Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Design of mobile phase composition for liquid chromatography with an internal pH gradient

Summary A method is suggested for calculating the mobile phase composition for ion exchange chromatography with an internal pH gradient. An internal pH gradient of the desired steepness can be achieved providing the titration curve of the sorbent is known. The procedure has been verified by use in the design of a mobile phase composed from few anionic buffers for generation of internal pH gradient on a microcolumn packed with a cation exchanger with carboxylic functional groups.     

Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.     

蛋白折叠液相色谱法中流动相对重组人干扰素-γ质量回收率的影响

蛋白折叠液相色谱法(PFLC)用于变性蛋白质复性并同时纯化时对流动相组成及其洗脱条件的要求远较通常的液相色谱法高。用端基为PEG-200的高效疏水作用色谱固定相对重组人干扰素-γ(rhIFN-γ)进行纯化并同时复性,详细研究了流动相组成、梯度洗脱模式和流速对rhIFN-γ质量回收率和活性的影响。分别以3.0 mol/L (NH4)2SO4 +0.05 mol/L KH2PO4(pH 7.0)和0.05 mol/L KH2PO4(pH 7.0)为流动相A和B,采用35 min非线性梯度洗脱时,所得rhIFN-γ的质量回收率最高。     

Multiple Step Gradient Analysis in Stationary Phase Optimised Selectivity LC for the Analysis of Complex Mixtures

Stationary phase optimised selectivity liquid chromatography (SOSLC) is an approach to tune a given LC separation by combining different stationary phases in a multi- segment column set-up. The presently available SOSLC optimisation procedure and algorithm are, however, only applicable to isocratic conditions. This is a severe limitation for the analysis of mixtures composed of components covering a broad hydrophobicity range. A strategy is described to circumvent this limitation. The components of a mixture are divided into different groups according to hydrophobicity as elucidated by a gradient analysis on a C₁₈ reversed-phase column. Each group separation is then individually optimised with a specific isocratic mobile phase composition using the original SOSLC strategy. The mobile phase composition thereby only differs in the percentage of organic modifier between the various groups. Finally, a combination of stationary phases that guarantees sufficient selectivity for all the groups is selected and the separation is performed by a multiple step gradient, whereby each level consists of the mobile phase composition applied for the SOSLC optimisation of the individual groups. The multi step gradient approach is demonstrated through the analysis of a mixture of 27 steroids covering a wide range of hydrophobicity.     

Mixed‐mode chromatography integrated with high‐performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target

A type of mixed‐mode chromatography was integrated with high‐performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed‐mode column, the silica beads were activated with γ‐(2,3‐epoxypropoxy)‐propytrimethoxysilane and coupled with 4‐mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.     

Anion exchange liquid chromatography--inductively coupled plasma-mass spectrometry detection of the Co2+, Cu2+, Fe3+ and Ni2+ complexes of mugineic and deoxymugineic acid

Phytosiderophores, such as mugineic and deoxymugineic acid, are produced by graminaceous plant species in response to Fe deficiency conditions normally experienced in calcareous and alkaline non-calcareous soils. As these phytosiderophores have the ability to form thermodynamically stable complexes with other metal cations present in the growing medium, they have also been implicated in the transport and bioavailability of these metals in the environment. However, routine analytical methodology to detect the various metal complexes formed by these phytosiderophores is lacking. Therefore, as these complexes are negatively charged over a wide range of pH values, anion exchange liquid chromatography (AE LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) was investigated as a means to separate and quantify these complexes. The metal-phytosiderophore complexes were prepared at pH 7 and separated by NaOH or NH4NO3 gradient elution on a Dionex AS11 anion exchange column. Of the metals tested only the Co2+ and Ni2+ complexes of mugineic and deoxymugineic acid were detected when using a 0-20mM NaOH gradient elution profile. However, the phytosiderophore complexes of Cu2+ and Fe3+ were also detected when using NH4NO3 as the mobile phase at pH 7. Base-assisted hydrolysis of the latter two complexes is proposed to explain their apparent 'instability' in the high pH NaOH mobile phase. The absolute detection limits of the developed methodologies for these metal complexes ranged from 0.1 to 2.8pmol. As phytosiderophore complexes with Cd2+ and Zn2+ were not detected, it was concluded that the dissociation kinetics of these metal-phytosiderophore complexes were too rapid for these complexes to be observed in the present chromatographic conditions.     

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。     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

Liquid Chromatographic Separation of Some Common Benzodiazepines and their Metabolites

Abstract

A series of benzodiazepines commonly encountered in forensic samples were separated using isocratic reversed-phase liquid chromatography. These compounds display a wide range of capacity factors on a C₁₈ stationary phase in a pH 8 phosphate buffer and methanol mobile phase. Clorazepate must be analyzed under basic mobile phase conditions to prevent its decomposition to N-desmethyldiazepam. The separation of common parent benzodiazepines such as chlordiazepoxide, diazepam and flurazepam from their corresponding metabolites was achieved under a variety of reversed-phase conditions.     

Monitoring free silanols on reversed-phase supports with peptide standards

Summary A series of synthetic peptide HPLC standards has been designed for use in monitoring free silanols on silica-based reversed-phase supports. The four standards (+ 1 to + 4 net charge) showed great versatility in monitoring silanol activity over a pH range of 2.0 to 7.0. We have developed a silanol monitoring system based on the sensitivity of the standards to ionic interactions with reversed-phase packings at pH 7.0. This monitoring procedure involves chromatographing the peptides at pH 7.0 with a combined acetonitrile and sodium perchlorate linear AB gradient and is generally applicable to reversed-phase packings exhibiting a wide range of silanol concentrations. These standards not only demonstrate the presence and extent of free silanols, they also allow the researcher to determine what changes must be made to the mobile phase composition to minimize ionic interactions. In addition, the standards, in conjunction with the double gradient monitoring system at pH 7.0, will aid researchers and manufacturers in the improvement and development of reversed-phase matrices.     

熊果酸与齐墩果酸高效液相色谱分离条件优化

描述了熊果酸、齐墩果酸色谱保留行为与流动相pH和有机溶剂体积分数之间的函数关系,并求解了各自模型中的9个参数,得到在流动相中熊果酸的电离常数pKa=4.8145,齐墩果酸电离常数pKa=4.9317。文章还对模型外推过程中存在的误差进行了校正,并运用校正后的模型预测熊果酸的容量因子,实验值接近预测值,误差小于5%。最后采用熊果酸和齐墩果酸的容量因子与流动相pH和有机溶剂组成之间的函数关系,进行了两者在同一流动相中分离条件的优化,获得了最佳的分离条件:流动相中甲醇体积分数为85.2%,pH为6.50。     

Separation of different oligomers by supercritical fluid chromatography using gradient elution

Summary In supercritical fluid chromatography (SFC), the solubilizing nature of the mobile phase enables the use of composition gradients (gradient elution) like in liquid chromatography. With this gradient technique, oligomer separations have been achieved over wide molecular weight ranges. For separating oligomers which absorb only at low wavelengths, a carbon dioxide/acetonitrile mobile phase was used which allows UV detection down to 200 nm. As an ideally suited detection assembly for the chromatography of oligomers, a combination of mass-sensitive and evaporative light-scattering detection is suggested.

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Trennung von verschiedenen Oligomeren durch Chromatographie mit überkritischen fluiden Phasen unter Verwendung der Gradientenelution

     

Chiral discrimination of phenoxypropionic acid herbicide enantiomers on teicoplanin phase: methanol dependence and eluent pH consideration

The chiral recognition mechanism for a series of d,l phenoxypropionic acid herbicides (PPAs) on a teicoplanin stationary phase was investigated in reversed phase liquid chromatography (RPLC) over a wide range of mobile phase pH and column temperature. The effect of methanol on the enantiomeric separation was studied by varying its fraction (v/v) in the mobile phase. The thermodynamic data indicated that the chiral recognition was controlled by the interaction between the anionic form of the solute and the teicoplanin phase while those with the neutral form played a minor role. In addition, it was demonstrated that the enhancement of the separation factor observed as the methanol fraction increased in the mobile phase was enthalpically controlled owing to stereoselective binding interactions. Such behavior was used to optimize the chromatographic conditions for separation of PPAs herbicides on teicoplanin.     

五种不同类型土壤中有机化合物土壤吸附系数的预测

为了验证土壤柱液相色谱法测定农药的土壤吸附系数的普遍适用性和预测的准确性,采用了5种组成及性质差异较大、具有一定代表性的土壤样品,对在甲醇 水流动相条件下测定的保留因子与土壤吸附系数间的相关性进行了研究。结果表明,土壤柱液相色谱法是预测农药的土壤吸附系数的一种有效方法,适于多种类型土壤中农药土壤吸附系数的预测。     

pH gradient reversed-phase liquid chromatography as a fractionation tool for the separation of peptides

Recent reports from our laboratory presented a comprehensive theory and demonstrated feasibility of reversed-phase liquid chromatography (RP-LC) employing the programmed gradient of pH of the mobile phase. The aim of that work was to explore the usefulness of the pH gradient-based approach in fractionation of peptides. The experiments were performed on a series of peptides separated at various LC conditions. Retention parameters of peptides in the pH gradient and in the simultaneous pH/organic modifier gradient RP-LC were compared. The best results were obtained with eluents comprising low but constant concentrations of organic modifier while gradient of pH in the mobile phase was developed several times during each chromatographic run. The elaborated LC conditions allowed controlling the elution of peptides not only according to their hydrophobic properties, but also taking into account their electronic properties, represented by isoelectric point (pI) values. The combination of isocratic (regarding organic modifier) LC mode with recurring eluent pH gradient is proposed as an effective fractionation method of peptide mixtures. Moreover, information on hydrophobicity and pI of the peptides, obtained by that approach, might be an additional peptides database matching constraint. Hence, a new tool for analytical and bioinformatics studies of peptides fractionation is proposed.     

Analysis of polymer additives in high-temperature liquid chromatography

A high-temperature liquid chromatographic technique is employed for the separation of commercially available polymer additives to enhance the resolution and speed. Separation efficiencies and elution behaviors for seven phthalate plasticizers and five antioxidants are evaluated at elevated column temperatures and with a thermal gradient. Diamondbond C18 (octadecylsilica), Zirchrom PS (zirconia-based polystyrene), and Zirchrom PBD (zirconia-based polybutadiene) columns are selected for the study because of their thermal stability. The temperature programming is controlled with a column oven in conjunction with an independent mobile phase preheater and a post-column effluent cooling assembly. Van't Hoff plots show that the reverse-phase liquid chromatography mechanism is maintained over a wide range of column temperatures. A 1% increase of acetonitrile in the mobile phase is estimated to have a comparable effect as a 7-7.5 degrees C column temperature increase on the retention time changes.     

Ionic liquid as a mobile phase additive in high‐performance liquid chromatography for the simultaneous determination of eleven fluorescent whitening agents in paper materials

In the present study, 11 4,4′‐diaminostilbene‐2,2′‐disulfonic acid based fluorescent whitening agents with different numbers of sulfonic acid groups were separated by using an ionic liquid as a mobile phase additive in high‐performance liquid chromatography with fluorescence detection. The effects of ionic liquid concentration, pH of mobile phase B, and composition of mobile phase A on the separation of fluorescent whitening agents were systematically investigated. The ionic liquid tetrabutylammonium tetrafluoroborate is superior to tetrabutylammomnium bromide for the separation of the fluorescent whitening agents. The optimal separation conditions were an ionic liquid concentration at 8 mM and the pH of mobile phase B at 8.5 with methanol as mobile phase A. The established method exhibited low limits of detection (0.04–0.07 ng/mL) and wide linearity ranges (0.30–20 ng/mL) with high linear correlation coefficients from 0.9994 to 0.9998. The optimized procedure was applied to analyze target analytes in paper samples with satisfactory results. Eleven target analytes were quantified, and the recoveries of spiked paper samples were in the range of 85–105% with the relative standard deviations from 2.1 to 5.1%. The obtained results indicated that the method was efficient for detection of 11 fluorescent whitening agents.     

The study of properties of HPLC determination of polycyclic aromatic nitrogen heterocycles

The study of the separation of polycyclic aromatic nitrogen heterocycles (PANHs) by reverse-phase liquid chromatography with an octadecyl stationary phase is presented. The retention behaviour of a mixture of PANHs was studied under different chromatographic conditions. A mixture of phosphate buffer/acetonitrile was used as mobile phase in isocratic and gradient modes. The effect of different pH mobile phase in the range from 2.5 to 6.5 has been investigated to describe retention changes of PANHs as a function of their acid/base properties. Different concentrations of phosphate buffer as a component of the mobile phase were used to study the effect of ionic strength. Very good RP-HPLC separation of 24 PANHs and 16 EPA polycyclic aromatic hydrocarbons (PAHs) was obtained without a pre-separation step in a test mixture and the extract of a real soil sample. Limits of detection of PANHs obtained by two detection techniques, ultraviolet-diode array detection (UV-DAD) and fluorescence detector (FD), are compared. The proposed method is tested with a real soil sample.