Ian A. Sutherland

Countercurrent chromatography in analytical chemistry (IUPAC Technical Report)

Countercurrent chromatography (CCC) is a generic term covering all forms of liquid-liquid chromatography that use a support-free liquid stationary phase held in place by a simple centrifugal or complex centrifugal force field. Biphasic liquid systems are used with one liquid phase being the stationary phase and the other being the mobile phase. Although initiated almost 30 years ago, CCC lacked reliable columns. This is changing now, and the newly designed centrifuges appearing on the market make excellent CCC columns. This review focuses on the advantages of a liquid stationary phase and addresses the chromatographic theory of CCC. The main difference with classical liquid chromatography (LC) is the variable volume of the stationary phase. There are mainly two different ways to obtain a liquid stationary phase using centrifugal forces, the hydrostatic way and the hydrodynamic way. These two kinds of CCC columns are described and compared. The reported applications of CCC in analytical chemistry and comparison with other separation and enrichment methods show that the technique can be successfully used in the analysis of plants and other natural products, for the separation of biochemicals and pharmaceuticals, for the separation of alkaloids from medical herbs, in food analysis, etc. On the basis of the studies of the last two decades, recommendations are also given for the application of CCC in trace inorganic analysis and in radioanalytical chemistry.

Preparative isolation and purification of ginsenosides Rf, Re, Rd and Rb1 from the roots of Panax ginseng with a salt/containing solvent system and flow step-gradient by high performance counter-current chromatography coupled with an evaporative light scattering detector.

Ginseng is a popular herb worldwide and has had varied uses in traditional Asian medicine for thousands of years. There are several different species of the herb, but all share the same constituents. Ginsenosides, the most extensively studied chemical components of ginseng, are generally considered to be one of the most important active ingredients of the plant. In this study, we have developed fast and efficient methodology for isolation of four known ginsenosides Rf, Rd, Re and Rb1 from Ginseng by high performance counter-current chromatography (HPCCC) coupled with evaporative light scattering detection (ELSD). The crude sample for HPCCC was purified firstly from a ginseng extraction using macroporous resin. The enriched saponin fraction (480 mg) was separated by using methylene chloride-methanol-5 mM aqueous ammonium acetate-isopropanol (6:2:4:3, v/v,) as the two-phase solvent system and yielded 10.7 mg of Rf, 11.0 mg of Rd, 13.4 mg of Re and 13.9 mg of Rb1. The purity of these ginsenosides was 99.2%, 88.3%, 93.7% and 91.8%, respectively assessed by HPLC-DAD-ELSD, and their structures were characterized by electrospray ionization mass spectrometry (ESI-MS) and compared with standards. Ammonium acetate was used to shorten the separation time and eliminate emulsification together with a flow step-gradient. The salt can be removed by re-dissolving the sample using acetone.

A NEW HYPOTHESIS ON PHASE DISTRIBUTION IN COUNTERCURRENT CHROMATOGRAPHY

This paper describes a new hypothesis on phase distribution in Countercurrent Chromatography, which, if proven to be true, will simplify the operation of CCC and greatly improve its predictability, efficiency, and reliability. It builds on the hydrodynamic distribution model first proposed by Ito[1] where “the radial force components act against the Archimedean force to establish a hydrostatic distribution of the two phases throughout the coil.” This study postulates that these forces do not necessarily have to oppose one another and, depending on the geometry of the helical/spiral coils and the direction of rotation, can be arranged to work together.

Counter-current chromatography: Applications to the separation of biopolymers, organelles and cells using either aqueous—organic or aqueous—aqueous phase systems

Counter-current chromatography is a form of liquid-liquid chromatography which uses low-speed centrifugation to hold one phase of an immiscible liquid pair stationary while the other is eluted through it. Two types of countercurrent chromatography are described: one suitable for preparative/analytical separation with aqueous-organic phase systems and the other for analytic fractionations using aqueous-aqueous phase systems. Applications of both processes are described, ranging from the purification of antibiotics, pesticides, and peptides to the fractionation of whole cells.

Stationary Phase Retention in CCC: Modelling the J‐Type Centrifuge as a Constant Pressure Drop Pump

Abstract To be able to design a J‐type centrifuge for a given need, a method of being able to predict peak elution is required. Predicting peak elution will also allow the user to optimise the process parameters for his or her needs. Such predictions require an accurate knowledge of the volume of the stationary phase retained in the coil for a given set of operating conditions. This paper builds upon an experimental relationship in that the stationary phase retention decreases proportionally to the square root of the mobile phase flow rate. Combining this experimental relationship with the hypothesis that the pressure drop across a coil is independent of mobile phase flow rate, and assuming that the mobile phase flow is laminar, the equation below is derived: Experimental evidence is presented supporting the above equation. The experimental evidence was gained using, a heptane–ethyl acetate–methanol–water (1.4:0.1:0.5:1) v/v phase system, in normal phase mode using three helical stainless steel coils. These stationary phase retention studies allowed the above equation to be tested under conditions of different rotational speeds and tubing internal diameter. The derived stationary phase retention characteristics from each retention study allowed pressure drop and Reynolds number data to be calculated. The pressure drop data shows that the pressure drop across a coil is constant and independent of the mobile phase flow rate.

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.

Countercurrent Chromatography (CCC) and its Versatile Application as an Industrial Purification & Production Process

Abstract This paper describes the versatile operation of CCC, its potential for scale up and compares its operational performance with HPLC as a generic preparative purification process. In the words of the UK Biology & Biotechnology Science Research Council (BBSRC), there is a need for a “generation of new, robust, and usable techniques for bioprocess intensification and simplification” and, in particular, technology that can be scaled from laboratory to process scale easily and cheaply without any fundamental change to the principle of separation. CCC offers the potential to do this.

Review of Progress Toward the Industrial Scale‐Up of CCC

Abstract Considerable advances have been made in the last two years on the industrial scale‐up of countercurrent chromatography. This paper briefly reviews the scale‐up progress being made by three groups, two in France and one in the UK before giving details of advances being made at Brunel Institute for Bioengineering, Brunel University in the UK on the scale‐up of their J‐type centrifuges.

Separation of patuletin-3-O-glucoside, astragalin, quercetin, kaempferol and isorhamnetin from Flaveria bidentis (L.) Kuntze by elution-pump-out high-performance counter-current chromatography.

Flaveria bidentis (L.) Kuntze is an annual alien weed of Flaveria Juss. (Asteraceae) in China. Bioactive compounds, mainly flavonol glycosides and flavones from F. bidentis (L.) Kuntze, have been studied in order to utilize this invasive weed, Analytical high-performance counter-current chromatography (HPCCC) was successfully used to separate patuletin-3-O-glucoside, a mixture of hyperoside (quercetin-3-O-galactoside) and 6-methoxykaempferol-3-O-galactoside, astragalin, quercetin, kaempferol and isorhamnetin using two runs with different solvent system. Ethyl acetate-methanol-water (10:1:10, v/v) was selected by analytical HPCCC as the optimum phase system for the separation of patuletin-3-O-glucoside, a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside, and astragalin. A Dichloromethane-methanol-water (5:3:2, v/v) was used for the separation of quercetin, kaempferol and isorhamnetin. The separation was then scaled up: the crude extract (ca 1.5 g) was separated by preparative HPCCC, yielding 12 mg of patuletin-3-O-glucoside at a purity of 98.3%, yielding 9 mg of a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside constituting over 98% of the fraction, and 16 mg of astragalin (kaempferol-3-O-glucoside) at a purity of over 99%. The pump-out peaks are isorhanetin (98% purity), kaemferol (93% purity) and quercitin (99% purity). The chemical structure of patuletin-3-O-glucoside and astragalin were confirmed by MS and ¹H, ¹³C NMR.

How to realize the linear scale-up process for rapid purification using high-performance counter-current chromatography.

This study used the isolation of six constituents from Selaginella tamariscina as an example to demonstrate how to achieve rapid and predictable linear scale-up processes in both normal- and reversed-phase high-performance counter-current chromatography. After systematic optimization of solvent systems, sample concentration, sample loading volume, rotation speed and flow rate on the analytical Mini-DE centrifuge, the optimized parameters obtained were directly transferred to the preparative Midi-DE centrifuge, with nearly the same purities, resolutions and elution times but with 50 times the throughput. Amentoflavone (446.7 mg, 97.8%), robustaflavone (21.6 mg, 89.4%), bilobetin (80.7 mg, 92.7%), hinokiflavone (15.1 mg, 85.5%), isocryptomerin (34.8 mg, 89.6%) and an apigenin-diglucoside (46.3mg, 96.4%) were obtained with amounts and purities shown in parentheses as analysed by HPLC. The process, therefore, offers an efficient and rapid method of obtaining sufficient quantities of target compounds with significantly increased throughput after a linear scale-up.