Using the liquid nature of the stationary phase. VI. Theoretical study of multi-dual mode countercurrent chromatography

Countercurrent chromatography (CCC) is a separation technique using a biphasic liquid system and centrifugal forces to maintain a support-free liquid stationary phase. Either one of the two phases can be the liquid stationary phase. It is even possible to switch the phase role during the separation. The dual-mode method is revisited recalling its theoretical background. The multi-dual mode (MDM) CCC method was introduced to enhance the resolution power of a CCC column. The theoretical study of the MDM method is validated by modeling the separation of two solutes. The basic hypothesis is that the forward step (partial classical elution) is followed by a backward step that returns the less retained solute to the column head. The equations show that the most important parameter to maximize resolution is not the number of MDM steps but the total volume of liquid phases used to elute the solutes. The model is validated calculating correctly the peak position of previously published MDM experiments.     

Closed-Loop Recycling Dual-Mode Counter-Current Chromatography with Specified Sample Loading Durations: Modeling of Preparative and Industrial-Scale Separations

We previously reported on a new counter-current chromatography (CCC) operating mode called closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC), which incorporates the advantages of closed-loop recycling (CLR) and dual-mode (DM) counter-current chromatography and includes sequential separation of compounds in the closed-loop recycling mode with the mobile x-phase and in the inverted-phase counter-current mode with the mobile y-phase. The theoretical analysis of several implementations of this separation method was carried out under impulse sample injection conditions. This study is dedicated to the further development of CLR DM CCC theory applied to preparative and industrial separations, where high-throughput operation is required. Large sample volumes can be loaded via continuous loading within a specified time. To simulate CLR DM CCC separations with specified sample loading durations, equations are developed and presented in “Mathcad” software.     

Resolution studies on counter-current chromatography using supercritical fluid carbon dioxide

Counter-current chromatography (CCC) is a form of liquid–liquid partition chromatography. It requires two immiscible solvent phases; the stationary phase is retained in the separation column, generally by centrifugal force, while the mobile phase is eluted. We recently replaced the mobile phase with supercritical fluid carbon dioxide (SF CO₂). Since the solvent strength of SF CO₂ can be varied by changing the temperature and pressure of the system, separation adjustments are thus more versatile. We investigated the pressure and temperature effects on resolution using water and low-carbon alcohol mixtures as the stationary phases. It was demonstrated that these special properties of SF CO₂ were indeed beneficial to the optimization of separations. In addition, the phase retention ratio was examined in terms of separation resolution. The results appeared very similar to those obtained from conventional traditional CCC. This study should be helpful for the future development of SF applications in CCC.     

Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency

Countercurrent chromatography (CCC) is a separation technique in which two immiscible liquid phases are used for the preparative purification of synthetic and natural products. In CCC the number of repetitive mixing and de-mixing processes, the retention of the stationary phase and the mass transfer between the liquid phases are significant parameters that influence the resolution and separation efficiency. Limited mass transfer is the main reason for peak broadening and a low number of theoretical plates along with impaired peak resolution in CCC. Hence, technical improvements with regard to column design and tubing modifications is an important aspect to enhance mixing and mass transfer.     

Estimation of chromatographic parameters on two silica-based columns connected in series under nonaqueous reversed phase liquid chromatographic conditions

Empirical equations were produced to relate important chromatographic parameters on two silica-based columns serially linked, in isocratic nonaqueous RP HPLC, to retention times and peak widths of the separated compounds on the individual columns. These equations were derived because the experimental data seemed to deviate from the values expected, applying basic chromatographic theoretical equations. The chromatographic parameters studied were retention time, peak width, resolution, number of theoretical plates, capacity factor, and separation factor. In addition, empirical linear relationships were produced for the estimation of the above mentioned parameters of the serial systems, in direct and reverse order, relating them to those obtained on each column, separately. The experimentally obtained values were in good agreement with those estimated by the derived equations.     

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.     

Separation of alkaloids from herbs using high-speed counter-current chromatography

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.     

Computer-assisted optimization of reversed-phase HPLC isocratic separation of neutral compounds

An appropriate optimization strategy should be used to find a desired resolution or selectivity with a minimum number of experiments in a limited time, which could assure the baseline separation of all target compounds. It was usually realized by means of a specialized computer program. In this paper, mapping optimization method and overlapping resolution mapping were compared for the optimization of a reversed-phase high-performance liquid chromatography (HPLC) isocratic separation of neutral compounds. The calculated resolutions and separation time of 7 to 10 experiments are fitted by different equations, which were used to build a contour plot with a minimum effective resolution and maximum retention time as a function of a mobile phase composition. The balance between resolution and analysis time could be easily realized by the overlapping of the final overlapping resolution mapping and analysis time mapping. The validity of the two methods was confirmed by some typical experiments. The models are simple, visual, and common without theoretical arithmetic.     

A novel approach to modelling counter-current chromatography

Literature lists a number of counter-current chromatography (CCC) models that can predict the retention time and to a certain extent the peak width of a solute eluting from a CCC column. The approach described in this paper distinguishes itself from previous reports by relating all model parameters directly to column dimensions and experimental settings. Most importantly, this model can predict a chromatogram from scratch without resorting to traditional calibration using empirical values. The model validation with experimental results obtained across a range of CCC instruments demonstrated that the solute retention time, peak width, and peak resolution could be predicted within reasonable accuracy. Additionally, the effect of several process parameters, such as mobile phase flow rate, rotational speed of the column or β-value, showed that the model is robust and applicable to a wide range of CCC instruments. Overall, this model proved to be a useful tool for parameter estimation and, most significantly, separation optimisation.     

Water-organic solvent systems in countercurrent chromatography: Liquid stationary phase retention and solvent polarity

Countercurrent chromatography (CCC) is a separation technique in which the stationary phase is a liquid. The liquid stationary phase retention is a critical problem in CCC. The retention of 18 organic solvents in a hydrodynamic CCC apparatus was measured with an aqueous mobile phase, the centrifuge spin rate and the mobile phase flow rate being constant, 800 rpm and 2 ml/min, respectively. Conversely, water retention was measured when the 18 solvents were the mobile phases. A direct relationship between the liquid stationary phase retention and the phase density difference was found. The liquid phase density difference is the most important parameter for stationary phase retention in a hydrodynamic CCC apparatus with coiled tubes. The chromatographic retention of formanilide was measured in biphasic systems and expressed as the formanilide partition coefficient. It is shown that the partition coefficient correlates with the Reichardt polarity index of the organic solvent when the liquid stationary phase retention volume does not.     

Characterisation of the surface Lewis acid-base properties of poly(butylene terephthalate) by inverse gas chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were applied to separate 3- and 4-sulfophthalic acid from a mixture. Conventional HSCCC was useful for the separation of up to several hundred milligram quantities of these positional isomers, while pH-zone-refining CCC was implemented successfully to separations at the multigram level. The conventional HSCCC separations were performed with a standard J-type HSCCC system that has a superior resolution but a lower level of retention of the stationary phase of the biphasic solvent system used (acidified n-butanol-water). The pH-zone-refining CCC separations were performed with an X-type HSCCC system (a cross-axis system) that has a higher capability for retention of the stationary phase. The purified positional isomers (over 99% pure as determined by HPLC) were characterized by 1H NMR and negative ion electrospray ionization mass spectrometry.     

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators.     

Advantages of a small-volume counter-current chromatography column

Counter-current chromatography (CCC) works with a support-free liquid stationary phase. This allows for preparative separations and purifications. However, there are serious technical constraints because of the need to keep a liquid stationary phase in a column. Centrifugal fields are used. A new commercial hydrodynamic 18 mL column made with a narrow-bore 0.8 mm Teflon tubing was evaluated by comparing it with older hydrodynamic CCC columns and a similar 19 mL column but made with 1.6 mm Teflon tubing. A small-volume CCC column allows for reliable and fast solute partition coefficient determination. When resolution is required, both high efficiency and liquid stationary phase retention are needed. Unfortunately, these two requirements bear technical contradictions. A column coiled with a narrow tubing bore will provide a high chromatographic efficiency while a column containing wider tubing bore will achieve higher stationary phase retention. In all cases, increasing the magnitude of the centrifugal field also increases the stationary phase retention. The solution is to build centrifuges able to produce high fields that will provide acceptable liquid phase retention with narrow-bore tubes. The new 18 mL 0.8 mm tubing bore column is able to rotate as fast as 2100 rpm generating a 240 × g field. The two older CCC columns cannot compete with the new one. However, the small 19 mL column with 1.6 mm bore tubing can be useful when fast results are desired without top resolution.     

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.     

Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications

Nowadays, centrifugal partition chromatography (CPC) separations can be routinely achieved at the laboratory scale. The solvent system selection has been made easy, as generic sets of solvent systems are described in publications and books. This approach, however, generally reduces the scope of optimization strategies for two important parameters: selectivity and sample solubility. This can be very limiting for the preparative separation of structurally similar compounds. Multiple dual-mode (MDM) CPC has been developed to provide an easy-to-use alternative technique to circumvent this problem. A MDM separation consists of a succession of dual-mode runs (i.e. multiple inversion of stationary and mobile phase) that can only be achieved because both chromatographic phases are liquids. This original elution mode is thus a semi-continuous process with a classical sample injection and which only requires a single CPC column. Underlying mechanisms of MDM were studied using a model mixture of acenaphthylene and naphthalene. A mixture of two synthetic pairs of diastereomers was then successfully submitted to MDM CPC, in the framework of the synthesis of biologically active compounds.     

Hydrophobicity of ionisable compounds studied by countercurrent chromatography

Countercurrent chromatography (CCC) is a liquid chromatography technique in which the stationary phase is also a liquid. The main chemical process involved in solute separation is partitioning between the two immiscible liquid phases: the mobile phase and the support-free liquid stationary phase. The octanol-water partition coefficients (P(o/w)) is the accepted parameter measuring the hydrophobicity of molecules. It is considered to estimate active principle partitioning over a biomembrane. It was related to the substance biological activity. CCC is able to work with an octanol stationary phase and an aqueous mobile phase. In this configuration, CCC is a useful and easy alternative to measure directly the P(o/w) of the molecules compared to other methods including the classical and tedious shake-flask method. Three ketones are used as model compounds to illustrate the CCC protocol of P(o/w) measurement. The focus of this work is put on ionisable molecules whose apparent P(o/w) is completely changed by ionization. β-Blockers, diuretics and sulfonamides are compound classes that were studied. Some of the experimentally determined P(o/w) coefficients of the molecular forms disagreed with calculated and experimental values available in the literature. The P(o/w) coefficients of the ionic forms and the acidity constants were also calculated using a theoretical model. Relationships between biological properties and hydrophobicity are also discussed.     

Test to evaluate countercurrent chromatographs. Liquid stationary phase retention and chromatographic resolution

Countercurrent chromatography (CCC) is a liquid chromatography (LC) technique with a special column able to retain a liquid stationary phase while the liquid mobile phase is pumped through. The coil planet centrifuge machines are made of open tube wound on spools. A simple test is proposed. The methanol-water (90:10, v/v)-heptane biphasic system is used with heptane as the mobile phase in the ascending or tail-to-head mode. The methanol-water stationary phase retention volume is measured at different flow-rates and rotor rotation speeds. After every machine equilibration, an alkylbenzene mixture is injected and the retention factors, peak efficiencies and resolution factors are measured or calculated for each solute. The wealth of information contained in the data set obtained is demonstrated. Four coil planet centrifuge machines of very different characteristics and one hydrostatic CCC machine with channels and ducts were submitted to the test. It was shown that the Sf, stationary retention factor, obtained with these machines was linearly dependent on the square root of F, the mobile phase flow-rate [Q. Du, C. Wu, G. Qian, P. Wu, Y. Ito, J. Chromatogr. A 835 (1999) 231-235]. It is shown that the slopes of the Sf versus F(1/2) lines could be related to a minimum rotor rotation, omega(mini), necessary to obtain the hydrodynamic equilibrium. The Sf and F parameters give the mobile phase linear velocity, u. It is shown that u is proportional to the square root of omega, the rotor rotation speed. The slope and intercept of the latter relationship also result in an omega(mini) value coherent with the first one. With the peak efficiencies and chromatographic resolution factors obtained for toluene and hexylbenzene, the parameters: number of plates per tubing turn, machine volume for one plate, and tubing length for one plate, were calculated and compared for the five machines. The internal diameter of the tubing used is shown to be a critical parameter acting on the machine volume and number of tubing turns.     

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.     

Preparative isolation and purification of senkyunolide-I, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong using counter-current chromatography

Three active compounds, senkyunolide-I, senkyunolide-H and ferulic acid (FA), were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by counter-current chromatography (CCC). Based on the principle of the partition coefficient values (k) for target compounds and the separation factor (α) between target compounds, the two-phase solvent system that contains n-hexane-ethyl acetate-methanol-water at an optimized volume ratio of 3:7:4:6 v/v was selected for the CCC separation, and the lower phase was employed as the mobile phase in the head-to-tail elution mode. In a single run, 400 mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg) and FA (4.4 mg) with the purities of 98, 93 and 99%, respectively. The CCC fractions were analyzed by high-performance liquid chromatography, and the structures of the three active compounds were identified by MS and (1)H NMR.