Numerical modeling of elution peak profiles in supercritical fluid chromatography. Part I—Elution of an unretained tracer

When chromatography is carried out with high-density carbon dioxide as the main component of the mobile phase (a method generally known as “supercritical fluid chromatography” or SFC), the required pressure gradient along the column is moderate. However, this mobile phase is highly compressible and, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat, cooling the column, which absorbs heat from the outside. The resulting heat transfer causes the formation of axial and radial gradients of temperature that may become large under certain conditions. Due to these gradients, the mobile phase velocity and most physico-chemical parameters of the system (viscosity, diffusion coefficients, etc.) are no longer constant throughout the column, resulting in a loss of column efficiency, even at low flow rates. At high flow rates and in serious cases, systematic variations of the retention factors and the separation factors with increasing flow rates and important deformations of the elution profiles of all sample components may occur. The model previously used to account satisfactorily for the effects of the viscous friction heating of the mobile phase in HPLC is adapted here to account for the expansion cooling of the mobile phase in SFC and is applied to the modeling of the elution peak profiles of an unretained compound in SFC. The numerical solution of the combined heat and mass balance equations provides temperature and pressure profiles inside the column, and values of the retention time and efficiency for elution of this unretained compound that are in excellent agreement with independent experimental data.     

Studies on the optimization of parameters of preparative liquid chromatographic columns for production of cardiac glycosides

Investigations aimed at maximization of the output of the production of cardiac glycosides from their mixtures using liquid chromatography are reported. The dependences of the yield of the process on column length, particle size and surface area of the packing and linear velocity of the mobile phase were examined. It has been established that a simultaneous decrease in the particle size and an increase in the column length and mobile phase flow-rate results in an improvement in the yield. However, such a procedure is limited by technological considerations. Optimum production conditions have been characterized.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Numerical modeling of the elution peak profiles of retained solutes in supercritical fluid chromatography

In supercritical fluid chromatography (SFC), the significant expansion of the mobile phase along the column causes the formation of axial and radial gradients of temperature. Due to these gradients, the mobile phase density, its viscosity, its velocity, its diffusion coefficients, etc. are not constant throughout the column. This results in a nonuniform flow velocity distribution, itself causing a loss of column efficiency in certain cases, even at low flow rates, as they do in HPLC. At high flow rates, an important deformation of the elution profiles of the sample components may occur. The model previously used to account satisfactorily for the retention of an unsorbed solute in SFC is applied to the modeling of the elution peak profiles of retained compounds. The numerical solution of the combined heat and mass balance equations provides the temperature and the pressure profiles inside the column and values of the retention time and the band profiles of retained compounds that are in excellent agreement with independent experimental data for large value of mobile phase reduced density. At low reduced densities, the band profiles can strongly depend on the column axial distribution of porosity.     

Rapid linear scale-up of a protein separation by centrifugal partition chromatography

The scaling up of the separation of two proteins with an aqueous two-phase system (ATPS) from 176 mg with a 500 ml laboratory scale centrifugal partition chromatography (CPC) column to 2.2g with a 6.25 litre pilot-scale column is presented. A model sample system of a mixture of lysozyme and myoglobin was chosen for this study using an ATPS system comprising 12.5% (w/w) PEG-1000:12.5% (w/w) K2HPO4. It was found that the maximum sample concentration possible without precipitation was 2.2mg/ml for each constituent. The optimisation of rotor speed, mobile phase flow rate and sample loading was performed on a laboratory-scale device. It was found that a centrifuge speed of 2000 rpm (224 'g'), 10 ml/min mobile phase flow rate with a 43 ml (10% of active column volume) sample volume gave optimum operating conditions. This was linearly scaled up to pilot scale by increasing mobile phase flow rate, fraction size and sample loading in the ratio of the system capacities (i.e. 12.5:1). Flow rate was therefore increased from 10 ml/min to 125 ml/min, fraction size from 10 ml to 125 ml and sample loading from 43 ml to 500 ml. Rotor speed however was reduced from 2000 rpm on the laboratory device to 1293 rpm on the pilot-scale device to maintain the same 224 'g' field in each chamber, as the pilot-scale CPC unit has a larger rotor radius than the laboratory one. Resolution increased from Rs=1.28 on the 500 ml rotor to Rs=1.88 on the 6.25 litre rotor, giving potential throughputs in batch mode of over 40 g/day.     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

高效液相色谱法分离双手性中心β-氨基酮旋光异构体

建立了使用AE. Lichrom AM 1手性色谱柱(250 mm×4.6 mm, 5 μm)直接拆分含有双手性中心的β-氨基酮类化合物的4个旋光异构体的方法。考察了流动相的极性大小、洗脱方式及流速等因素对异构体拆分的影响,确定了分离β-氨基酮类化合物异构体的最佳分离条件: 流动相为正己烷/异丙醇,洗脱模式为二元高压梯度洗脱,流速为1 mL/min。在上述色谱条件下,完成了4种含双手性中心的β-氨基酮类化合物的4个旋光异构体的基线分离(Rs＞1.5)。结果表明该方法可快捷、简便地分离双手性中心β-氨基酮类化合物的4个旋光异构体。     

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.     

线性非理想条件下液相色谱柱末端峰形规律

色谱流出曲线的二阶中心矩μ２和三阶中心矩μ３以及描述峰形非对称程度的偏态系数∑ｋ＝μ３／μ１．５２是反映色谱峰形的重要参数。从液相色谱过程动力学方程出发，运用电子计算机证明了在线性非理想条件下高效液相色谱体系中不同保留值组分在柱末端峰形的分布基本一致的结论。     

线性非理想条件下液相色谱柱末端峰形规律

 色谱流出曲线的二阶中心矩μ２和三阶中心矩μ３以及描述峰形非对称程度的偏态系数∑ｋ＝μ３／μ１．５２是反映色谱峰形的重要参数。从液相色谱过程动力学方程出发，运用电子计算机证明了在线性非理想条件下高效液相色谱体系中不同保留值组分在柱末端峰形的分布基本一致的结论。     

A new application of stopped-flow chiral HPLC: inversion of enantiomer elution order

A newly developed procedure to reverse the enantiomer elution order of compounds resolved on chiral stationary phases (CSPs) for HPLC is presented. The optimized analytical protocol is based on the effect of temperature on enantioselectivity and does not involve any changing in mobile phase composition or type of CSP. In essence, the approach entails variable temperature chromatography at two temperatures. The enantiomer separation is performed at a low column temperature, with stopping the flow prior to elution of the less retained enantiomer. Then, the column temperature is changed with the peaks trapped inside the column, followed by elution with the same mobile phase in reverse direction. Under these conditions, the more pronounced loss in free energy of binding for the more strongly bound enantiomer results in an inversion of the elution order. This procedure may be applied to each enantiomer pair that is separated by chiral HPLC under an appreciable enthalpy-control.     

pH-zone-refining counter-current chromatography: Origin,mechanism, procedure and applications

Since 1980, high-speed counter-current chromatography (HSCCC) has been used for separation and purification of natural and synthetic products in a standard elution mode. In 1991, a novel elution mode called pH-zone refining CCC was introduced from an incidental discovery that an organic acid in the sample solution formed the sharp peak of an acid analyte. The cause of this sharp peak formation was found to be bromoacetic acid present in the sample solution which formed a sharp trailing border to trap the acidic analyte. Further studies on the separation of DNP-amino acids with three spacer acids in the stationary phase revealed that increased sample size resulted in the formation of fused rectangular peaks, each preserving high purity and zone pH with sharp boundaries. The mechanism of this phenomenon was found to be the formation of a sharp trailing border of an acid (retainer) in the column which moves at a lower rate than that of the mobile phase. In order to facilitate the application of the method, a new method was devised using a set of retainer and eluter to form a sharp retainer rear border which moves through the column at a desired rate regardless of the composition of the two-phase solvent system. This was achieved by adding the retainer in the stationary phase and the eluter in the mobile phase at a given molar ratio. Using this new method the hydrodynamics of pH-zone-refining CCC was diagrammatically illustrated by three acidic samples. In this review paper, typical pH-zone-refining CCC separations were presented, including affinity separations with a ligand and a separation of a racemic mixture using a chiral selector in the stationary phase. Major characteristics of pH-zone-refining CCC over conventional HSCCC are as follows: the sample loading capacity is increased over 10 times; fractions are highly concentrated near saturation level; yield is improved by increasing the sample size; minute charged compounds are concentrated and detected at the peak boundaries; and elution peaks are monitored with a pH flow meter for compounds with no chromophore. Since 1994, over 70 research papers on pH-zone-refining CCC have been published with the trends increasing in the recent years.     

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.     

Influence of column radial heterogeneity on peak fronting in linear chromatography.

Using numerical calculations of elution peak profiles, an explanation of the fronting behavior of elution peaks in linear chromatography was found in certain radial distributions of the mobile phase flow velocity and local bed efficiency. Fronting peaks are observed only if the flow velocity is higher in the wall region than in the center part of the column and the local efficiency is lower near the wall than in the center. By contrast, tailing or symmetrical peaks are observed if only the flow velocity or the local efficiency are radially heterogeneous. The degree of peak fronting increases with increasing amplitude of the radial distributions. The influence of the radial heterogeneity of the flow velocity on the degree of peak fronting is more severe for high than for low efficiency columns. An equation is suggested to correlate peak fronting behavior for columns of different efficiencies and a procedure proposed for the estimation of the radial distributions of the flow velocity and the local efficiency by analyzing some characteristics of asymmetric peaks.     

The gradient volume principle for fast evaluation of optimum conditions for isocratic analysis

Summary The paper describes by simple experiments in a pragmatical way by easy rules of thumbs gradient optimization. Besides selection of the stationary phase and initial and final conditions the two other important variables are program time and eluent flow rate. It is demonstrated, that when the product of both, the gradient volume, is kept constant, the solutes are always eluted with the same eluent composition at column outlet. At constant gradient volume, peak broadening depends on flow rate and on the eluent properties (viscosity) at which the solutes elute, and on the time the solutes spend in the column. Because peak broadening increases with increasing gradient volume, the peak capacity in gradient elution shows an optimum at gradient volumes around 15 empty column volumes (program times 45 to 60 min at flow rates of 1 ml/min with standard columns).Gradient elution can also be used for fast evaluation of optimum eluent composition for isocratic analysis. This procedure requires a calibration of the equipment for determination of eluent composition at column outlet. The sample is chromatographed in a standard gradient run of 10 to 15 empty column volumes. The eluent composition at which the solute of interest elutes during the gradient is used for isocratic analysis, where the k' value of this solute will then be around 2.Part of Ph. D. Thesis H. Elgass, Saarbrücken, 1978, present address Hewlett-Packard, Waldbronn, FRG. In part presented at Eastern Analytical Symposium, New York, 1982.     

Migration of neutral solutes by double stepwise gradient elution in capillary electrochromatography

Characteristics of electroosmotic flow (EOF) and the migration of neutral solutes under double stepwise gradient elution in capillary electrochromatography were studied systematically. EOF velocity proved to be the function of operation time changing with the introduction of the second mobile phase. Accordingly, the retention of components also changed. The migration of neutral solutes was studied under the following three situations; A, components eluted when the column was filled only with the first kind of mobile phase; B, solutes eluted still in the first kind of mobile phase while at that time two kinds of mobile phase coexisted in the column and C, samples eluted in the second kind of mobile phase. Equations to describe the retention times of components under these three kinds of conditions were deduced and applied to predict the retention times of 12 aromatic compounds. Relative errors between experimental and calculated values were below 5.0%, which proved the reliability of the equations. In addition, parameters that might affect the retention time of solutes, such as the transferring time of mobile phase vials, the capacity factors of components and EOF velocities two steps were studied systematically.     

An estimation of the column efficiency made by analyzing tailing peak profiles

It has been shown previously that most columns are not radially homogeneous but exhibit radial distributions of the mobile phase flow velocity and the local efficiency. Both distributions are best approximated by fourth-order polynomial, with the velocity in the column center being maximum for most packed columns and minimum for monolithic columns. These distributions may be an important source of tailing of elution peaks. The numerical calculation of elution peaks shows how peak tailing is related to the characteristics of these two distributions. An approach is proposed that permits estimations of the true efficiency and of the degree of column radial heterogeneity by inversing this calculation and using the tailing profiles of the elution peaks that are experimentally measured. This method was applied in two concrete cases of tailing peak profiles that had been previously reported and were analyzed by applying this new inverse approach. The results obtained prove its validity and demonstrate that this numerical method is effective for deriving the true column efficiency from experimental tailing profiles.     

Structural radial heterogeneity of a silica-based wide-bore monolithic column

The radial distribution of the main characteristics (elution time and standard deviation) of the elution profiles of a flat injected band recorded at the exit of a monolithic column were determined. These distributions provide the radial distributions of the average mobile phase velocity, the elution time and the maximum height of the peak of an analyte, the column efficiency and the analyte concentration. The band profiles were measured at the exit of a 10-mm i.d., 100-mm long silica-based monolithic column. An on-column local electrochemical amperometric detector allowed the recording of the elution profiles at different spatial positions throughout the column cross-section. The local spatial distribution of the mobile phase velocity does not follow a piston-flow behavior but exhibits radial heterogeneity. The local efficiency near the wall is lower than that near the column center. The radial distribution of the maximum concentration of the peaks varies throughout the column exit section, partially due to the radial variations of the column efficiency. These results might explain the rather large value of the A term of the Van Deemter or the Knox equations reported previously for monolithic columns.     

Optimization of capillary parameters for gas chromatography

An HETP equation for the capillary column is developed that takes into account the dependence of gaseous diffusion on pressure, the compressibility of the mobile phase, together with the unique relationship between mobile phase velocity, and the resistance to mass transfer in the stationary phase. The equation is used to develop a procedure for column optimization and expressions are derived that allow the optimum column radius and optimum column length to be calculated for a given fixed inlet pressure. It is shown that fast, simple separations are optimally achieved using relatively short small diameter columns. Conversely, optimum performance for the separation of complex mixtures requiring higher efficiencies requires the use of long columns with relatively large diameters.     

Efficiency and Resolution in Countercurrrent Chromatography

Abstract

Efficiency and resolution have been studied with a CCC apparatus classified by Y. Ito¹ as a HDES type J, and known as the HSCCC or the Multilayer Coil Separator Extractor. It has been shown that the efficiency decreases when increasing the linear velocity of the mobile phase in the column, which can be done by increasing the flow rate, or by decreasing the volume of the mobile phase in the column. The two ways of changing the linear velocity are not equivalent, since, at constant linear velocity, the efficiency increases when the volume of the mobile phase is well balanced with that of stationary phase. For each flow rate there is a minimum value for the volume of the mobile phase, and the relationship is linear. Working under these conditions, changing both the flow rate and the volume of the mobile phase leads to an efficiency which goes through a minimum at medium flow rates.

The resolution does not show this minimum: it is best at low flow rates and low volumes of mobile phase, and its variations are more important at low flow rates. This means that, if the resolution is good at moderate flow rates, it will remain good at higher flow rates. Combining three equations, it has been possible to predict the resolution when working with the minimum volume of mobile phase for a given flow rate.