturonic acid fraction (>90%). The separation took less than 2h demonstrating that CPC is a promising technique for the separation of these sugars with potential for application within an integrated, whole crop biorefinery.

Scalable Technology for the Extraction of Pharmaceutics (STEP): The transition from academic knowhow to industrial reality

This paper addresses the technological readiness of counter-current chromatography (CCC) instruments to become platform technology for the pharmaceutical industry. It charts the development of the prototype technology since its inception in 1966, through conceptual improvements in the 1980s that led to higher speed separations in hours as opposed to days. It then describes the engineering improvements that have led to the development of high performance counter-current chromatography with the potential for scale-up to process scale for manufacturing products in industry with separation times in minutes rather than hours. A new UK Technology Strategy Board high value manufacturing £1.5 m research programme to take CCC through to technology readiness level 8 (i.e. as platform technology for continuous 24 × 7 operation by industry) is introduced. Four case studies are given as examples of successes from its expanding applications portfolio, which is mainly confidential. Finally, the hurdles for the uptake of new technology by industry are highlighted and the following potential solutions given: rapid method development, automation, continuous processing and instrument reliability and robustness. The future challenge for the CCC community will be to address these development needs urgently if CCC is to become the platform technology it deserves to be.