Prediction of experimental parameters in combined isocratic and gradient elution reversed phase high performance liquid chromatography (RP-HPLC) using acetonitrile as organic modifier

The use of gradient elution in RP-HPLC is increasing. Aqueous buffers, such as phosphate at pH 1.7, with acetonitrile as the organic modifier are particularly advantageous as eluents with linear elution strength (LES). In the isocratic mode, under the experimental conditions used, fairly good linearity exists between logk' and x_B (x${}_{\text{B}}^{\text{º}}$ is the volume ratio of the organic modifier, i.e. acetonitrile) with a correlation coefficient of 0.9995. The relationship between isocratic and gradient elution parameters, such as x_B, x${}_{\text{B}}^{\text{º}}$ (starting composition in gradient elution), m (slope of the linear gradient), is transparent and the parameters of interest can be predicted fairly accurately using simple equations. The proposed system could be used in elaboration of a general retention index system for organic molecules in RP-HPLC.     

Analytical strategy for the assessment of the protein glycation status in uremic patients by high-performance liquid chromatography.

We propose a newly integrated procedure for the analysis of furosine (early glycation product) and pentosidine (glycoxidation end-product) in plasma proteins and the simultaneous assessment of advanced glycation end-product (AGE) peptides and free pentosidine in plasma. In order to determine furosine and protein-linked pentosidine, plasma proteins were hydrolyzed in 8 M HCl and each analyte was purified by solid-phase extraction. Furosine was determined by ion-pair RP-HPLC methodology with isocratic elution and spectrophotometric detection at 280 nm and pentosidine by ion-pair RP-HPLC by using gradient elution and fluorimetric detection at 335/385 nm. To assess free pentosidine concentration and simultaneously evaluate the AGE peptides, an aliquot of plasma sample was diluted and ultrafiltered by using Centricon 10 M(r) < or = 10,000) ultrafiltration membranes. Free pentosidine and AGE peptides were analysed by ion-pair RP-HPLC, by using gradient elution and fluorimetric detection at 385 nm upon excitation at 335 nm. The HPLC methodology has been successfully used for the determination of glycation and glycoxidation protein status in uremic patients.     

Simultaneous Determination of Maprotiline,Desipramine, and Moclobemide by Reversed-Phase High-Performance Liquid Chromatography and Statistical Optimization

Abstract

A method for separation of three antidepressants, maprotiline, desipramine, and moclobemide, by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated. To find optimal conditions and estimate the impact of individual parameters on the separation, a complete set of 2³ interdependent relationships of the mobile phase composition, temperature, and the volume flow rate were examined. Full separation of the investigated components from a laboratory mixture was achieved on a Supelcosil LC-18 (120 mm × 4.6 mm, 5 µm) column, using two solvent systems, 3% ammonium ion in water/ethanol and acetonitrile, and alternating isocratic gradient–isocratic elution modes. Relevance of the proposed method for therapeutic drug monitoring is anticipated.     

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.     

Application of retention modelling to the simulation of separation of organic anions in suppressed ion chromatography

The ion-exchange separation of organic anions of varying molecular mass has been demonstrated using ion chromatography with isocratic, gradient and multi-step eluent profiles on commercially available columns with UV detection. A retention model derived previously for inorganic ions and based solely on electrostatic interactions between the analytes and the stationary phase was applied. This model was found to accurately describe the observed elution of all the anions under isocratic, gradient and multi-step eluent conditions. Hydrophobic interactions, although likely to be present to varying degrees, did not limit the applicability of the ion-exchange retention model. Various instrumental configurations were investigated to overcome problems associated with the use of organic modifiers in the eluent which caused compatibility issues with the electrolytically derived, and subsequently suppressed, eluent. The preferred configuration allowed the organic modifier stream to bypass the eluent generator, followed by subsequent mixing before entering the injection valve and column. Accurate elution prediction was achieved even when using 5-step eluent profiles with errors in retention time generally being less than 1% relative standard deviation (RSD) and all being less than 5% RSD. Peak widths for linear gradient separations were also modelled and showed good agreement with experimentally determined values.     

Separation of polycyclic aromatic hydrocarbons under isocratic conditions by a column switching technique

Summary A simple and efficient method of separating a 20-component PAH-mixture (RSM 1647 standard mixture +benzene, toluene, perylene and coronene) by RP-HPLC is described. Separation was by using two Superspher-100 RP-18 cartridges thermostatted at different temperatures under isocratic conditions with water-acetonitrile eluent. The analysis time with complete resolution of all components can be reduced to 15 min.     

人参皂甙的反相高效液相色谱多台阶梯度优化方法

建立了一种反相高效液相色谱多台阶梯度分离人参皂甙的方法.该方法以乙腈-水溶液为流动相,通过一系列等度实验,获得了8种人参皂甙Rg1,Re,Rf,Rg2,Rb1,Rc,Rb2和Rd的色谱保留参数,发现两参数保留方程不适合用于人参皂甙这种天然产物的分离条件的优化,而三参数保留方程的高精度才可满足预测的要求.在三参数保留方程的基础上,通过计算确定了8种人参皂甙(包括3台阶梯度)的液相色谱分离条件.通过实验对此优化条件进行了验证,实验结果显示了较好的预测精度和分离度.将本方法用于分离人参皂甙,分析时间短且分离度高,显示了等度台阶梯度优化方法对确定色谱分离条件的优越性.     

Lead speciation by HPLC-ICP-AES and HPLC-ICP-MS

Speciation of inorganic lead (Pb2+) and several trialkyllead species (trimethyllead chloride [TML], triethyllead chloride [TEL], and triphenyllead chloride [TPhL]) is investigated using high-performance liquid chromatography (HPLC) with detection by both inductively coupled plasma emission spectroscopy (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS). Reversed-phase, ion-pairing, and ion-exchange HPLC modes are studied. Optimal chromatographic conditions for ICP-AES detection include a reversed-phase separation utilizing a step gradient from 10 to 70% methanol. However, the gradient has been found to destabilize the plasma when using ICP-MS detection. An isocratic separation with a 30% methanol mobile phase has been found to be the best compromise between plasma stability and chromatographic resolution. Detection limits using ICP-MS detection are 3 orders of magnitude improved over ICP-AES detection.     

TLC as a pilot technique for the optimization of gradient HPLC II. Use of data obtained from RP-18 plates for the prediction of gradient programs in reversed-phase liquid chromatography

Summary A method is described and experimentally verified permitting the determination of the optimal gradient programs in HPLC on the basis of isocratic TLC data. The proposed method has many advantages and can also be used in reversed-phase systems. A satisfactory agreement between the theoretical and experimental of V_R/V_O values has been confirmed. Some discrepancies for more strongly retained solutes are, probably due to the respective equilibrium and nonequilibrium state of the column in isocratic and gradient elution.Part I: Ref. [1].     

Retention models for isocratic and gradient elution in reversed-phase liquid chromatography

One- and multi-variable retention models proposed for isocratic and/or gradient elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under gradient elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated elution times as well as the transferability of retention data among isocratic and gradient elution modes are also examined and discussed.     

Two-dimensional ion chromatography using tandem ion-exchange columns with gradient-pulse column switching

A two-dimensional ion chromatography (2D-IC) approach has been developed which provides greater resolution of complex samples than is possible currently using a single column. Two columns containing different stationary phases are connected via a tee-piece, which enables an additional eluent flow and independent control of eluent concentration on each column. The resultant mixed eluent flow at the tee-piece can be varied to produce a different eluent concentration on the second column. This allows analytes strongly retained on the first column to be separated rapidly on the second column, whilst maintaining a highly efficient, well resolved separation of analytes retained weakly on the first column. A group of 18 inorganic anions has been separated to demonstrate the utility of this approach and the proposed 2D-IC method provided separation of this mixture with resolution of all analytes greater than 1.3. Careful optimisation of the eluent profiles on both columns resulted in run times of less than 28 min, including re-equilibration. Separations were performed using isocratic or gradient elution on the first column, with an isocratic separation being used on the second column. Switching of the analytes onto the second column was performed using a gradient pulse of concentrated eluent to quickly elute strongly retained analytes from the first column onto the second column. The separations were highly repeatable (RSD of 0.01–0.12% for retention times and 0.08–2.9% for peak areas) and efficient (typically 8000–260,000 plates). Detection limits were 3–80 ppb.     

利用HCI程序确定儿茶酚化合物的反相高效液相色谱最佳分离条件

有效地确定了反相高效液相色谱分离儿茶酚化合物的最佳条件。在水和甲醇的二元流动相里分别加入乙酸缓冲液,利用基于ln k=ln kw +SF, k=A+B/F, ln k=L+MF+NF2 (F是流动相中有机物甲醇的体积分数)等保留因子的一次或二次方程式的塔板理论得到色谱分离结果;利用保留原理得到等度和梯度洗脱的最佳条件。得出最佳初始流动相是含0.1%乙酸的水和含0.1%乙酸的甲醇(体积比为75∶25)的混合溶液;梯度洗脱条件：初始流动相保持15 min,然后用10 min的时间将上述二元流动相的体积比线性变换成50∶50,直到完成全部分离。通过实验证实该计算结果与实验值相近。     

Monitoring lipase-catalyzed methanolysis of sunflower oil by reversed-phase high-performance liquid chromatography: elucidation of the mechanisms of lipases

Reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at 210 nm was used to monitor the formation of the major compounds during the lipase-catalyzed transesterification reaction of sunflower oil with methanol. Individual triacylglycerols, diacylglycerols, monoacylglycerols as well as fatty acids and their corresponding methyl esters were separated using acetonitrile/acetone as a mobile phase and a combined linear gradient-isocratic-step gradient-isocratic elution procedure. Another relatively short method consisting of a linear gradient elution followed by an isocratic elution gave similar results, yet with lower resolution. HPLC/mass spectrometry with an ion trap analyzer and atmospheric pressure chemical ionization source was used for the identification of the individual compounds. Individual calibration curves obtained with UV detection at 210 nm were found to be of use for quantitative analyses of double-bond containing methyl esters and acylglycerols. The use of the RP-HPLC methods in the elucidation of the mechanisms of three immobilized lipases, namely Lipozyme TL IM, Lipozyme RM IM and Novozym 435, in biodiesel production was described.     

Effect of electric field on liquid chromatographic separation of peptide digests. Combining capillary separation techniques

A system is described which allows operation of a range of capillary based liquid phase separations including capillary electrophoresis, isocratic and gradient capillary electrochromatography, isocratic and gradient capillary liquid chromatography and electrically assisted gradient capillary liquid chromatography. The system was coupled to electrospray ionization mass spectrometry in the electrically assisted capillary liquid chromatography mode to investigate the effect of applied voltage on the selectivity in peptide mapping separations. Analyses were performed on tryptic digests of recombinant human growth hormone and tissue plasminogen activator. The results show a small but useful effect on selectivity that can be used to fine tune specific separations.     

梯度液相色谱保留时间公式在梯形梯度洗脱研究中的应用

根据前期得到的梯度液相色谱保留时间计算公式,在不指定溶剂强度模型形式的前提下,探讨了梯形梯度洗脱的一些特点。对于溶质在梯形梯度坡度上流出时的情形,推导得到溶质流出色谱柱所对应的流动相组成(φ_R)随梯度斜率(B)变化的表达式。该公式表明,在该情形中φ_R将会随着B值的增加而增加。对于溶质在梯形梯度最后一个等度区间流出时的情形,如果初始和终止流动相组成保持不变而仅有梯度的斜率发生变化时,从理论上证明了溶质保留时间(t_R)与梯度斜率的倒数(1/B)之间呈线性关系。实验中以C18色谱柱为固定相,甲醇-水为流动相,联苯为样品,测定了不同流动相组成以及梯形梯度条件下的保留时间,所得到的实验值与理论值吻合,从而验证了理论方法的正确性。     

Optimization of an isocratic reversed phase liquid chromatographic system for the separation of fourteen steroids using factorial design and computer simulation

An optimization strategy for an isocratic reversed phase high performance liquid chromatographic system (RP-HPLC) is described. Factorial design and a computer program are used to predict the retention time and resolution of fourteen steroids. An optimized rapid (less than 25 min) isocratic RP-HPLC system for the satisfactory separation of cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17 alpha-hydroxyprogesterone, progesterone, androstenedione, testosterone, estrone, estradiol, estriol, prednisone acetate and dexamethasone acetate has been developed using this strategy through eight experiments.     

Calligonum polygonoides Linnaeus Extract: HPLC‐EC and Total Antioxidant Capacity Evaluation

Flavonoids in Calligonum polygonoides Linnaeus extract were separated, detected, and identified by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) with electrochemical detection (EC) in combined isocratic and gradient elution using a glassy carbon or a boron doped diamond electrode. Ultrasonication coupled with a microwave‐assisted technique was developed to optimize the extraction of the phenolic compounds. The total antioxidant capacity was quantified using the DPPH^. method and voltammetry. The RP‐HPLC‐EC led to the detection of nine different flavonoids: catechin, delphinidin, fisetin, myricetin, epicatechin, kuromanin, rutin, callistephin and procyanidin A2, in a single run by direct injection of the sample extract solution.     

Automated instrumental method for on-line fraction analysis and peak deconvolution in gradient multicomponent overloaded high performance liquid chromatography

An automated method for deconvolution of overloaded band profiles in gradient elution is described. The instrumental set-up consists of a pseudo-bidimensional HPLC system, where overloaded band profiles generated in the first direction are sampled on the second one. The method, previously employed under isocratic conditions, has been now extended to gradient elution, where one has to face the problem of band compression (and possibly band interference) during gradient, which decreases the time-window available along the first direction for sampling the overloaded profiles. The effect of the gradient steepness on the problem of defining a minimum number of sampling points to reconstruct single component bands from overloaded profiles is investigated. This approach is particularly useful in the framework of using inverse method (IM) to determine adsorption isotherm parameters for preparative gradient elution. In fact, it allows for the gathering of the information necessary to run IM calculations with minimum efford, also in cases where the individual component forming the mixture have different UV spectra.     

New approach to linear gradient elution used for optimisation in reversed-phase liquid chromatography

A new mathematical treatment concerning the gradient elution in reversed-phase liquid chromatography when the volume fraction psi of an organic modifier in the water-organic mobile phase varies linearly with time is presented. The experimental ln k versus psi curve, where k is the retention factor under isocratic conditions in a binary mobile phase, is subdivided into a finite number of linear portions and the solute gradient retention time tR is calculated by means of an analytical expression arising from the fundamental equation of gradient elution. The validity of the proposed analytical expression and the methodology followed for the calculation of tR was tested using eight catechol-related solutes with mobile phases modified by methanol or acetonitrile. It was found that in all cases the accuracy of the predicted gradient retention times is very satisfactory because it is the same with the accuracy of the retention times predicted under isocratic conditions. Finally, the above method for estimating gradient retention times was used in an optimisation algorithm, which determines the best variation pattern of psi that leads to the optimum separation of a mixture of solutes at different values of the total elution time.     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。