GUESSmix-guided optimization of elution–extrusion counter-current separations

Rational strategies for the optimization of separations are vital to any chromatographic technique. In counter-current separations (CS), once a suitable solvent system is selected for a given separation, the operator is faced with the task of optimizing the separation through the manipulation of those adjustable operation parameters allowed for by the current CS technology. This study employed a mixture of 21 natural products of varying polarity, molecular mass, and functionality, termed the GUESSmix, as a tool to assess the effectiveness of optimization strategies. The behavior of the GUESSmix was observed in the hexane/ethyl acetate/methanol/water 4:6:4:6 (HEMWat +3) solvent system. The effect of operation parameters on both the elution and extrusion stages of a recently introduced CS methodology, termed elution–extrusion counter-current chromatography (EECCC), was investigated. The resulting chromatograms were plotted with K-based reciprocal symmetry plots (ReS and ReSS), which allow comparison of the K values of significant peaks and assessment of resolution of eluting compounds in the interval 0 ≤ K ≤ ∞. The operation parameters studied were: (1) the effect of temperature controlled water circulation around the centrifuge; (2) the combination of flow rate and revolution speed; (3) sample loading capacity; (4) the direction of rotation either agreeing with or opposing the direction of coil winding; (5) injection before equilibration, a practice that saves operator time and reduces solvent consumption. The GUESSmix was found to be a highly useful reference mixture to compare and contrast stationary phase retention volume ratios, resolution, K-values, peak shapes, and extrusion characteristics between CS experiments. EECCC is shown to be a robust technique that may be enhanced with appropriate temperature, rpm, flow rate, sample loading, direction of rotation, and injection timing. Plotting ReS[S] chromatograms enables systematic study of CS parameters, which are not reproducibly represented in retention time or volume plots.     

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.     

Universal counter-current chromatography modelling based on counter-current distribution

There is clearly a need for a model which is versatile enough to take into account the numerous operating modes and pump out procedures that can be used with counter-current chromatography (CCC). This paper will describe a universal model for counter-current chromatography based on counter-current distribution. The model is validated with real separations from the literature and against established CCC partition theory. This universal model is proven to give good results for isocratic flow modes, as well as for co-current CCC and dual flow CCC, and will likely also give good results for other modes such as intermittent CCC.     

Role of counter-current chromatography in the modernisation of Chinese herbal medicines

This review focuses on the growing popularity of using counter-current chromatography (CCC), with its liquid stationary phase, as one of the prime methods for isolating compounds from Chinese herbal medicines (CHMs). 198 publications are reviewed covering 108 different plant species from 56 plant families. These describe the isolation of 354 different molecules across a wide range of polarities, chemical classes and molecular weights (in the range 100–1000 Da). The suitability of CCC for the separation of active compounds from CHM, the phase systems used, how CCC has developed in China, compounds isolated, CCC instrumentation, performance, operational issues and innovations, all supported by detailed cross-referencing, are described. It is concluded that CCC is making an increasingly important contribution to the modernisation of Chinese herbal medicines.     

手性逆流色谱的研究进展

总结了手性逆流色谱的5个特点,系统地介绍了逆流色谱的手性分离以及高速逆流色谱手性分离中氨基酸衍生物、环糊精衍生物、手性有机酸、多糖衍生物、牛血清白蛋白等手性选择剂的应用。     

Enantiomer separation by counter-current chromatography. Optimisation and drawbacks in the use of L-proline derivatives as chiral selectors

Several L-proline and (4R)-hydroxy-L-proline derivatives were evaluated as chiral selectors (CSs) in the separation of enantiomers by counter-current chromatography (CCC). A variety of biphasic solvent systems, all of organic/aqueous nature, were tested in order to determine the appropriate distribution for CSs and racemates (N-(3,5-dinitrobenzoyl)-(+/-)-leucine and (+/-)-ketoprofen). Successful separations of DNB-(+/-)-leucine in analogous experimental conditions allow the comparative study of the enantioselectivity displayed by the considered CSs. The low solubility of certain CSs limits their applicability for preparative purposes even for improved enantioselectivity. The effect that the nature and pH of the buffer solutions used as a component of the solvent system have on the separation was also studied.     

Advanced separation methods of food anthocyanins,isoflavones and flavanols

In recent years, increasing knowledge of the positive health effects of food polyphenols has prompted the need to develop new separation techniques for their extraction, fractionation and analysis. This article provides an updated and exhaustive review of the application of counter-current chromatography, high performance liquid chromatography, capillary electrophoresis, and their hyphenation with mass spectrometry to the study of food polyphenols. Flavonoids constitute the largest class of polyphenols, widely spread in the plant kingdom and common in human diet which has been the most widely studied with respect to their antioxidant and biological activities. The main subgroups are anthocyanins, catechins, isoflavones, flavonols and flavones. They are reported to exhibit antioxidant, anti-carcinogenic, anti-inflammatory, anti-atherogenic, anti-thrombotic, and immune modulating functions, among others. Since red fruit anthocyanins, soy isoflavones and flavanols from grapes and teas are currently the most used phenolic compounds for producing new nutraceuticals and functional foods, this review is focused on these three flavonoid groups.     

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.     

Adsorption of DNA onto anionic lipid surfaces

Currently self-assembled DNA delivery systems composed of DNA multivalent cations and anionic lipids are considered to be promising tools for gene therapy. These systems become an alternative to traditional cationic lipid–DNA complexes because of their low cytotoxicity lipids. However, currently these nonviral gene delivery methods exhibit low transfection efficiencies. This feature is in large part due to the poorly understood DNA complexation mechanisms at the molecular level. It is well-known that the adsorption of DNA onto like charged lipid surfaces requires the presence of multivalent cations that act as bridges between DNA and anionic lipids. Unfortunately, the molecular mechanisms behind such adsorption phenomenon still remain unclear. Accordingly a historical background of experimental evidence related to adsorption and complexation of DNA onto anionic lipid surfaces mediated by different multivalent cations is firstly reviewed. Next, recent experiments aimed to characterise the interfacial adsorption of DNA onto a model anionic phospholipid monolayer mediated by Ca^2 + (including AFM images) are discussed. Afterwards, modelling studies of DNA adsorption onto charged surfaces are summarised before presenting preliminary results obtained from both CG and all-atomic MD computer simulations. Our results allow us to establish the optimal conditions for cation-mediated adsorption of DNA onto negatively charged surfaces. Moreover, atomistic simulations provide an excellent framework to understand the interaction between DNA and anionic lipids in the presence of divalent cations. Accordingly,our simulation results in conjunction go beyond the macroscopic picture in which DNA is stuck to anionic membranes by using multivalent cations that form glue layers between them. Structural aspects of the DNA adsorption and molecular binding between the different charged groups from DNA and lipids in the presenceof divalent cations are reported in the last part of the study. Although this research work is far from biomedical applications, we truly believe that scientific advances in this line will assist, at least in part, in the rationaldesign and development of optimal carrier systems for genes and applicable to other drugs.     

Preparative separation of 1,3,6-pyrenetrisulfonic acid trisodium salt from the color additive D&C Green No. 8 (pyranine) by pH-zone-refining counter-current chromatography

In developing analytical methods for batch certification of the color additive D&C Green No. 8 (G8), the U.S. Food and Drug Administration needed the trisodium salt of 1,3,6-pyrenetrisulfonic acid (P3S) for use as a reference material. Since P3S was not commercially available, preparative quantities of it were separated from portions of a sample of G8 that contained ～3.5% P3S. The separations were performed by pH-zone-refining counter-current chromatography using dodecylamine (DA) as the hydrophobic counterion. The added DA enabled partitioning of the polysulfonated components into the organic stationary phase of the two-phase solvent system used, 1-butanol-water (1:1). Thus, a typical separation that involved 20.3g of G8, using sulfuric acid as the retainer acid and 20% DA in the stationary phase and 0.1M sodium hydroxide as the mobile phase, resulted in ～0.58 g of P3S of greater than 99% purity. The identification and characterization of the separated P3S were performed by elemental analyses, proton nuclear magnetic resonance, high-resolution mass spectrometry, ultra-violet spectra, and high-performance liquid chromatography.