Injection profiles in liquid chromatography. I. A fundamental investigation

This is a fundamental experimental and theoretical investigation on how the injection profile depends on important experimental parameters. The experiments revealed that the injection profile becomes more eroded with increased (i) flow rate, (ii) viscosity of the eluent, (iii) size of the solute, (iv) injection volume and (v) inner diameter of the injection loop capillary. These observations cannot be explained by a 1D-convection-diffusion equation, since it does not account for the effect of the parabolic flow and the radial diffusion on the elution profile. Therefore, the 1D model was expanded into a 2D-convection-diffusion equation with cylindrical coordinates, a model that showed a good agreement with the experimental injection profiles dependence on the experimental parameters. For a deeper understanding of the appearance of the injection profile the 2D model is excellent, but to account for injection profiles of various injection volumes and flow rates in preparative and process-chromatography using computer-optimizations, a more pragmatic approach must be developed. The result will give guidelines about how to reduce the extra-column variance caused by the injection profile. This is important both for preparative and analytical chromatography; in particular for modern analytical systems using short and narrow columns.     

On the minimization of the band-broadening contributions of a modern, very high pressure liquid chromatograph

The contributions of the volume of sample injected, the mobile phase flow rate, the inner diameter of the needle seat capillary and that of the connector capillary, the heat exchanger, and the detector cell volume to the widths of bands eluted from the 1290 Infinity HPLC instrument were investigated in depth. Four sample volumes (0.16, 0.80, 4.0, and 20 μL), three flow rates (0.04, 0.4, and 4.0 mL/min), two needle seat capillary I.D. (100 mm × 115 and 140 μm), three sets of connector capillary I.D. (350 mm × 80, 115, and 140 μm placed upstream the column, and 220 mm × 80, 115, and 140 μm downstream the column), two UV detector cell volumes (0.8 and 2.4 μL), and the presence/absence of the heat exchanger (1.6 μL) between the inlet connector capillary tube and the column were combined to generate up to 4 × 3 × 2 × 3 × 2 × 2=288 system configurations for this instrument. For each configuration, 5 consecutive injections were performed in order to assess the injection-to-injection repeatability, providing 1440 elution band profiles which are analyzed. The results demonstrate that the band broadening contribution of the instrument depends mostly on the detector cell volume and on the inner diameter of the needle seat capillary tube. The impact of these two contributions is particularly important at high flow rates (4 mL/min). Best efficiencies are obtained with a small sample volume, below 1 μL, which avoids volume overload of the instrument, or with large sample volumes, which maximize the radial concentration gradients of the sample across the instrument channels, in the vicinity of the anfractuosities of the channel walls. The injection of large sample volumes reveals the imperfection of current injection systems, the performance of which is remote from the one expected to provide an ideal rectangular injection (～+4 μL(2)). Although the present behavior of the instrument is satisfactory, serious improvements would become necessary to operate the next generation of more efficient columns that might be commercialized soon.     

Performance of experimental sample injectors for high-performance liquid chromatography microcolumns

An experimental injector for HPLC microcolumns and a 3-nl conductivity detector connected directly to the injector outlet with a 19-nl tube were used to study injector dispersion, guide the design of improved injectors, and suggest appropriate injection techniques. With regard to the small injection volumes required when no on-column concentration technique is used, we show that in some circumstances: (i) there are two volumes to be considered, the sample volume (that which is intended to be injected) and the effective injection volume (that which contains all the sample after it has completely emerged from the injector). Due to dispersion, the latter is often many times the former. An injector performance factor is defined as the ratio of the two volumes. (ii) A smaller sample chamber volume in an injector does not necessarily produce a proportionately smaller effective injection volume, in which case there is a reduction of peak height that degrades sensitivity without a commensurate reduction in peak width that would improve resolution. (iii) Adjusting the geometry of the sample chamber and stator passage can significantly improve injector performance, as illustrated for sample volumes from 2 nl to 1 microl. (iv) In some cases, reducing the diameter of an injector passageway in an attempt to reduce dispersion actually causes performance to worsen.     

Evaluation of loop and pump injection systems in preparative liquid chromatography

The usefulness of pump or loop injectors in preparative liquid chromatography was evaluated. The concentration distribution of samples flowing through a UV detector cell, directly connected to the injector, was recorded at various flow velocities, at various diameters of loop tubing, and using various methods of injection [injection with sample volumes equal to a fraction or total loop volume, etc.]. The most advantageous methods were found to be either use of a loop injector to inject only a fraction of its total volume or use of a pump. Both of these methods ensure a almost rectangular concentration distribution.     

Novel three-dimensional in-cavity transient temperature measurements in injection molding and fluid-assisted injection molding

This report presents novel experimental set-ups which allow the measurement of three-dimensional temperature fields in the depths of injection molding cavities throughout molding cycles. Two specific molds equipped with two types of temperature measuring devices, i.e., a mesh type device consisting of thermocouples mounted on metal wires and a tubular type device consisting of tubular needles guiding embedded micro-thermocouples inside the cavities, have been designed and built. Temperature distributions in both injection molding and fluid assisted injection molding processes, including gas and water assisted injection molding, were measured and recorded by a data acquisition system on a personal computer. It was found that the tubular type device induced much less flow disturbance and provided accurate temperature profiles. Among the processes, the water assisted injection molding process required the least time to cool the parts, followed by gas assisted injection molding and conventional injection molding. In addition, the experimental results also suggested that the shear heating by viscous dissipation in the runners leads to a significant increase in melt temperature. A precise measurement of the in-depth temperature profile can be helpful to better understand the molding phenomena, to validate numerical simulation results, as well as to optimize the parameters for the molding processes.     

Constrained optimization of a preparative ion-exchange step for antibody purification

Today, the optimization of chromatographic separation is usually based on experimental work and rule of thumb. The process and analytical technology (PAT) initiative, of the US Food and Drug Administration, has provided the opportunity of using model-based approach when designing downstream processing of pharmaceutical substances. A nonlinear chromatography model was used in this study to optimize a preparative ion-exchange separation step involving two components. Separation was simulated with the general rate model employing Langmuir kinetics. Optimization was performed with an indirect method allowing constraints on the purity, thus avoiding sub-optimization, which can lead to noisy objective functions. The six decision variables used in the optimizations were flow rate, loading volume, initial salt concentration in the elution, final salt concentration in the linear elution gradient and the two cut points. A graphical representation of the effect of the decision variables on the objective function was used to verify that the optimization had converged to the true optimum. The optimal operating points, using productivity and yield separately as objective functions, were found and compared with the product of productivity and yield as objective function. The optimum obtained with this objective function had a lower productivity, than the productivity function, but much higher yield, which makes it a good substitute for a cost function.     

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.     

Strategy for developing and optimizing liquid chromatography methods in pharmaceutical development using computer-assisted screening and Plackett-Burman experimental design

We describe a three-step method development/optimization strategy for HPLC assay/impurity methods for pharmaceuticals, which include multiple-column/mobile phase screening using a system equipped with a column-switching device, further optimization of separation by using multiple organic modifiers in the mobile phase, and multiple-factor method optimization using Plackett-Burman experimental designs. In the first two steps, commercially available chromatography optimization software, DryLab, was used to perform computer simulations. This allows the method developer to evaluate each condition (one column/mobile phase combination) with retention data from two scouting gradient runs. This approach significantly reduces the number of runs in method development. After a satisfactory separation was obtained, we used a method optimization step with Plackett-Burman experimental designs. The purpose of the 16-injection set experiments was to evaluate nine method factors with regard to method precision, accuracy, sensitivity and specificity. The results provided logical justifications in selecting method parameters such as column temperature, detection wavelength, injection volume, and sample solvent, etc. In data analysis, instead of the traditional mathematical manipulations, we used the graphical methods to examine and present data by creating the so-called main effect plots. Because replicates of design points were not run, the data did not allow the testing of statistical significance. However, it provided visual presentations in a way that is easy to understand for the method developer and end user alike.     

Easy and accurate high-performance liquid chromatography retention prediction with different gradients, flow rates, and instruments by back-calculation of gradient and flow rate profiles

Isocratic retention data should make a suitable foundation for an accurate, cross-instrument LC retention prediction system. Our previous work suggested that in order to accurately calculate (or "project") gradient retention times on a wide range of HPLC systems using a single set of isocratic retention data, the precise shape of both the gradient and flow rate profiles produced by each instrument must be properly taken into account. However, accurate measurement of these system properties is difficult and time-consuming. In this work, we describe an approach that uses the measured gradient retention times of a set of standard solutes spiked into the sample along with their known isocratic retention vs. eluent composition relationships to determine the effective gradient and flow rate profiles by back-calculation. Retention "projections" of 20 other solutes using these back-calculated profiles, under various chromatographic conditions typical of metabolomics experiments, were remarkably accurate (as good as 0.23% of the gradient time, R2 up to 0.99996), being very near the level of retention reproducibility. Our calculations suggest that this level of accuracy will allow a quadrupole MS to identify 38-fold more compounds out of a simulated mixture of 7307; it would allow an FTICR-MS to improve its identification rate nearly two-fold with the same mixture. Moreover, very little effort is required of the user. This approach provides a simple way to correct for all instrument-related factors affecting retention, allowing dramatically streamlined and improved retention projection across gradients, flow rates, and HPLC instruments.     

10°锥角台锥型液相色谱柱尺寸等比例放大和柱内谱带流型的可视化研究

摘要利用改进的可视化装置, 研究了10°锥角的台锥型液相色谱柱内的谱带流型与柱参数变化的关系. 将有机玻璃柱管加工成内圆台外方型的一体结构, 选择折射率一致的色谱固定相硅胶和流动相环己烷, 使整个色谱柱成为高度清晰的透明体, 能直接观察柱中彩色样品谱带的动态三维流型. 研究结果表明, 在实验条件范围内, 流动相流速对谱带流型无影响, 填料的形状和性质对塞子状流型有一定程度的影响. 比较了柱长为5cm和等比例放大后柱长为10cm锥型柱内的流型, 发现放大后的锥型柱内仍然保持塞子状流型, 总柱效等比例增加. 表明继续按比例放大成为工业规模色谱柱后仍能保持塞子状流型.     

Comparison of activated chromatography resins for protein immobilization

The adsorption of bovine serum albumin (BSA) to an immobilized camelid‐derived antibody fragment was investigated using six different activated resins, of which two are prototypes. The resins differed in base material, coupling chemistry and particle size. The adsorption, washing and desorption stage of the affinity chromatography process were taken into account. Dynamic binding capacities at 10% breakthrough ranged between 0.76 and 4.8 mg BSA/mL resin. The washing volume ranged between 2.9 and 10 column volumes. One of the resins did not concentrate BSA, while the highest concentration was 13‐fold. We present a method to rank and weigh the properties of the resins to find the optimal resin to meet specific requirements. For three of the resins the adsorption flow rate was varied, while the washing and desorption flow rate was kept the same. The dynamic binding capacity decreased with increasing flow rate, as expected. For one resin, the washing volume remained constant, but for the others it decreased with increasing adsorption flow rate. The number of column volumes required to purify a given amount of BSA increases with increasing flow rate, which indicates that higher flow rates do not necessarily speed up the process.     

计算机辅助高效液相色谱梯度洗脱的优化

本文介绍了计算机辅助反相高效液相色谱梯度洗脱优化分离的方法。首先采用混合设计法，通过９个预试验建立目标函数，然后运用双因素（初始流动相组成Ｃ和梯度时间Ｔ）扫描技术，由计算机给出优化分离条件，实验结果和预示完全一致。     

Optimization of two-factor(pH and ion concentration)simultaneous selection in HPLC using a statistical method

A computer-assisted method is presented for simultaneous optimization of pH and ion con-centration selection for the optimal separation in reversed-phase HPLC.The method is based on apolynomial estimation from nine preliminary experiments according to two-factor rectangular design.This is followed by a two-dimension computer scanning technique.Resolution is used as the selectioncriterion.Good agreement was obtained between predicted data and experimental results.     

Simulation of droplet heating and desolvation in an inductively coupled plasma — Part I

The total desolvation rate of sample droplets in an argon inductively coupled plasma (Ar ICP) is investigated through the development of a two-phase continuum flow computer model. The desolvation model is supplemented by equations used to determine the trajectories of particles through the plasma. The model is used to calculate the behavior of aerosol droplets from a direct injection high efficiency nebulizer (DIHEN), a micronebulizer used to inject microliter quantities of samples that are toxic, expensive, or of limited volume. We use the combination of desolvation and transport models to present the first predicted spatial distribution of droplet concentrations and evaporation rates in an ICP flow. These data are compared with the behavior of a DIHEN spray in an environment with no net argon gas flow to determine the importance of gas flow rates to overall droplet concentration profiles in the ICP. In addition, two separate techniques (Stokes’ equation and the direct simulation Monte Carlo treatment) for determining droplet trajectories are contrasted.     

Statistical scanning method for the optimization of gradient elution high performance liquid chromatography

Summary A computer-assisted method is presented for the optimization of separation in gradient elution reversed-phase HPLC. The method is based on a polynomial estimation from nine preliminary experiments according to a two-factor (initial solvent composition C and gradient time T) rectangular design. This is followed by a two-dimension computer scanning technique. Resolution is used as the selection criterion. Good agreement was obtained between predicted data and experimental results.     

Finite-element analysis of craze deformation

The finite-element method is applied to a craze model in order to estimate surface displacements and stresses of an isolated craze in a thin rectangular plate. The limitation of assuming linear-elastic behavior of the craze fibrils is demonstrated by comparing computed profiles with published experimental ones. Accurate finite-element estimates of craze surface stresses are obtained from the experimental displacement profile. The analysis is extended to the three-dimensional case but the difference between the craze displacements at the surface of the plate and those at midthickness is too small to justify the considerable increase in computer time in comparison with that required for two-dimensional analysis.     

Flow-rate dependence of gel permeation chromatography

Analysis of polystyrene standards by gel permeation chromatography over a wide range of flow rates revealed two sources of error in volume measurement. These errors arise from solvent evaporation in the siphon chamber and from solvent continuing to flow into the siphon during discharge. Appropriate corrections are discussed, and a vapor feedback loop to eliminate the solvent evaporation error is described. The flow rate dependence of the GPC calibration curve, expressed in the corrected elution volumes, appears different from that reported in the literature. The corrected flow rate dependence of peak elution volumes is in agreement with the expectation of diffusion and exclusion theories.     

Improvement of lipoprotein separation with a guard channel prior to asymmetrical flow field-flow fractionation using fluorescence detection

In this article, a simple experimental approach to improve lipoprotein separation and detection in flow field-flow fractionation (FlFFF) is detailed. Lipoproteins are globular particles composed of lipids and proteins in blood serum and their roles include transferring fats and cholesterols through blood vessels throughout the body. Especially, presence of small, dense low-density lipoproteins (LDL) is associated with cardiovascular risk. Two experimental approaches were explored in this study: an increase in the reproducibility of LDL particle separation by implementing a guard channel prior to an asymmetrical FlFFF (AFlFFF) channel in order to deplete small molecular weight serum proteins and reducing the required injection volume of a serum sample by implementing fluorescence detection. The guard channel was made of a simple hollow fiber module so that the serum sample can be washed with the help of radial flow prior to injection into the AFlFFF channel. The channel was tested with protein standards and serum samples to ensure precision of the retention time and the protein recovery rate. A fluorescent phospholipid dye was utilized to label lipoprotein particles before separation for fluorescence detection, which resulted in a reduction of the required injection volume of serum.     

Theoretical and experimental aspects of the response of stripping voltammetry in flow injection systems

The coupling of stripping voltammetry with flow injection systems offers significant advantages over conventional stripping methods. Equations solved for conventional stripping voltammetry, with steady-state deposition current, are not applicable to flow injection systems. Theoretical equations for the peak current under flow injection conditions are derived. As the total charge passed during the deposition step, ∫i dt, is independent of the shape of the sample plug, the stripping peak current is independent of the degree of dispersion. As a result, precise control of the dispersion or deposition period may not be required. The peak current is predicted to be directly proportional to the sample volume. Experimental results are incorporated to support the theoretical conclusions. The effects of experimental variables such as flow rate, length of tubing, deposition period, or sample volume are presented using cadmium ion as test species.