A new non-synchronous preparative counter-current centrifuge—the next generation of dynamic extraction/chromatography devices with independent mixing and settling control,which offer a step change in efficiency

A new and significantly more robust design of non-synchronous coil planet centrifuge is introduced where the degree of mixing between two immiscible phases can be changed independently from the “g” field required to separate out the phases. A hypothesis that an optimum ratio between the speed of the bobbin and the speed of the rotor can be found to optimise the efficiency of the separation for a given force field is upheld for an intermediate polarity phase system. This paves the way for extensive further research to find the optimum non-synchronous conditions for a range of different phase systems that are desirable for the separation of large molecules, proteins and biologics but can tend to emulsify in the standard “J” type centrifuge systems currently available and routinely in use for aqueous organic phase systems. A step change of up to 30% in resolution and 90% in plate efficiency is demonstrated.     

Suspension Separation in a Centrifuge with Biconical Rotor

Separation of cellulose nitrate suspensions in a centrifuge with biconical rotor was studied. An empirical equation was derived, enabling calculation of the efficiency of purification of process water and wastewater to remove the solid phase in centrifuges of this kind.     

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.     

Rapid linear scale-up of a protein separation by centrifugal partition chromatography

The scaling up of the separation of two proteins with an aqueous two-phase system (ATPS) from 176 mg with a 500 ml laboratory scale centrifugal partition chromatography (CPC) column to 2.2g with a 6.25 litre pilot-scale column is presented. A model sample system of a mixture of lysozyme and myoglobin was chosen for this study using an ATPS system comprising 12.5% (w/w) PEG-1000:12.5% (w/w) K2HPO4. It was found that the maximum sample concentration possible without precipitation was 2.2mg/ml for each constituent. The optimisation of rotor speed, mobile phase flow rate and sample loading was performed on a laboratory-scale device. It was found that a centrifuge speed of 2000 rpm (224 'g'), 10 ml/min mobile phase flow rate with a 43 ml (10% of active column volume) sample volume gave optimum operating conditions. This was linearly scaled up to pilot scale by increasing mobile phase flow rate, fraction size and sample loading in the ratio of the system capacities (i.e. 12.5:1). Flow rate was therefore increased from 10 ml/min to 125 ml/min, fraction size from 10 ml to 125 ml and sample loading from 43 ml to 500 ml. Rotor speed however was reduced from 2000 rpm on the laboratory device to 1293 rpm on the pilot-scale device to maintain the same 224 'g' field in each chamber, as the pilot-scale CPC unit has a larger rotor radius than the laboratory one. Resolution increased from Rs=1.28 on the 500 ml rotor to Rs=1.88 on the 6.25 litre rotor, giving potential throughputs in batch mode of over 40 g/day.     

Compact type-I coil planet centrifuge for counter-current chromatography

A compact type-I coil planet centrifuge has been developed for performing counter-current chromatography. It has a revolution radius of 10 cm and a column holder height of 5 cm compared with 37 and 50 cm in the original prototype, respectively. The reduction in the revolution radius and column length permits application of higher revolution speed and more stable balancing of the rotor which leads us to learn more about its performance and the future potential of type-I coil planet centrifuge. The chromatographic performance of this apparatus was evaluated in terms of retention of the stationary phase (S_f), peak resolution (R_s), theoretical plate (N) and peak retention time (t_R). The results of the experiment indicated that increasing the revolution speed slightly improved both the retention of the stationary phase and the peak resolution while the separation time is remarkably shortened to yield an excellent peak resolution at a revolution speed of 800 rpm. With a 12 ml capacity coiled column, DNP-DL-glu, DNP-β-ala and DNP-l-ala were resolved at R_s of 2.75 and 2.16 within 90 min at a flow rate of 0.4 ml/min. We believe that the compact type-I coil planet centrifuge has a high analytical potential.     

In situ fabricated porous filters for microsystems

Porous filters were fabricated inside a microchannel using emulsion photo-polymerization. The porosity of the filter is dependent on the composition of the pre-polymer mixture. The morphology and flow resistance of four filters with varying water and cross-linker concentrations was studied. The porous filter was used to separate samples, such as whole blood to cell/serum components, based on size. The efficiency of cell/serum separation by the porous filter was tested by performing a glucose-6-phosphate dehydrogenase assay and was found to be comparable to separation by centrifuge. The porous filter thus mimics the functionality of the centrifuge, with the added benefits of no power requirements and ability to handle small sample volumes.     

Multi-stage mixer-settler planet centrifuge. Preliminary studies on partition of macromolecules with organic-aqueous and aqueous-aqueous two-phase solvent systems

A rotary-seal-free planetary centrifuge holds a separation column which consists of multiple partition units (ca. 200) connected in series with transfer tubes. In the cavity of each partition unit the transfer tube extends to form a mixer which vibrates to stir the contents under an oscillating force field generated by the planetary motion of the centrifuge. Consequently, solutes locally introduced at the inlet of the column are subjected to an efficient partition process in each partition unit and separated according to their partition coefficients. The mixer tube equipped with a flexible silicone rubber joint was found to produce excellent results for partition with viscous polymer phase systems. The capability of the method was demonstrated on separation of cytochrome c and lysozyme using a PEG-aqueous dibasic potassium phosphate-aqueous two-phase solvent system.     

Optimization of Solvent Mixture Compositions for High Speed Countercurrent Distribution

Abstract

Seven solvents considered appropriate for use with relatively sensitive biosynthetic products undergoing separation by multilayer coil planet centrifuge high speed countercurrent distribution (HSCCD) have been evaluated in a new and systematic procedure for optimal solvent selection. the new method was illustrated by a complete HSCCD separation of five closely related dipeptides.     

Feasibility of scaling from pilot to process scale

The pharmaceutical industry is looking for new technology that is easy to scale up from analytical to process scale and is cheap and reliable to operate. Large scale counter-current chromatography is an emerging technology that could provide this advance, but little was known about the key variables affecting scale-up. This paper investigates two such variables: the rotor radius and the tubing bore. The effect of rotor radius was studied using identical: length, beta-value, helix angle and tubing bore coils for rotors of different radii (50 mm, 110 mm and 300 mm). The effect of bore was researched using identical: length, helix angle and mean beta-value coils on the Maxi-DE centrifuge (R=300 mm). The rotor radius results show that there is very little difference in retention and resolution as rotor radius increases at constant bore. The tubing bore results show that good retention is maintained as bore increases and resolution only decrease slightly, but at the highest bore (17.5 mm) resolution can be maintained at very high flow rates making it possible for process scale centrifuges to be designed with throughputs exceeding 25 kg/day.     

Protein separation with aqueous-aqueous polymer systems by two types of counter-current chromatographs.

Two different types of counter-current chromatographs, the cross-axis coil planet centrifuge (X-axis CPC) and horizontal flow-through coil planet centrifuge (horizontal CPC), were evaluated for protein separation with an aqueous-aqueous two-phase polymer system. The sample solution, containing 10-200 mg each of cytochrome c, myoglobin, ovalbumin and hemoglobin in 2 ml of each phase was eluted with the lower phase. In both instruments, the effects of the flow-rate, revolution speed, and parameter beta (helical diameters of the multilayer coil) on the protein separation were investigated. The best results were obtained from the X-axis CPC operated at 750 rpm and a flow-rate of 2.0 ml/min using a multilayer coil with a small helical diameter (beta = 0.25-0.60). Four protein samples were well resolved in less than 5 h.     

New angle rotor coil planet centrifuge for counter-current chromatography. I. Analysis of acceleration

Simple mathematical analysis has been performed on a new type of synchronous planetary motion to elucidate the acceleration field. Using generalized formulae derived from the above analysis, distribution of the centrifugal force vectors produced by all types of the synchronous planetary motion is computed and expressed in the same format to facilitate comparative studies. The results of the above analysis predict high performance of the new angle rotor coil planet centrifuge in counter-current chromatography.     

New angle rotor coil planet centrifuge for counter-current chromatography. II. Design of the apparatus and studies on phase retention and partition capability

A new angle rotor coil planet centrifuge (psi = 25 degrees), which produces a Type J-L synchronous planetary motion, has been constructed to examine its capability in terms of stationary phase retention and solute partitioning. Studies on phase distribution diagrams obtained from various two-phase solvent systems indicated that the present system can be adapted to a wide variety of solvent systems by adjusting the centrifugal conditions. Excellent partition capability of the apparatus was successfully demonstrated in separations of dinitrophenyl amino acid samples with chloroform-acetic acid-0.1 N hydrochloric acid (2:2:1).     

Chemical processes in a plasma centrifuge

Investigations on separating hydrogen isotopes have been carried out in a system with plasma rotating in crossed E × B fields— a plasma centrifuge. The processing of chemical reactions in the plasma phase in a strong centrifugal field was simulated. High radial separation factors (up to 140) have been obtained for the decomposition of water vapor with simultaneous separation of hydrogen and oxygen. Because of spatial separation of the dissociation products there was no need for their special quenching.     

Countercurrent Chromatography Using a Toroidal Coil Planet-Centrifuge: A Comparative Study of the Separation of Organelles Using Aqueous Two-Phase Partition

Abstract

A toroidal coil planet centrifuge is described and compared with other countercurrent chromatography (CCC) and countercurrent distribution (CCD) techniques. The basis of separation is partition in aqueous two-phase polymer systems, with each method assessed by fractionating rat liver organelles. The size and ease of operation of the toroidal coil planet centrifuge gave significant advantages over conventional CCD systems achieving equivalent resolution in a fraction of the time.     

Counter-current chromatography separation scaled up from an analytical column to a production column

An analytical separation was performed on an analytical J-type counter-current chromatography (CCC) instrument using a 5.4 ml column, with a 1 ml/min mobile phase flow rate. This separation had a resolution of 0.69 and was achieved in 10 min. The same separation was performed using two 2300 ml columns connected in series at a flow rate of 850 ml/min using a production scale J-type centrifuge. This production scale separation was also obtained in 10 min with a resolution of 0.71. This represents an 850 times increase in productivity. This paper presents these separations and the underlying scale up theory.     

Chromatography of AC-ASP-TYR-MET-GLY-TRP-MET-ASP-NH2 on the Horizontal flow-Through Coil Planet Centrifuge and the High-Speed Multi-Layer Coil Planet Centrifuge

Abstract

The peptide Ac-Asp-Tyr-Met-Gly-Trp-Met-Asp-NH₂ was purified by countercurrent chromatography in the horizontal flow-through coil planet centrifuge. The solvent system used was ammonium acetate, pH 8.5 and n-butanol (1:1 by volume). The high pH served to maintain the peptide in solution. When the upper phase was utilized as the mobile phase better separation of the peptide from impurities resulted. The peptide was also chromatographed in a new apparatus, the high-speed multi-layer coil planet centrifuge. With the lower phase mobile and at a higher temperature, the peptide was fractionated very rapidly in 30 min compared to 7 hr on the other instrument.     

Large-scale separation of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by pH-zone-refining countercurrent chromatography

pH-Zone-refining countercurrent chromatography was successfully applied to the separation of salvianolic acid B from the Chinese medicinal plant, Salvia miltiorrhiza Bunge, using a multilayer coil planet centrifuge. A 2.0 g quantity of sample was separated using the following two-phase solvent system: methyl tert-butyl ether (MtBE)-water, 10 mM TFA in organic stationary phase and 10 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by HPLC and ESI-MS. The separation yielded 572 mg of the main component of salvianolic acid B with a purity of 94.1%.     

Automated cellular sample preparation using a Centrifuge-on-a-Chip

The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allows enrichment of cancer cells from spiked blood samples at the mL min(-1) scale, followed by fluorescent labeling of intra- and extra-cellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge-analogue may open opportunities in automated, low-cost and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource poor settings.     

Preparative isolation and purification of celastrol from Celastrus orbiculatus Thunb. by a new counter-current chromatography method with an upright coil planet centrifuge

A new counter-current chromatography (CCC) method with an upright coil planet centrifuge, which holds four identical multilayer coil columns in the symmetrical positions around the centrifuge axis, was applied to the isolation and purification of celastrol from the roots of Celastrus orbiculatus Thunb. The crude celastrol was obtained by elution with light petroleum from ethanol extracts using 15 cm x 5 cm i.d. silica gel flash chromatography. Preparative CCC with a two-phase system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v) was successfully performed, yielding 798 mg celastrol at 99.5% purity from 1020 mg of the crude sample in one step separation.     

Separation of apple procyanidins into different degrees of polymerization by high-speed counter-current chromatography

Apple procyanidins were fractionated by high-speed counter-current chromatography in a one-step operation from apple condensed tannins using a type-J multilayer coil planet centrifuge. The separation of procyanidins was performed with a two-phase solvent system composed of methyl acetate-water (1:1) by eluting the upper phase at a flow-rate of 1.0 ml/min. Each fraction was examined by time-of-flight mass spectrometry. Procyanidins were separated according to their degrees of polymerization.