Computer-assisted method development and optimization in high-performance liquid chromatography

This paper reports the use of DryLab, a computer simulation software package, to assist in the development and optimization of a reversed-phase high-performance liquid chromatographic (HPLC) method for the separation of a model drug candidate and its degradation products. Prior to the optimization process, columns with various bonded phases are evaluated for their chromatographic performance using the sample of interest. Simultaneous optimization of two separation variables and the use of resolution maps to predict the optimal conditions are illustrated. Options to optimize column conditions (column length and flow-rate) to further reduce run time are briefly discussed. The accuracy of DryLab-predicted retention times and resolution is compared with experimental values. The DryLab software used in this study provided satisfactory predictions for the selected model, with average errors of less than 3.5 and 11.8% for retention time and resolution, respectively.     

A better global resolution function and a novel iterative stochastic search method for optimization of high-performance liquid chromatographic separation

HPLC optimization strategy consists of four elements; experimental design, retention modeling, quality criteria function, and optimum search method. In this paper we present a simple, superior alternative to general classes of classical resolution functions (S function) and a novel optimum search algorithm (iterative stochastic search, ISS) for HPLC optimization. Comparison of S with general classes of resolution-based quality criteria functions (Rs, Rp, and Rmin) shows superior features such as correct assessment of favorable separation conditions, preservation of peak pair contributions, elimination of arbitrary cut-off values, and a unique capability to interpret absolute significance of function values through a simple inequality. The proposed ISS algorithm is more robust than standard methods and it is easily applicable to hyperdimensional optimization. ISS also shows clear advantages in its ability to correctly identify the global optimum (instead of local optimum), with higher precision, with more efficient use of computation cycles, and with easier implementation. Successful application of S and ISS to HPLC optimization was demonstrated in the separation of representative functionalities (sugars, alcohols, and organic acids) present in microbial fermentations. Both the optimal and pathological (worst) conditions were successfully predicted and experimentally verified.     

A straightforward means of coupling preparative high-performance liquid chromatography and mass spectrometry

Flow splitting to a mass spectrometer is a common way of coupling a highly specific detector to preparative or semi-preparative high-performance liquid chromatography (HPLC) purification of combinatorial libraries, drug metabolites, and characterizable impurities. The sensitive mass spectrometer consumes only a small fraction of the analyte while providing online structure-specific detection, and its output can thus be used to trigger collection of the desired fraction. Coupling mass spectrometry to preparative HPLC is difficult due to the susceptibility of the detector to fouling under conditions of high analyte concentration or solute amount, or to changes in solvent composition. We report here on a device, the mass rate attenuator (MRA), which automatically produces split ratios over a range of 100:1 to 100 000:1 under programmable user control. The MRA is a flow-control device that periodically gates a small aliquot from one liquid stream into another. The design allows the user to set the frequency of the gating without interruption of the HPLC flow stream. The MRA also allows control of the volume of the aliquot that is transferred between the flow streams. This additional control, compared to passive splitting devices, facilitates optimization of the tubing connecting the separation, detection and collection events. We demonstrate that such optimization can reduce the volume of the collected fraction without compromising recovery, thus reducing the time spent in evaporating solvents to reclaim purified products.     

制备液相色谱法分离纯化碘帕醇

基于制备液相色谱法,开发与优化了碘帕醇的分离纯化工艺,制备得到高纯度碘帕醇样品。实验首先在分析水平发展碘帕醇的反相分离方法,考察了两种不同键合量的反相C18固定相、柱温和上样量对碘帕醇的保留、分离度和峰形等的影响。结果表明,碘帕醇在键合量为13.7%的反相C18-1分析柱（250 mm×4.6 mm,10 μm）上保留较好,且可与杂质有效分离;柱温升高,碘帕醇保留变弱,和杂质之间的分离度降低,最终选用20~25℃作为分离纯化的温度;上样量增加,碘帕醇出峰时间提前,不利于前杂的去除。在制备水平上,以水和甲醇为洗脱剂,在20℃条件下使用装填C18-1固定相的制备柱（270 mm×50 mm,10 μm）对碘帕醇进行分离纯化,制备的碘帕醇样品的色谱纯度可达98.97%,回收率为93.44%,各项有关物质均符合限量规定。该方法可以在保证高回收率的条件下有效降低杂质水平,为碘帕醇分离纯化生产工艺的开发提供新方法。     

Application of optimization procedures for the separation of anabolic compounds by high-performance liquid chromatography

A simple systematic approach is presented for optimizing high-performance liquid chromatographic separations of anabolics with multi-component isocratic mobile phases. A computer program was obtained and adjusted for use with an IBM-compatible XT personal computer. The program requires experimental retention data with three quaternary solvent mixtures to calculate the optimum solvent composition using a geometric model of a prism. For each possible composition of the mobile phase the set of retention data can be calculated. Applications are shown for mixtures of anabolic compounds using a mobile phase composed of methanol, tetrahydrofuran and acetonitrile. The predicted retention data agreed very well with the experimental data.     

Preparative separation of lithospermic acid B from Salvia miltiorrhiza by polyamide resin and preparative high-performance liquid chromatography

Adsorption on polyamide resin was investigated as a means of separating lithospermic acid B (LAB) from a crude extract of the roots of the traditional Chinese medicine Salvia miltiorrhiza Bunge ("Danshen"). Variables affecting adsorption capacity (solution pH, contact time on resin, initial LAB concentration) were studied. Adsorption was strongly dependent upon the initial concentration of LAB and pH. In all conditions, the polyamide resin gave optimal adsorption of LAB at an initial concentration of 2.66 mg/mL and pH <3.0. The adsorption isotherm correlated well with the Langmuir-type adsorption isotherm. Maximal adsorption capacity was calculated to be 380 mg/g at pH 2.0 and 25°C. LAB purity of 85.30% could be obtained by polyamide resin adsorption followed by elution with 70% ethanol solution, and the recovery was 87.1%. After preparative HPLC, the maximum HPLC purity obtained was 99.28% with a recovery of 75.2%. This method provides an efficient and low-cost method for LAB purification for industrial applications.     

Factors affecting the separation and loading capacity of proteins in preparative gradient elution high-performance liquid chromatography.

The optimum conditions for the purification of proteins by gradient elution in reversed-phase liquid chromatography were studied, with emphasis on the column length. Because of the strong dependence of the retention of proteins on the mobile phase composition, very short columns can be used successfully to perform analytical separations. A similar conclusion is extended to preparative separations. Columns with different lengths and diameters were used. The dependence of the loading capacity for touching band separation on the column length, diameter and volume was studied, in addition to the regeneration time between successive runs, the starting mobile phase composition and the necessary column efficiency.     

Automation of the optimization of the separation of protamines by high-performance liquid chromatography

An automatic system for optimizing the conditions for the chromatographic separation of protamines has been developed using Nelder and Mead's modified simplex method. It is proposed to use penalty functions in order to take into account nonrigid constraints on the parameters to be optimized. The optimum conditions for the separation of the protamines from sturgeon gonads have been determined. It has been established that the greatest influence on the separation of the protamines is exerted by the concentration of the ion-pair reagent — trifluoroacetic acid. The system described permits the optimization of any chromatographic separations and the determinations to be performed thanks to the application of penalty functions to the optimization parameter.     

Automation of the optimization of the separation of protamines by high-performance liquid chromatography

An automatic system for optimizing the conditions for the chromatographic separation of protamines has been developed using Nelder and Mead's modified simplex method. It is proposed to use penalty functions in order to take into account nonrigid constraints on the parameters to be optimized. The optimum conditions for the separation of the protamines from sturgeon gonads have been determined. It has been established that the greatest influence on the separation of the protamines is exerted by the concentration of the ion-pair reagent — trifluoroacetic acid. The system described permits the optimization of any chromatographic separations and the determinations to be performed thanks to the application of penalty functions to the optimization parameter.Moscow Institute of Applied Biotechnology. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 663–669, September–October, 1990.     

Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides

Biologically active peptides synthesized by the solid phase methodology of Merrifield were purified by reversed-phase high-performance liquid chromatography using newly developed preparative radially compressed cartridges fitting Waters Assoc . Prep LC 500 liquid chromatograph. Cartridges were handpacked with Vydac C18, C4 or diphenyl derivatized silicas (pore size 300 A) of different particle sizes (10-20 micron). Large scale purification of gram amounts of gonadotropin releasing hormone analogs (agonist and antagonist) as well as amidated human pancreatic tumor growth hormone releasing factor (a 40-peptide) illustrate the resolutive power of this technique applied to the isolation of more than 300 synthetic peptides in our laboratory over the last two years. Difficult separations were achieved by changing supports (C18, C4, diphenyl) as well as mobile phase composition: (triethylammonium phosphate pH 2.25 or 6.5, 0.1% trifluoroacetic acid, ammonium acetate pH 6.5 and acetonitrile). Protected amino acids and peptides amenable to normal-phase chromatography on Vydac spherical underivatized silica were purified economically by the reversed-phase mode. It is understood that this general, convenient and versatile strategy may be applicable to the preparative scale isolation of any other class of compounds usually separated on reversed-phase high-performance liquid chromatography.     

A simplex optimization of the experimental parameters in preparative liquid chromatography

Summary A study of the optimization of the experimental conditions for the purification or extraction of pure compounds by liquid chromatography is presented. Optimum values of the parameters of overloaded elution are derived for maximum production rate, using a Simplex algorithm and the procedure previously described for the simulation of the elution profiles of binary mixtures. The mobile phase flow velocity and the sample size have been optimized together in a first step, simulating the procedure followed in practice, when a column is available. In a second part, the influence of the column length and the average particle size of the packing material on the column performance as well as the trade-offs between the production rate and the yield are discussed.There are three major conclusions in this work. First, the optimum experimental conditions are often very different, depending whether one is primarily interested in the first or in the second eluted component of a mixture. Second, the column efficiency under analytical conditions is very important: it is traded-off for high flow rates, hence short cycle time and increased production rate. Third, the production rate depends strongly on the maximum pressure at which the equipment can be operated. Finally, the optimum production rate varies rather smoothly with the mobile phase velocity and the sample size, so a high yield (70% or more) can usually be obtained with a limited loss in production rate (30 to 60%).     

Parallel preparative high-performance liquid chromatography with on-line molecular mass characterization

High-throughput chemistry (HTC) is now an integral part of the lead discovery process in many pharmaceutical and related chemical companies. As this process becomes refined or improved, with the integration of systems with enhanced capabilities, and the requirement for quality compounds of high purity increases, purification is often considered a bottleneck. Although a wide range of purification techniques is available, high-performance liquid chromatography (HPLC) is usually the preferred method of purification to produce high-purity compounds. Parallel preparative HPLC with robust UV-guided fraction collection has been shown to reduce the bottleneck and complement the parallel synthesis systems. However, despite the success of this technique, post-purification analysis of fractions to identify the target compound adds an additional level of complexity. This paper describes the interfacing of the Biotage Parallex with the MUX interface on a single quadrupole mass spectrometer, thus combining robust UV-guided fractionation with on-line MS characterization.     

Rapid separation of lactucin and lactucopicrin from Cichorium glandulosum by medium-pressure preparative liquid chromatography and quantitative analyses by high-performance thin-layer chromatography

Medium-pressure preparative liquid chromatography (MPPLC) was used to isolate and prepare lactucin and lactucopicrin from the whole herb of Cichorium glandulosum. After extracting the methanolic extract of the whole plant with petroleum ether and ethyl acetate several times to obtain ethyl acetate extract, the crude products, namely, lactucin and lactucopicrin were separated using MPPLC and thin-layer chromatography (TLC) tracking, and their purity rates reached more than 80%. The qualitative and quantitative analyses of lactucin and lactucopicrin were carried out by high-performance thin-layer chromatography (HPTLC) on the whole herb of C. glandulosum. The contents of lactucin and lactucopicrin were determined by scanning at 256 nm in the whole herb of C. glandulosum. The R_F values of lactucin and lactucopicrin were 0.42 ± 0.05 and 0.65 ± 0.05 with linear ranges of 0.498–2.988 and 0.499–2.994 μg/band, respectively. The correlation coefficients were 0.9938 and 0.9946, respectively, thereby showing a good linear relationship. The average recoveries were 99.96% and 99.52%, and the relative standard deviations (RSDs) were 2.49% and 2.45%, respectively. The crude products, namely, lactucin and lactucopicrin, can be isolated with high purity from the whole herbs of C. glandulosum by MPPLC. The lactucin and lactucopicrin contents of C. glandulosum can be determined rapidly and accurately by HPTLC.

     

Two-dimensional preparative liquid chromatography system for preparative separation of minor amount components from complicated natural products

An on-line comprehensive two-dimensional preparative liquid chromatography system was developed for preparative separation of minor amount components from complicated natural products. Medium-pressure liquid chromatograph (MPLC) was applied as the first dimension and preparative HPLC as the second one, in conjunction with trapping column and makeup pump. The performance of the trapping column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure from the view of medium pressure liquid chromatograph. Satisfactory trapping efficiency can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The instrument operation and the performance of this MPLC × preparative HPLC system were illustrated by gram-scale isolation of crude macro-porous resin enriched water extract of Rheum hotaoense. Automated multi-step preparative separation of 25 compounds, whose structures were identified by MS, ¹H NMR and even by less-sensitive ¹³C NMR, could be achieved in a short period of time using this system, exhibiting great advantages in analytical efficiency and sample treatment capacity compared with conventional methods.     

Chiral separation of atropine by high-performance liquid chromatography

The separation and quantitation of the enantiomers and also the determination of the enantiomeric purity are now current and indispensable tasks for the pharmaceutical analysis. Among the various techniques, liquid chromatography remains the best modality owing to several advantages. High speed, sensitivity, and reproducible results make LC the method of choice in almost all laboratories. Phases that contain alpha1-acid glycoprotein as chiral selector are suitable for separation of charged and uncharged enantiomers with widely different structure. Atropine is widely used as parasympatolytic, anticholinergic and antiemetic drugs. It is one of the preferred antidote for immediate management of toxicity associated with nerve agents. Stereoselective separation was achieved with a prepacked alpha1-acid glycoprotein column without any derivatization procedure. The liquid chromatography system is coupled to mass spectrometry with an atmospheric pressure chemical ionization interface in the positive-ion mode. The chromatographed analytes are detected in selective ion monitoring after optimisation using factorial experimental design. Small amount of enantiomeric composition can be evaluated either by MS or by UV spectrometry (less than 5%).     

Computer aided optimization in high-performance liquid chromatography

In reversed-phase liquid chromatography the composition of the mobile phase is the key to a successful separation. Lengthy and uncertain trial and error techniques can be replaced by more efficient computer-guided approaches to find the optimum composition.     

Purification of erucic acid by preparative high-performance liquid chromatography and crystallization.

Erucic acid (C22:l fatty acid) has been found to be useful in the treatment of adrenoleukodystrophy (ALD). It appears to work by reducing the blood levels of very-long-chain fatty acids (VLCFAs) which destroy the myelin sheaths of the nerves. Erucic acid was purified by reversed-phase high-performance liquid chromatography (HPLC) on columns packed with YMC C18 (10-20 microns, 120 A). Using ethanol-water as the mobile phase, the recovery of erucic acid was 69% and the purity was more than 97% as measured by gas chromatography. The amount of saturated VLCFAs was found to be within the limits specified for ALD treatment. The production rate (yield per 8 h shift) was low, however. Using methanol-water instead of ethanol-water as the mobile phase, a ninefold increase in the production rate was achieved. The recovery of erucic acid was 65% and the purity of erucic acid was 98%. All other purity specifications were met. By performing a low-temperature crystallization after the preparative HPLC step, the production rate was increased a further 142%. This represents a 22-fold increase in production rate over the ethanol-water method. The crystalline erucic acid was found to be 99% pure. All other purity requirements were met. The yield for the combined process (HPLC plus crystallization) decreased to 55%, however.