Toroidal Coil Countercurrent Chromatography: A Fast Simple Alternative to Countercurrent Distribution Using Aqueous Two Phase Partition: Principles,Theory, and Apparatus

Abstract

The principles, theoretical basis and equipment for continuous two phase toroidal coil chromatography are described. Rat liver homogenates were subjected to analytical subcellular fractionation by toroidal coil chromatography in a phase mixture of 3.3% (w/w) dextran T500, 5.4% (w/w) poly(ethylene glycol) 6000, 10 mM sodium phosphate-phosphoric acid buffer, pH 7.4, in 0.26 M sucrose containing 0.05 mM Na₂EDTA and 1 mM ethanol. The distribution of organelles, as reflected by their marker enzymes, was compared to that obtained by discrete counter-current partition in a 17 transfer apparatus. Toroidal coil chromatography showed enhanced resolution of certain organelles. In particular, almost complete separation of plasma membrane from endoplasmic reticulum was achieved and some resolution of plasma membrane from lysosomes was obtained. It is concluded that toroidal coil chromatography offers a potentially useful alternative approach to organelle separation techniques.     

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode.

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.     

Kinetics of monodisperse iron oxide nanocrystal formation by "heating-up" process

We studied the kinetics of the formation of iron oxide nanocrystals obtained from the solution-phase thermal decomposition of iron-oleate complex via the "heating-up" process. To obtain detailed information on the thermal decomposition process and the formation of iron oxide nanocrystals in the solution, we performed a thermogravimetric-mass spectrometric analysis (TG-MS) and in-situ magnetic measurements using SQUID. The TG-MS results showed that iron-oleate complex was decomposed at around 320 degrees C. The in-situ SQUID data revealed that the thermal decomposition of iron-oleate complex generates intermediate species, which seem to act as monomers for the iron oxide nanocrystals. Extensive studies on the nucleation and growth process using size exclusion chromatography, the crystallization yield data, and TEM showed that the sudden increase in the number concentration of the nanocrystals (burst of nucleation) is followed by the rapid narrowing of the size distribution (size focusing). We constructed a theoretical model to describe the "heating-up" process and performed a numerical simulation. The simulation results matched well with the experimental data, and furthermore they are well fitted to the well-known LaMer model that is characterized by the burst of nucleation and the separation of nucleation and growth under continuous monomer supply condition. Through this theoretical work, we showed that the "heating-up" and "hot injection" processes could be understood within the same theoretical framework in which they share the characteristics of nucleation and growth stages.     

Simultaneous correlation chromatography,a new technique applied to calibration in high-performance liquid chromatography

Simultaneous correlation chromatography (SCC) is introduced as a technique capable of analyzing several different samples simultaneously on the same chromatographic column. The theoretical basis of the technique is outlined and a computer simulation demonstrates the feasibility of the method. The advantages and potential of the technique are discussed. The technique is applied experimentally in a calibration procedure for high-performance liquid chromatography. During the calibration, unknown sample and calibration standards are processed under the same conditions, resulting in very accurate calibration. Other applications are outlined.     

Some theoretical considerations on preparative separation with orthogonal pressurised planar electrochromatography

In this work, separation of multicomponent mixtures containing components with the same and different electrophoretic mobility by using orthogonal pressurised planar electrochromatography is studied theoretically. Additionally, a simple way for determination of a maximum amount of mixture causing volume overload of flat/planar columns used in this technique is presented. In the next stage, effects of change in different parameters on process performance by simulation case studies are investigated. A comparative study of separation productivity of orthogonal pressurised planar electrochromatography with the continuous and periodic modes of mixture delivery and column chromatography is carried out.     

Methods to adjust for the interference of N2O on delta13C and delta18O measurements of CO2 from soil mineralization

In this paper we present an overview of the present knowledge relating to methods that avoid interference of N2O on delta13C and delta18O measurements of CO2. The main focus of research to date has been on atmospheric samples. However, N2O is predominantly generated by soil processes. Isotope analyses related to soil trace gas emissions are often performed with continuous flow isotope ratio mass spectrometers, which do not necessarily have the high precision needed for atmospheric research. However, it was shown by using laboratory and field samples that a correction to obtain reliable delta13C and delta18O values is also required for a commercial continuous flow isotope ratio mass spectrometer. The capillary gas chromatography column of the original equipment was changed to a packed Porapak Q column. This adaptation resulted in an improved accuracy and precision of delta13C (standard deviation(Ghent): from 0.2 to 0.08 per thousand; standard deviation(Lincoln): from 0.2 to 0.13 per thousand) of CO2 for N2O/CO2 ratios up to 0.1. For delta18O there was an improvement for the standard deviation measured at Ghent University (0.13 to 0.08 per thousand) but not for the measurements at Lincoln University (0.08 to 0.23 per thousand). The benefits of using the packed Porapak Q column compared with the theoretical correction method meant that samples were not limited to small N(2)O concentrations, they did not require an extra N2O concentration measurement, and measurements were independent of the variable isotopic composition of N2O from soil.     

Control of column temperature in reversed-phase liquid chromatography

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.     

Prediction of measurement uncertainty in isotope dilution gas chromatography/mass spectrometry

An equation is theoretically derived which describes the relative standard deviation (RSD) of the amount ratios of analyte to its isotope-labeled variant in gas chromatography/mass spectrometry (GC/MS) using the stable isotope dilution method. The determination of methyltestosterone is taken as an example. The uncertainty equation proposed is justified by comparing the theoretical RSD values with the experimental RSD values obtained by replication over a wide range of analyte amount. The detection limit and quantitation limit are estimated from the continuous plot (precision profile) of the theoretical RSD against analyte amount.     

A critical review of vacuum-assisted headspace solid-phase microextraction for environmental analysis

Vacuum-assisted headspace solid-phase microextraction (Vac-HSSPME) is an emerging analytical technique, which further advances HSSPME by providing lower detection limits of analytes with poor volatility at shorter extraction times. This review discusses the theoretical aspects and possibilities of the Vac-HSSPME technique for analysis of environmental samples. Optimization of key parameters, currently available equipment and methods for quantification of organic pollutants in water and soil are considered. Key problems and limitations of the technique are discussed along with possible approaches for its future development. The technique has a well-developed theory, which could be used for modeling of the extraction process, faster method development, and optimization. Wider application of the technique is limited by the lack of automation, which, however, seems possible to develop and implement by manufacturers of commercial multi-purpose autosamplers for gas chromatography instruments. It has been shown that Vac-HSSPME allows decreasing cross-contamination of samples from the laboratory air, which is advantageous for identification and quantification of trace environmental pollutants. Simple equipment for the technique makes it possible to apply for on-site sample preparation and analysis of environmental samples.     

Controlled-cycle pulsed liquid-liquid chromatography. A modified version of Craig's counter-current distribution

A new liquid-liquid chromatography technique developed from a combination of controlled-cycle operation and a pulsed-mixing technique is suggested and validated. The controlled-cycle pulsed liquid-liquid chromatography (CPLC) system operates without involving a centrifuge and consists, of a series of multistage units, and a method for imparting pulsation motion to the liquids inside the units (the pulsation cycle). This chromatography technique can be considered as an improved continuous form of Craig's counter-current distribution method, or, alternatively, as a form of droplet chromatography with the cycling mode of operation. The theoretical model has been designed to account for the effects of the basic parameters influencing the CPLC operation. The theoretical model's suitability was proved by direct comparison between the experimental and model responses. The CPLC devices containing 1, 2, 4 and 5 multistage columns (each column was divided into 26 stages) have been designed, fabricated and tested; experiments were conducted to test the chromatographic behavior of organic (monocarboxylic) and mineral acids. The mass transfer rate in the stages depends on the nature of both--phase and sample systems: the highest values were achieved in experiments with acetic acid by using the octane/water biphasic system, where an equilibrium concentration distribution between stationary and mobile phases in the stages was attained. The results obtained demonstrated the potential of the new technique for preparative and industrial scale separations.     

Estimation of the instrumental band broadening contribution to the column elution profiles in open tubular capillary liquid chromatography

In comparison with conventionally packed HPLC columns, from a theoretical point of view, open capillary liquid chromatography (OTLC) systems offer a number of advantages like high plate numbers and short analysis times. On the other hand, drastic changes have to be made to the instrumentation. In particular, the contribution to band broadening by the chromatographic equipment must be considerably reduced. In the present study an OTLC system was developed and evaluated, which yields satisfactory results for 26 μm i. d. columns. The determination of the contribution of the chromatographic equipment to the total band broadening is discussed.     

Preparation of eicosapentaenoic acid ethyl ester from fish oil ethyl esters by continuous batch chromatography

Fish oils are rich in eicosapentaenoic acid, which has the wide‐ranging biological activities. The rapid and efficient separation of eicosapentaenoic acid ethyl ester from fish oils ethyl ester is still regarded as a challenge. In this study, we described an effective and flexible chromatography for eicosapentaenoic acid ethyl ester preparation, named continuous batch chromatography, which combined the batch chromatography with the continuous chromatographic operation mode. After continuous batch chromatography experiment, the recovery of eicosapentaenoic acid ethyl ester was 82.01%, the average relative purity and the relative highest purity of eicosapentaenoic acid ethyl ester were 97.82 and 98.98%. The productivity of continuous batch chromatography was 5.48 times higher than that of batch chromatography, while the solvent consumption of eicosapentaenoic acid ethyl ester was 78% of the batch chromatography. This study provided a reference for the separation of the targeted chemical component from multi‐component mixtures.     

气相色谱分析过程的计算机仿真模拟与优化

以色谱法的ＬＥＳ模型作为气相色谱分析过程的计算机仿真模拟与优化的理论基础，提出了把两次多阶程度升温实验保留时间作为基本数据进行模拟优化的方法，拓宽了模拟优化方法在复杂组分样品中的应用。软件编程采用了计算数学方法，提高了模拟优化的精度和速度，节省了分析样品的时间。     

连续旋转环状色谱的数学模型参数分析

连续旋转环状色谱用数学模型模拟时,模拟结果的精确度和所采用参数的“灵敏”程度有关。为此,探讨了相平衡常数、相间传质系数、弥散返混系数等对色谱峰质量浓度最高点位置角度（保留值）、分离度、峰宽和产率等参数的灵敏度并对它们逐一进行了分析。认为相平衡关系对分离效果最为敏感。筛选吸附量大、选择性好的吸附剂是改进操作效果的最佳途径。     

A Computer-based, Interactive, Multimedia Software System for Teaching and Independent Student Learning of Liquid Chromatography

This paper describes the design and development of an interactive, simulation-based education software system for the exploration of key concepts in liquid chromatography. Evaluation results of student use of the software will be presented.Software modules have been designed to supplement, but not replace, classroom and laboratory learning about liquid chromatography, providing students with theory, visual aids, and interactive simulations. Combined with animations and narration, the modules complement the foundation on which chromatography is taught in a general, analytical, or organic chemistry setting. Furthermore, this software allows the student to interact with the illustrated chemistry lessons, which is especially important in instances where the concepts taught transcend typical classroom material.This work serves to connect what one observes experimentally in a chromatography experiment to changing parameters such as capacity factor, number of theoretical plates, height equivalent of a theoretical plate, column length, resolution, retention time, and selectivity factor. Simulated separations are portrayed in an animated fashion as a chromatogram developing over time as a function of one or more of these variables. Finally, the software has real sample, mobile phase, column type, flow rate, and detection type options allowing the selection and simulation of solving a realistic chromatography problem. Hypothetical unknown mixtures can be separated and the sample components identified after simulating the separation of known samples.     

Hairdressing shish-kebabs; comprehensive survey and theoretical discussion of overgrowth crystallization

The platelet component of shish-kebabs crystallizes after the core, during cooling or storage below the formation temperature of the core. Three basic platelet morphologies were previously identified which were mutually interconvertible, a process we have termed “hairdressing”. In this paper we show that these three categories are special cases of a continuous range of overgrowth spacings. Crystallization at high temperatures gives widely spaced overgrowths and, as the crystallization temperature is reduced, so the overgrowth spacing decreases gradually. In the extreme case (only obtainable by quenching) the overgrowths become so close as to overlap and appear continuous. We also report a variety of further effects which were caused by exposing the shish-kebabs, while in solvent, to temperatures above their initial formation temperature. A new theoretical approach is described which considers the depletion of material available to form new overgrowths during crystallization. Two versions of this theory are presented (one a computer simulation and one analytic); interpreting our results on the basis of this theory we show that shish-kebabs crystallize at high temperatures even when quenched (90°C and above except in a few exceptional circumstances) and we are able to explain all the features of shish-kebab crystallization that we have observed.     

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.     

Thermal expansion and impurity effects on lattice thermal conductivity of solid argon

Thermal expansion and impurity effects on the lattice thermal conductivity of solid argon have been investigated with equilibrium molecular dynamics simulation. Thermal conductivity is simulated over the temperature range of 20-80 K. Thermal expansion effects, which strongly reduce thermal conductivity, are incorporated into the simulations using experimentally measured lattice constants of solid argon at different temperatures. It is found that the experimentally measured deviations from a T(-1) high-temperature dependence in thermal conductivity can be quantitatively attributed to thermal expansion effects. Phonon scattering on defects also contributes to the deviations. Comparison of simulation results on argon lattices with vacancy and impurity defects to those predicted from the theoretical models of Klemens and Ashegi et al. demonstrates that phonon scattering on impurities due to lattice strain is stronger than that due to differences in mass between the defect and the surrounding matrix. In addition, the results indicate the utility of molecular dynamics simulation for determining parameters in theoretical impurity scattering models under a wide range of conditions. It is also confirmed from the simulation results that thermal conductivity is not sensitive to the impurity concentration at high temperatures.