Cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography. I. Apparatus and studies on stationary phase retention in short coils

Using a new cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius, the retention of the stationary phase for nine solvent systems was studied with short coils mounted at two different locations on three holders with 5-, 15- and 25-cm hub diameters. Coils mounted 10 cm to the left of the center of a holder produced a much improved retention of most of the solvent systems compared with the same coils mounted at the center of the holder. In the lateral coil position the retention was found to be affected by the direction of the planetary motion and the head-tail elution mode. This phenomenon may be attributed to the effect of the lateral force field acting asymmetrically between the upper and lower halves of the coil.     

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography. I. Hydrodynamic distribution of two solvent phases in a helical column subjected to two types of synchronous planetary motion

Hydrodynamic distribution of two-phase solvent systems in a rotating helical column subjected to centrifugal fields produced by two different types of synchronous planetary motion has been studied by the use of the combined horizontal flow-through coil planet centrifuge. With continuous elution of the mobile phase, the simpler type of motion resulted in low retention of the stationary phase in the column whereas a more complex motion, which produces a quasi-radial centrifugal field varying in both intensity and direction, yielded high stationary phase retention for commonly used solvent systems having a wide range of hydrophobicity. These solvent systems display highly complex modes of hydrodynamic interaction in the coil according to their particular physical properties.     

Large-Scale Preparative Counter-Current Chromatography with a Coil Planet Centrifuge

Abstract

Development of the large-scale preparative countercurrent chromatographic schemes has been continued by increasing the diameter of the separation column. A 0.55 cm i.d. FEP tube was coaxially coiled around the holder (7.5 cm, 10 cm or 15 cm in diameter) of a horizontal flow-through coil planet centrifuge (15 cm revolutional radius). Performance of each column was evaluated on the separation of dinitrophenyl amino acid samples with a two-phase solvent system composed of chloroform, acetic acid, and 0.1N hydrochloric acid (2:2:1) by using both aqueous and nonaqueous phases as the mobile phase. Experiments with the short preliminary columns (114 ml capacity) revealed that the hydrodynamic distribution of the two solvent phases was sensitively affected by the helical diameter of the column. However, by choosing the proper elution mode of the mobile phase, satisfactory results were obtained with the helical diameters of 7.5 cm and 15 cm at a high flow rate of 500 ml/h under a moderate revolutional speed of 300 rpm. With the long coiled columns (750 ml capacity), the preparative capability of the present scheme was successfully demonstrated on separations of the 1g-quantity sample mixture under optimized operational conditions. Overall results indicated that the sample-loading capacity of the present scheme can be further increased by the use of longer and/or larger-diameter columns.     

Performance of short-chain alcohols versus acetonitrile in the surfactant-mediated reversed-phase liquid chromatographic separation of β-blockers

Organic solvents are traditionally added to micellar mobile phases to achieve adequate retention times and peak profiles, in a chromatographic mode which has been called micellar liquid chromatography (MLC). The organic solvent content is limited to preserve the formation of micelles. However, at increasing organic solvent contents, the transition to a situation where micelles do not exist is gradual. Also, there is no reason to neglect the potentiality of mobile phases containing only surfactant monomers instead of micelles (high submicellar chromatography, HSC). This is demonstrated here for the analysis of β-blockers. The performance of four organic solvents (methanol, ethanol, 1-propanol, and acetonitrile) was compared in mobile phases containing the anionic surfactant sodium dodecyl sulphate in the MLC and HSC modes. The association of the organic solvent molecules with micelles gives rise to a significant loss in the elution strength of the organic solvent; whereas upon disruption of micelles, it tends to that observed in the hydro-organic mode. The elution behaviour of the β-blockers was modelled to predict the retention times. This allowed the detailed exploration of the selectivity and resolution of the chromatographic systems in relatively wide ranges of concentration of surfactant and organic solvent. The best performance in terms of resolution and analysis time was achieved using HSC with acetonitrile, being able to base-line resolve a mixture of eight β-blockers. Ethanol also provided a good separation performance, significantly improved with respect to methanol and 1-propanol. In contrast, the hydro-organic mode using acetonitrile or any of the short-chain alcohols could not succeed with the separation of the β-blockers, owing to the poorer selectivity and wider peaks.     

Selectivity and efficiency of different reversed-phase and mixed-mode sorbents to preconcentrate and isolate aroma molecules

A comparison of the ability of different sorbent systems, including mixed-mode resins and reversed-phase sorbents, to extract and isolate volatile molecules from hydroalcoholic medium has been carried out by means of the determination of liquid–solid distribution coefficients. Eighteen volatile compounds covering a wide range of physicochemical properties (acids, bases and neutrals) and chemical functionalities, and thirteen different sorbents have been tested. LiChrolut EN and Isolute ENV (both polymeric with high surface area) showed the highest retention capability for nearly all analytes at all pHs tested. Exceptions were 2,3,5-trimethylpyrazine, most efficiently extracted with Strata XC at acidic pH, and indole best retained with Oasis MCX and Strata XC at any pH. Although nearly all basic compounds were most selectively extracted with cationic mixed-mode resins at acid pHs and 3-mercaptohexyl acetate and m-cresol show maxima α at pH 10 with Oasis MAX, the α values obtained have been relatively low, which suggests that retention is not particularly driven by ionic forces. The study has also shown that selectivity depends on the pH, the exact kind of mixed-mode sorbent and on the polarity of the analyte. High selectivity towards ionogenic compounds can be obtained by combining retention in mixed mode, a rinsing with a non-polar solvent and further elution with a solvent containing a neutralizing agent. However, not all the ionogenic molecules seem to be retained in ionic mode in the conditions tested and the complete elution of some analytes can be difficult, which suggests that analyte-specific isolation procedures should be analyzed case by case.     

Error analysis and performance of different retention models in the transference of data from/to isocratic/gradient elution

The transferability of retention data among isocratic and gradient RPLC elution modes is studied. For this purpose, 16 beta-blockers were chromatographed under both isocratic and gradient elution with acetonitrile-water mobile phases. Taking into account the elution mode where the experimental data come from, and the mode where the retention should be predicted, the following combinations are possible: isocratic predictions from (i) isocratic or (ii) gradient experimental designs; and gradient predictions from (iii) isocratic or (iv) gradient data. Each of these possibilities was checked using three retention models that relate the logarithm of the retention factor: (a) linearly and (b) quadratically with the volume fraction of organic solvent, and (c) linearly with a normalised mobile phase polarity parameter. The study was carried out under two different perspectives: a straightforward examination of the prediction errors and the analysis of the uncertainties derived from the variance-covariance matrix of the fitted models. The best combinations of prediction mode and model were: (i)-(b), (ii)-(c), (iii)-(b), and (iv)-(a) or (c).     

Retention prediction in ternary solvent reversed-phase liquid chromatography systems based on the variation of retention with binary mobile phase composition

An extension of the treatment adopted in a recent paper [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33] was used to derive expressions describing the variation of solute retention k with composition in ternary reversed phase liquid chromatography, RP-LC, solvent systems. The equation of the partition model obtained in this way for a ternary mobile phase was identical to that previously derived using the solubility parameter concept. This equation as well as two new expressions of In k versus organic modifiers content were tested in a variety of ternary solvent systems in order to examine the possibility of predicting retention behavior of solutes under ternary solvent mixture elution conditions from known retention characteristics in binary mobile phases. It was demonstrated the superiority of both new equations derived in this paper to that previously proposed and applied to date in ternary solvent mixtures.     

Preparative separation of bioactive constitutes from Zanthoxylum planispinum using linear gradient counter‐current chromatography

Counter‐current chromatography is a chromatographic technique with a support‐free liquid stationary phase. In the present study, a successful application of linear gradient counter‐current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n‐hexane/ethyl acetate/methanol/water quaternary solvents, in terms of “HEMWat” or “Arizona” solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter‐current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n‐hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter‐current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.     

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

Retention behavior and solvent optimization of organochlorine pesticides in solid-phase extraction

Summary Plots of capacity factor and retention time vs. elution solvent composition were proved to be useful to interpret the retention behavior of Kepone and its metabolites in a reversed-phase solid-phase extraction and to optimize the elution solvent. The percent recovery of Kepone was largely improved when the solvent was optimized. The standard deviations of the results of extractions were also improved upon the optimization of the solvent.     

Lipid retention of novel pressurized extraction vessels as a function of the number of static and flushing cycles,flush volume,and flow rate

Lipid retention in novel pressurized solvent extraction vessels was examined as a function of static and flushing cycles, flushing flow rate and flush volume in an effort to decrease lipid elution after extraction. Results indicate that none of these variables plays a pivotal role in lipid retention within this system. Regardless of which variable was altered, lipid elution remained virtually constant at (55 ± 7) %. Findings suggest that lipid elution has more to do with the high flow rate at which extracts are purged from the system at the end of the static cycle.     

Computer-aided development of a high-performance liquid chromatographic method for the determination of hydroxyanthraquinone derivatives in Chinese herb medicine rhubarb.

With computer simulation predicting separation in reversed-phase gradient elution, a method to separate and determine five hydroxyanthraquinone derivatives having a wide range of polarity in extract of Chinese herbal medicine rhubarb has been developed. The software DryLab was used to model the retention behavior of the compounds as a function of gradient conditions, based on data from two scouting gradient runs. Under the optimized conditions, i.e. column, Zorbax RX-C18, 5 microm, 15x0.46 cm; solvent A, 36 mM triethylamine phosphate (TEAP), pH=2.5; solvent B, ACN; gradient, 36/36/80/80% B at 0/5.5/20.5/25.5 min; flow-rate, 1.00 ml/min; temperature, ambient, the method was successfully applied to monitor the quality of rhubarb from different sources. The effect of sample preparation procedures on extraction efficiency was also examined.     

Large-scale preparative countercurrent chromatography for separation of polar compounds

Gram quantity separations of polar compounds (tryptophyl-leucine and valyl-tyrosine) have successfully been accomplished with the use of a horizontal coil planet centrifuge. Two columns of different length fluorinated ethylene propylene tubing but of same internal diameter (0.55 cm) were coaxially coiled around a holder 7.5 cm or 15 cm in diameter and used to assess the preparative capabilities of the apparatus in terms of stationary phase retention and sample peak resolution. Optimal operating conditions derived from preliminary studies with the short column were applied to a column 7 times in length and volume. Volume capacities were 114 ml and 750 ml respectively. A hydrophilic solvent system of n-butanol, acetic acid and water (4:1:5) was used with both the aqueous and non-aqueous phases being used as the mobile phase. Preliminary studies revealed that the hydrodynamic distribution of the two phases was independent of the helical diameter while peak resolution was sensitive to both helical diameter and rpm setting.     

Separation of copoly(styrene/acrylonitrile) samples according to composition under reversed phase conditions

Summary Copoly(styrene/acrylonitrile) samples (S/AN) have been repeatedly separated according to composition by gradient HPLC with alkane hydrocarbons as a starting eluent A and dichloromethane (DCM) or tetrahydrofuran (THF) as a solvent B. In these systems, retention increased with AN content of the copolymers. The chemical nature of the column packings used had almost no influence on the retention of S/AN samples. The present paper shows thatn-pentane andn-heptane, when used in a given volumetric gradient with DCM+20% methanol as a solvent B, lead to identical solution characteristics of S/AN on silica columns. A similar result was obtained on C18 columns withn-heptane or cyclohexane, whereas gradient elution with toluene as a starting eluent caused insufficient resolution. Reversed phase separation of S/AN copolymers could be achieved on polystyrene gel columns through gradients with methanol as a starting eluent and DCM or THF as a solvent B. In both systems, retention decreased with increasing AN content of the copolymers. The elution characteristics were almost linear in the range 0–20 wt% AN. This behaviour can be understood in the context of polymer solubility: in both systems, the solubility borderline of S/AN has a distinct maximum at about 25 wt% AN. Reversed phase separation was achieved at the lefthand slope of these curves where the dissolution of a sample with a higher AN content requires less DCM or THF solvent than the dissolution of copolymers which are poorer in AN. This idea predicts that samples with more than 25 wt% AN should elute later than S/AN whose composition is near to the solubility maximum. This indeed was found with a copolymer containing 36.2 wt% AN.     

Overpressured layer chromatography in comparison with thin-layer and high-performance liquid chromatography for the determination of coumarins with reference to the composition of the mobile phase

The retention behaviour of fifteen closely related coumarins in normal-phase overpressured layer chromatography (OPLC) was studied with the aim of comparing the retentions with those in normal-phase thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) when optimization of the mobile phase was carried out according to the PRISMA system. The mobile phase optimization was carried out on TLC plates in unsaturated chambers. The resulting mobile phases were transposed to off-line, non-equilibrated OPLC and further to HPLC. The retention in TLC was measured at 37 selectivity points and in OPLC and HPLC at 13 points. Capacity factors (k′) and separation factors () were calculated in order to study the retention behaviour in the different systems. Two- and three-dimensional evaluations of k′ against selectivity points showed similar retention behaviours for the coumarins in TLC, OPLC and HPLC. The values for TLC, OPLC and HPLC showed similar patterns in the three-dimensional evaluations. The retention behaviour at different solvent strengths was also examined. According to quadratic regression, k′ showed a dependence on the change in solvent strength. OPLC, which can be considered as a “planar column” technique, and TLC are closely related methods, whereas HPLC shows a different behaviour in the elution process with regard to solvent strength.     

毛细管电色谱柱重复性考察及实验条件的选择

成功地研制了７５μｍ内径偶联式和７５,１００μｍ内径非偶联式毛细管电色谱柱。在自制电色谱柱上获得了小于２．１２％的保留时间ＲＳＤ值,考察了ｐＨ值、有机溶剂浓度对保留行为的影响,并用以指导实验条件的选择。比较了两种方式电色谱柱的峰形。     

High‐performance liquid chromatography enantioseparation of polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases under multimodal elution

An investigation on the high‐performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns—namely, Lux Cellulose‐1, Lux Cellulose‐2, Lux Cellulose‐4, and Lux Amylose‐2—and two immobilized columns—namely, Chiralpak IC and IA—were used under normal, polar organic, and reversed‐phase elution modes. Moreover, Chiralcel OJ was considered under normal‐phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent‐induced enantiomer elution order reversal occurred under normal‐phase mode. All the considered 4,4′‐bipyridines were enantioseparated at the multimilligram level.     

Separation of basic solutes by reversed-phase capillary electrochromatography

The separation of basic solutes at low pH by capillary electrochromatography (CEC) has been investigated. The feasibility of separation of basic solutes by CEC was demonstrated. Influence of operational parameters, solvent composition, pH, temperature on retention and selectivity of the separation of a mixture of basic, neutral and acidic drug standards has been investigated. The observed elution behavior has been modeled to account for both chromatographic retention and differential electrophoretic mobility of the solutes. This model was verified experimentally. It is demonstrated in this work that the elution window of solutes in reversed-phase CEC is expanded to range from -1 to infinity.     

液相色谱梯度洗脱中的谱带压缩效应

谱带压缩效应是梯度洗脱区别于等度洗脱的重要特征。经典的范德姆特(van Deemter)理论塔板高度方程基于等度洗脱推导得到,因此不能对谱带压缩效应进行描述。在梯度洗脱中,保留因子(k)会随流动相组成(φ)的改变而发生变化,这就使得对梯度洗脱机理的研究要比等度洗脱复杂许多。该文对近10年来谱带压缩效应的研究进展,特别是溶剂强度模型(即描述ln k与φ关系的数学表达式)的非线性特征对谱带压缩因子(G)的影响进行了述评,指出为了更好地认识谱带压缩效应需要将这种非线性因素考虑在内。