Computer-assisted optimization of reversed-phase HPLC isocratic separation of neutral compounds

An appropriate optimization strategy should be used to find a desired resolution or selectivity with a minimum number of experiments in a limited time, which could assure the baseline separation of all target compounds. It was usually realized by means of a specialized computer program. In this paper, mapping optimization method and overlapping resolution mapping were compared for the optimization of a reversed-phase high-performance liquid chromatography (HPLC) isocratic separation of neutral compounds. The calculated resolutions and separation time of 7 to 10 experiments are fitted by different equations, which were used to build a contour plot with a minimum effective resolution and maximum retention time as a function of a mobile phase composition. The balance between resolution and analysis time could be easily realized by the overlapping of the final overlapping resolution mapping and analysis time mapping. The validity of the two methods was confirmed by some typical experiments. The models are simple, visual, and common without theoretical arithmetic.     

应用扩展的重叠分辨率法优化液相色谱条件分离二茂铁类化合物

本文对优化液相色谱分离条件的重叠分辨率法进行了改进与扩展：首先利用全范围的二元线性梯度淋洗进行溶剂强度的优化，再通过重叠分辨率法进行了溶剂选择性的优化，并且建立了计算机系统，使此方法程序化。利用此方法建立了分离Ｎ－二茂铁甲酰基－Ｎ＇－芳基硫脲类衍生物的最佳流动相体系。     

Ion-Interaction Chromatographic Separation of Free Amino Acids

Abstract

An extensive study of the HPLC separation of 20 free amino acids by the addition of alkanesulfonate salts to the mobile phase was previously reported (1). This paper describes modifications in the procedure that improves the separation and resolution of the 20 free amino acids. Mobile phase variables (type and concentration of alkanesulfonate salt, organic modifier concentration, mobile phase pH, and mobile phase ionic strength), and stationary phase variables (particle size, type of packing) which can affect amino acid separation, resolution and selectivity were studied. Two stationary phases were compared, the 5 μm Hamilton PRP-1 and Phase Separations 3 μm, ODS-2. Longer chain alkanesulfonate salts (octane and decanesulfonate salts) were evaluated as mobile phase additives. A mobile phase gradient of increasing per cent organic modifier was necessary for separating complex mixtures of polar and nonpolar-basic amino acids. It is now possible to separate 19 of 20 free amino acids with this ion-interaction chromatographic procedure.     

125I-Labeled Peptide Mapping and High-Performance Liquid Chromatography I-Peptide Separation of Protein I of Four Strains of Neisseria Gonorrhoeae

Abstract

¹²⁵I-labeled α-chymotryptic peptides of the principal outer membrane proteins (P.Is) of four strains of Neisseria gonorrhoeae were separated and visualized by two-dimensional (2-D) ¹²⁵I-peptide mapping and by high-performance liquid chromatography (HPLC) coupled with a Beckman Biogamma counter. In addition, ¹²⁵I-peptides were recovered from the HPLC separation and re-separated by the 2-D ¹²⁵I-peptide mapping system. The results indicated that the 2-D ¹²⁵I-peptide mapping procedure was best suited for comparative analyses of α-chymotryptic digests whereas the HPLC system, which is able to detect many more peptides than the 2-D system, is ideally suited for preparative separation of the ¹²⁵I-peptides. ¹²⁵I-peptides separated by HPLC could be recovered, rerun on the 2-D system, and the location of each peptide ascertained. The coupling of these two procedures allows for the isolation of specific ¹²⁵I-labeled peptides for further immunological and structural analyses of these outer membrane proteins.     

Rapid and Complete Separation of Inorganic Polyphosphates from Monomer to 35-Mer by Hplc

Abstract

The on-line coupling system of high-performance liquid chromatography (HPLC) and flow injection analysis (FIA) was developed for the rapid separation and sensitive detection of inorganic polyphosphates. Effects of gradient elution conditions on the chromatographic behavior were studied. The column temperature as well as a chloride concentration gradient were found to be very effective for the improvement of resolution(1). To minimize analysis time and to maximize resolution, the gradient elution conditions were optimized by use of a computer-assisted retention prediction system(2). More than 35 kinds of inorganic polyphosphates could be separated completely within 200 min under the optimum conditions as shown in Fig. 1.     

Optimization of supercritical fluid chromatographic separation of nitroaromatics by the overlapping resolution mapping scheme

The use of an interpretative optimization method to predict optimum conditions for the supercritical fluid chromatographic separation of nitroaromatics is demonstrated. The scheme utilizes the overlapping resolution mapping (ORM) procedure for multivariate prediction of the pressure gradient and temperature in order to separate eight nitroaromatics. Using the ORM scheme, only seven experiments were conducted before a global optimum was revealed. All the peaks were satisfactorily separated using experimental conditions derived from the scheme.     

High Resolution Separation of Urinary Organic Acids by High Performance Liquid Chromatography

Abstract

Reverse phase, anion exchange, and two-dimensional HPLC techniques were studied in order to increase resolution of organic urinary acids for eventual quantitative measurements. Reverse phase HPLC with a phosphate buffer/acetonitrile gradient yielded a separation of over 85 components in forty minutes and a peak area reproducibility of better than 5%. Connecting two reverse phase columns together resulted in the separation of 110 components. Anion exchange chromatography was determined to be of little use in resolving urinary acids in a resonable time except as the first stage in two-dimensional chromatography where fractions from the anion exchange column were injected into a reverse phase column. Over 139 components were separated by this two-dimensional method.     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Computer-Assisted Modeling, Prediction, and Multifactor Optimization of Hydrophobic Compounds in Liquid Chromatography

The traditional overlapping resolution mapping (ORM) scheme was modified for application in HPLC with the flow rate of the mobile phase as a new factor. The mobile phase composition and a flow rate were taken as parameters to influence effective resolution and elution time. Chromatograms were obtained for the separation of the 16 compounds for ten trial conditions. Computer-assisted mathematical simulation was used to obtain optimum conditions for resolution and separation time of the compounds. The validity of the model developed was confirmed by experiments.     

二氢吡啶类药物和肺保三效片组分高效液相色谱流动相的优化

本文使用重叠分辨图谱(ORM)法预示了HPLC分离二氢吡啶类药物和肺保三效片中组分的最佳流动相组成。实验表明ORM法是一种迅速、多用途的优化方法。     

Ion Exchanger Liquid Chromatography of N-Acetylaspartic Acid and Some N-Acetylaspartyl Peptides

Abstract

A high performance liquid chromatographic (HPLC) procedure for the rapid separation of N-acetylaspartic acid, N-acetyl-aspartyl-glutamic acid and N-acetylaspartyl-glutamyl-aspartic acid is described. The procedure utilizes a pellicular ion-exchange column support, and ionic strength gradient with mobile phase solutions buffered to pH 5.0 and a UV detector operated at 210 nm. Reproducibility and quantitative capabilities are also discussed. The method has been used for a tentative estimation of N-acetylaspartic acid and N-acetylaspartyl peptides in a rat brain synaptosomal extract.     

High Performance Liquid Cbromatographic Separation of Globin Chains on a Large-Pore C4 Column

Abstract

Excellent resolution of human and baboon globin chains may be obtained by HPLC on a Vydac large-pore C₄ column. The procedure is rapid and uses a gradient between aqueous trifluoroacetic acid and trifluoroacetic acid in acetonitrile. The common human γ chains are easily separable from each other as are some α- and β-chain variants from the normal chains and from each other.     

The Separation of 24 OPA-AA Of Natural Origin and Quantitative Analysis of Tyrosine by Means of HPLC

Abstract

The separation of a mixture of 24 amino-acids (AA) of natural origin is carried out by column HPLC μ. Bondapak C18 10 μm, following pre-column derivatization using orthophthalaldehyde (OPA). Because of the large numbers of AA to separate, the use of a linear gradient making use of the diversity of the AA polarities ensures the resolution of the mixture in less than 45 min.

The gradient is established through the progressive addition of acetonitrile to monopotassic phosphate solution (pH 6.80). The thioisoindol derivatives, which are thus formed and wich absorb greatly at 340 nm, are detected with a spectrophotometer. Quantitative analysis of tyrosine is carried out by means of the internal calibration method with two calibration substances: glutathion and 6amino caproic acid.

This analysis, which is directly applicable in industrial surroundings to the separation of primary AA, is both reproducible and sensitive.     

Two-dimensional chromatography of complex polymers 6. Method development for (meth)acrylate-based copolymers

The free-radical copolymerisation of various acrylates and methacrylates resulting in complex copolymers for cosmetic applications were investigated using different chromatographic techniques including HPLC and on-line coupled two-dimensional (2D) liquid chromatography. The complete separation of all polymerisation products was achieved by gradient HPLC. A computated optimisation procedure, using the Polymer Chromatographic Model allowed us to design a step mobile phase gradient to improve resolution of homopolymer chromatographic separation. By combining gradient HPLC and SEC (Size Exclusion Chromatography) in a fully automated two-dimensional chromatography setup, the complex distributions of chemical composition and molar mass could be simultaneously described and fingerprinted.     

五味子提取物高效液相色谱分析方法的优化

针对五味子乙醇提取物的复杂体系,借助于复杂样品分析系统软件(CSASS),根据组分在4次简单线性梯度下42个峰的保留时间,快速准确地计算出各组分的液相色谱保留参数a,c值和峰形参数σ,W1/2。借助这些参数,对五味子色谱的分离情况进行高精度仿真预测。在此基础上,应用移动重叠分辨分离图和谱图仿真技术,发展了计算机辅助的五味子提取物的高效液相色谱全局优化方法。在优化条件下,五味子提取物的高效液相色谱分析可在40 min内完成,且常量成分和部分低含量成分都能够得到较好的分离。所建立的方法已成功地用于五味子中化合物保留时间及峰形的预测,并在此基础上对其色谱分离条件进行优化。     

Separation of digitalis cardenolides by HPLC

The separation of Digitalis cardenolides has been carried out by HPLC on an adsorbent column. By choice of suitable mobile phase, isocratic elution permitted resolution of mixtures of (a) aglycones, (b) secondary glycosides, and (c) primary glycosides, while gradient elution provided a means of resolving more complex mixtures of these cardiac steroids. HPLC could therefore be used in the quality control of cardiotonic drugs replacing the TLC tests for related compounds currently used, and by suitable calibration could replace the colorimetric assay procedure normally used for such drugs.     

Separation of Dihydrofolate Reductase Inhibitors by RP-HPLC and Comparison with Capillary Zone Electrophoresis

The dihydrofolate reductase inhibitors (DHFRIs) are a group of antibiotic compounds with closely related chemical structures. A previous attempt to separate the eight compounds by CZE was successful, but only at low pH (2.1) and high buffer concentration (250mM phosphate). As a result, baseline noise was high. Additionally, baseline resolution was not quite achieved and the separation took 25 minutes. However, the size-based separation indicated that reversed-phase HPLC might be a good alternative. A three-dimensional overlapping resolution mapping (OR_sM) scheme utilised pH, flow rate and percentage organic modifier (for both methanol and acetonitrile). Clear generalised outcomes were observed under isocratic conditions. At higher pHs, where the analytes were largely unionised, retention was excessive. At higher percentages of MeOH or ACN, the more-difficult-to-separate components were poorly resolved. On the other hand, low pHs (2.5 in 50mM phosphate) with low percentages of the organic modifiers (7% ACN or 11% MeOH) but high flow rates (2.3mLmin^–1) yielded better than baseline resolution in 17 minutes. A partially optimised gradient run (at pH 6.5) again yielded far better than baseline resolution in 12 minutes, but required 4 minutes re-equilibration. Hence, the HPLC separations are superior to the CZE separation in all of runtime (40%), resolution and limit of detection (down by 6).     

Combination of orthogonal array design and triangle overlapping resolution mapping scheme for resolution optimization in micellar electrokinetic chromatography.

This paper describes the application of a combined orthogonal array design and overlapping resolution mapping to the optimization of miceller electrokinetic chromatography for the separation of 10 substituted benzenes. The most important factors were first determined according to an OA16(2(15)) through 16 pre-designed experiments; a second set of experiments was carried out according to a triangle overlapping resolution mapping scheme, in which 7 pre-planed experiments were executed and global optimum conditions for the separation were obtained.     

Liquid chromatographic analysis of the anticancer alkaloid luotonin A and some new derivatives in human serum samples

The quantitation of the natural cytotoxic and anti‐inflammatory alkaloid luotonin A and five recently synthesized derivatives is described, constituting the first report of a HPLC method for the analysis of these compounds in human serum samples. The conditions for the chromatographic separation were optimized and the method was validated for the analysis of these compounds in biological samples according to international guidelines. An RP‐HPLC method with fluorimetric detection and a C₁₈ stationary phase was applied. Different ACN/water mobile phases were assayed, including 0–4% of a mobile phase modifier such as tetrahydrofuran, dioxane or tert‐butyl methyl ether. Isocratic and gradient elution conditions are compared. The influence of pH on the efficiency and resolution of the separation was also considered. The developed method was applied to the determination of luotonins in pooled human serum samples by gradient elution RP‐HPLC using a simple cleanup procedure. The proposed chromatographic method exhibits satisfactory analytical figures of merit, with LOD from 1.0×10^?10 to 2.0×10^?10 M, intraday and interday precision below 6% except for the concentration level closest to LOD, and good agreement between experimental and theoretical concentrations. Therefore, the developed method is suitable, reliable, rapid, and simple.     

Separation of Bufotalin and Cinobufotalin by Preparative Liquid Chromatography

Abstract

Complete resolution of bufadienolides by traditional column chromatography, or by thin-layer chromatography (TLC), is quite difficult. Separation of various bufadienolide conjugates by high performance liquid chromatography (HPLC) has been described, but not for resolution of the corresponding genins. A preparative HPLC procedure has been developed for resolving the difficultly separable bufotalin (1) and cinobufotalin (2). A Lobar B column packed with LiChromprep Si-60 was used to separate these bufadienolides employing a recycling procedure.