Principle and Potential of Gradient Elution in Planar Chromatography

It was the intention of this paper to show some recent developments in thin layer chroma-togrphy aiming at increasing the separation efficiency by instrumental means and techniquesusing existing separation layers. Gradient elution in the normal phase is a most efficient wayto achieve this goal. Multi-dimensional separations by coupling gradient elution column liquid chromatography inthe reversed phase with AMD gradient elution in normal phase is suitable to achieve newdimensions of separation numbers. Assuming that the N numbers reported for HPLC translateinto separation numbers near 100, by coupling the two kinds of chromatography, separationnumbers around 500 become practically usable.     

Theoretical background of short chromatographic layers. Optimization of gradient elution in short columns

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.     

High-performance chromatofocusing using linear and concave pH gradients formed with simple buffer mixtures. II. Separation of proteins

The separation of proteins using high-performance chromatofocusing with linear or concave pH gradients formed using simple mixtures of buffering species in the elution buffer is investigated experimentally. The separation achieved is comparable to that using polyampholyte elution buffers with these types of systems. More specifically, protein band widths at one half of the band height in the range between 0.1 and 0.025 pH units were observed, and good resolution was achieved of protein variants differing by a single amino acid residue in separation times of 30 min or less. An especially useful elution buffer is investigated that contains only four buffering species and that produces a linear pH gradient in the range between pH 9.5 and 6.0 when used together with a particular high-performance column packing made specifically for chromatofocusing. This elution buffer and column packing combination is evaluated by using it for the chromatofocusing of equine myoglobin and human hemoglobin variants. Additional applications are described in which a polyethyleneimine derivatized silica column packing and a pH gradient that is concave in shape are used for the separation of proteins in an E. coli cell lysate.     

Characterization of unstable ion-exchange chromatographic separation of proteins.

Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within +/-0.1 unit and the ionic strength within +/-0.002 mol/l in order to meet the criteria (+/-5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.     

Separation of free sterols by high temperature liquid chromatography

Increasing the column temperature accelerates markedly elution in HPLC. The separation of five free sterols was studied on three packing materials that can withstand high temperatures. These stationary phases included graphitic carbon, a polymeric C18 silica, and a zirconia-based adsorbent. Measurements of retention data were made at up to 150 degrees C with mobile phases of different compositions. Since the columns tested afford different retention mechanisms, a variety of elution patterns were observed, with some being more advantageous than others for certain sterol separations. Effects observed include some selectivity improvements and some elution order reversals. The separation of free sterols in selected fruit juices is also presented. Albeit at the expense of a longer analysis time, the graphitic carbon column produced the best separation of the sterols in this study.     

Separation of antidepressants by capillary electrophoresis with in-line solid-phase extraction using a novel monolithic adsorbent

The separation of three selective serotonin reuptake inhibitors (SSRIs) by capillary electrophoresis (CE) with fully integrated solid-phase extraction (SPE) is described. Polymeric monolithic SPE modules were prepared in situ within a fused silica capillary from either butyl methacrylate-co-ethylene dimethacrylate or 3-sulfopropyl methacrylate-co-butyl methacrylate-co-ethylene dimethacrylate. Using a 1 cm SPE module placed at the inlet of the capillary, a mixture of sertraline, fluoxetine and fluvoxamine was extracted from aqueous solution by applying a simple pressure rinse. Under pressure-driven conditions, efficient elution was possible from both SPE materials investigated using 50 mM phosphate buffer, pH 3.5 in acetonitrile (20/80, v/v). Two different strategies were investigated for the efficient elution and subsequent CE separation. Injection of an aqueous sample plug directly into the non-aqueous elution/separation buffer was found to be unsuitable with poor elution profiles observed in the electrodriven mode. Alternatively, a sample plug equivalent to several capillary volumes could be injected by pressure followed by filling the capillary with the non-aqueous elution/separation buffer from the outlet end using a combination of pressure and electrodriven flow. Using a neutral monolith, efficient elution/separation was not possible due to an unstable electroosmotic flow (EOF), however, by adding the ionisable monomer, 3-sulfopropyl methacrylate to the SPE module to increase and stabilise the EOF, it was possible to achieve efficient elution from the SPE module, followed by baseline separation by CE using a 200 mM acetate buffer, pH 3.5 in acetonitrile (10/90, v/v). With enrichment factors of over 500 achieved for each of the analytes this demonstrates the potential of in-line SPE-CE for the sensitive analysis of these drugs.     

分子印迹手性整体柱的制备及对非对映异构体的分离

 采用原位分子印迹技术 ,单步制备了一种辛可宁印迹的手性整体柱。为了提高柱效和选择性 ,选择了相对低极性的甲苯 /十二醇复合致孔体系。在等度及梯度洗脱条件下 ,非对映异构体辛可宁与辛可尼丁被完全分离。等度洗脱中相对较宽的峰可以在梯度洗脱中得到改善。同时考察了流动相中醋酸浓度、流速以及温度对分离的影响。由于柱中存在大的流通孔 ,大大降低了分离过程中的柱压降 ,从而使这种柱能够在相对高的流速下使用。提高温度可以提高分离因子 ,在 60℃获得最大分离因子 5 40。     

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.     

Determination of Pollutant Phenols by Capillary High‐Performance Liquid Chromatography with UV Detection

This paper describes a liquid chromatographic method using a reversed phase capillary column coupled to an UV detector for the quantitation of thirteen pollutant phenols. Chromatographic separation was carried out with gradient elution at 25.0 ± 0.1°C. The two major anisocratic elution modes (gradient elution and temperature programming) were evaluated. The detection limit range was 10–81 pg (100 nL injected). The chromatographic method combined with liquid‐liquid extraction was applied to analysis of these compounds in river water. Recoveries of 75–103% were achieved for most of them.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

采用QSPR模型预测手性二芳基甲烷衍生物保留因子与分离因子

对手性化合物的保留因子和分离因子进行定量结构-特征关系(QSPR)研究, 对于预测保留因子和分离因子甚至对映体的洗脱顺序都起着重要作用. 本文选择手性二芳基甲烷衍生物为研究对象, 采用VolSurf程序计算分子结构参数, 并分别在其与保留因子以及分离因子间建立模型, 采用测试集外部检验、留多法交叉验证和Y随机性检验等方法对分离因子模型的鲁棒性进行了评估, 结果令人满意. 对变量进行分析显示, 分子的球形性, 中等能级的亲水区、亲水-亲脂平衡、两亲矩、合适的氢键给体和受体均有利于异构体在手性固定相上的保留; 一对对映体的高能级的亲水区、低能级的疏水区、两亲矩、合适的氢键给体和受体以及阴离子区之间大的差异对对映体在手性固定相上的分离是有利的. 利用这些模型, 可以轻松地预测对映体的保留因子和分离因子, 甚至洗脱顺序.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Characterization of voltage degradation in dynamic field gradient focusing

Dynamic field gradient focusing (DFGF) is an equilibrium gradient method that utilizes an electric field gradient to simultaneously separate and concentrate charged analytes based on their individual electrophoretic mobilities. This work describes the use of a 2-D nonlinear, numerical simulation to examine the impact of voltage loss from the electrodes to the separation channel, termed voltage degradation, and distortions in the electric field on the performance of DFGF. One of the design parameters that has a large impact on the degree of voltage degradation is the placement of the electrodes in relation to the separation channel. The simulation shows that a distance of about 3 mm from the electrodes to the separation channel gives the electric field profile with least amount of voltage degradation. The simulation was also used to describe the elution of focused protein peaks. The simulation shows that elution under constant electric field gradient gives better performance than elution through shallowing of the electric field. Qualitative agreement between the numerical simulation and experimental results is shown. The simulation also illustrates that the presence of a defocusing region at the cathodic end of the separation channel causes peak dispersion during elution. The numerical model is then used to design a system that does not suffer from a defocusing region. Peaks eluted under this design experienced no band broadening in our simulations. Preliminary experimental results using the redesigned chamber are shown.     

Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.     

Development of an ion chromatographic gradient retention model from isocratic elution experiments

When facing separation problems in ion chromatography, chromatographers often lack guidelines to decide a priori if isocratic elution will give enough separation in a reasonable analysis time or a gradient elution will be required. This situation may be solved by the prediction of retention in gradient elution mode by using isocratic experimental data. This work describes the development of an ion chromatographic gradient elution retention model for fluoride, chloride, nitrite, bromide, nitrate, sulfate and phosphate by using isocratic experimental data. The isocratic elution retention model was developed by applying a polynomial relation between the logarithm of the retention factor and logarithm of the concentration of competing ions; the gradient elution retention model was based on the stepwise numerical integration of the corresponding differential equation. It was shown that the developed gradient elution retention model was not significantly affected by transferring data form isocratic experiment. The root mean squared prediction error for gradient elution retention model was between 0.0863 for fluoride and 0.7027 for bromide proving a very good predictive ability of developed gradient elution retention model.     

Development of a micro-total analysis system (μ-TAS) for the determination of catecholamines

In this study, the first micro-total analysis system (μ-TAS) for catecholamines (dopamine, epinephrine, and norepinephrine) analysis in which preconcentration, separation, and determination steps were integrated on a microchip was developed. Electrophoresis microchips in a variety of channel lengths and designs were produced in borofloat glass for the μ-TAS studies. Chambers for the preparation of monolithic disks were formed in the microchips at the intersection of the injection and separation channels. Vinyl phenylboronic acid–ethylene glycol dimethacrylate polymers were prepared as monolithic disks in these chambers with a depth of 0.05 mm and a diameter of 2.1 mm. The microchips could be used more than 50 times if mechanical problems such as plugging or fracturing did not occur. Adsorption and elution of catecholamines were realized electrokinetically, with catecholamines determined via laser-induced native fluorescence detection following elution and electrophoretic separation. The most promising results were obtained with 100 mM phosphate buffer (pH 2) for elution with 25% propanol added to the separation buffer (100 mM phosphate, pH 3).     

Superheated water chromatography of low molecular weight polyethylene glycols with ultraviolet detection.

Superheated water chromatography (SWC) with ultraviolet detection was applied to the separation of low molecular weight polyethylene glycols (PEGs). PEG oligomers could be detected sensitively when the detection wavelength was set at 190 nm. The effect of column temperature on the separation of PEG oligomers was investigated. The elution time of all PEG oligomers decreased with increase in the column temperature; linear relationships were obtained between ln k and 1/T. A temperature-programmed SWC separation enabled the baseline separation of a PEG 200 sample within 50 min.     

Rational method for designing efficient separations by chromatography on polystyrene-divinylbenzene resins eluted with aqueous ethanol

A rational method for designing separation processes by chromatography with polystyrene-divinylbenzene (PS-DVB) resins of different particle diameters (10-400microm) was developed. As model samples, catechin and epigallocatehin gallate (EGCG) were chosen and the mobile phase was an ethanol-water mixture. Linear gradient elution experiments were carried out with different gradient slopes, and the peak ethanol concentration was plotted against the normalized gradient slope. The plots were similar regardless of particle diameter. From these plots the two parameters describing the distribution coefficient K as a function of ethanol concentration I were determined. The K-I curves obtained were verified by isocratic elution experiments, and the conditions at which a baseline separation of the two polyphenols is possible was sought, and confirmed experimentally. Stepwise elution experiments were also designed and performed successfully on the basis of the K-I curve.     

Separation of purine bases, nucleosides and nucleotides by a column-switching technique combining reversed-phase and anion-exchange high-performance liquid chromatography

An on-line two-stage column chromatographic technique is described which combines reversed-phase and anion-exchange chromatography for the separation of purine nucleic acid components. The elution program applied, consisting of two gradient programmes, provides a separation of bases and nucleosides on the octadecyl silica column and a separation of the nucleotides on the anion-exchange column to which they have been switched at the beginning of the elution. This method is easy to modify for special problems and can be used when establishing a complete profile of purines.     

Ion-exchange separation of nucleic acid constituents by high-performance liquid chromatography

The high-performance liquid chromatographic separation of a large variety of nucleic acid constituents on a silica-based, weak-anion exchange column was accomplished. Using this technique it was possible to achieve some relatively difficult separations, such as the separation of 2'-, 3'-, and 5'-AMP, and the separation of a mixture of ribo- and deoxyribo-nucleosides and -nucleotides. A number of other separations are demonstrated by isocratic or gradient elution. These include the separation of a mixture of nucleoside monophosphates, the separation of a mixture of nucleoside mono-, di-, and triphosphates, the separation of a mixture of nucleosides and bases, and the separation of a mixture of nucleotide oligomers. These chromatographic separations were accomplished using relatively simple experimental procedures at ambient temperatures and involved relatively short analysis times. Excellent separations were obtained, in most cases, by adjustment of buffer concentration and pH, or by addition of an organic modifier. In some cases, it was necessary to use gradient elution to achieve optimum resolution.