Literature | MOLNÁR-INSTITUTE

Journal of Chromatography A · 1998

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development: I. Application to 14 different samples using computer simulation

John W. Dolan, Lloyd R. Snyder, N. M. Djordjevic, D. W. Hill, D. L. Saunders, L. Van Heukelem, T. J. Waeghe

The optimized reversed-phase HPLC separation of 14 different samples is reported, based on simultaneous changes in temperature and gradient steepness. Four experimental runs are required for each sample, following which preferred conditions can be predicted using computer simulation software (DryLab). The overall accuracy and effectiveness of this method development approach is discussed, with particular attention to the use of resolution maps provided by the software. These maps are useful for maximizing resolution for the total sample, for optimizing the separation of a smaller number of selected sample compounds, and as an initial step in the separation of more demanding samples.

Journal of Chromatography B: Biomedical Sciences and Applications · 1997

Changing reversed-phase high performance liquid chromatography selectivity Which variables should be tried first?

L. R. Snyder

When carrying out HPLC method development, it is often necessary to vary the relative retention of the sample (values of α) by changing some experimental variable, e.g., solvent type, pH, etc. The choice of which variable will be most suitable for a change in selectivity depends on two conflicting goals: (a) the attainment of maximum changes in α for the better control of resolution and (b) the avoidance of practical problems associated with the use of a given variable to optimize selectivity. This study provides a quantitative evaluation of different variables for their effect on selectivity (α). Various practical problems which must be balanced against this ability of a variable to change values of α are also discussed. The selection of any two variables for their simultaneous use in controlling α is also examined.

Food Microbiology · 2016

Inhibition mechanism of Listeria monocytogenes by a bioprotective bacteria Lactococcus piscium CNCM I-4031

Taous Saraoui, Papa Abdoulaye Fall, Françoise Leroi, Jean-Philippe Antignac, Sylvain Chéreau, Marie France Pilet

Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3–4 log CFU g−1 after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent.

Journal of Chromatography · 1990

High-performance liquid chromatographic computer simulation based on a restricted multi-parameter approach: I. Theory and verification

J. W. Dolan, D. C. Lommen, L. R. Snyder

A computer program (DryLab MP®) is described for the simulation of HPLC separations where two or more variables (e.g., temperature and pH) are changed simultaneously. It is assumed that preliminary method development has resulted in a mobile phase of appropriate strength, such that 1 < k′ < 20 for all bands in the chromatogram. If it is desired to simulate separation as a function of changes in the values of n variables, then one or two additional runs are carried out for each variable —changing only that variable. Retention times for each of the latter runs are entered into the computer, and predictions of separation as a function of all conditions are now possible. Because simultaneous changes in two or more variables can lead to significant interaction effects and less accurate predictions, the software evaluates each simulation for possible errors. Allowed conditions must be capable of predicting values of α with an accuracy better than ± 2% (1 S.D.). The present computer program has a number of possible applications during and following method development, as discussed in the following paper (Part II).

Journal of Chromatography · 1987

Band-spacing in reversed-phase high-performance liquid chromatography as a function of solvent strength: A simple and fast alternative to solvent optimization for method development

M. A. Quarry, R. L. Grob, Lloyd R. Snyder, John W. Dolan, M. P. Rigney

Numerous reports have described the use of solvent optimization for isocratic reversed-phase high-performance liquid chromatography method development. Solvent optimization involves the use of different solvents (usually methanol, acetonitrile and tetrahydrofuran) to control band-spacing for maximum resolution of the sample. Here, we examine an alternative approach, based on variation of the concentration of organic solvent in the mobile phase (solvent strength). This procedure is less powerful than classical solvent optimization, but it nevertheless possesses a significant ability to effect changes in band-spacing. It is also much more easily carried out. Many samples do not require solvent optimization, and in these cases, a change in solvent strength may be the more practical approach. The retention data required for solvent-strength optimization are most conveniently collected by using two initial gradient runs. The application of gradient retention data for developing a final isocratic separation is facilitated by the use of commercial software. The advantages and limitations of gradient-retention data for this purpose are examined.

Journal of Chromatography · 1990

High-performance liquid chromatographic computer simulation based on a restricted multi-parameter approach: II. Applications

L. R. Snyder, J. W. Dolan, D. C. Lommen

A computer program (DryLab® MP) is described that allows restricted multi-parameter mapping for any number (or kind) of separation variables, based on only a few experiments. Multi-parameter computer simulation can be used to develop an high-performance liquid chromatographic method from the beginning, or it can be used to enhance a method developed by other means; e.g., by trial-and-error, single-parameter mapping, etc. The software can also be used to evaluate (and improve) method ruggedness. Finally, various problems (column-to-column variability, change of retention with ambient temperature fluctuations, experimental errors, etc.) that are commonly encountered during routine operation can be handled in the same general way. Examples of these various applications are given.

Journal of Chromatography A · 2024

Application of chiral stationary phases for the separation of vitamin A acetate isomers

Nicole Schräder, Wan Xia Zhu, Alexander Jaekel, Mo Legelli, Daniel Meyer, Kevin Streckel, Michaela Wirtz, Stefan Lamotte

The separation of vitamin A acetate isomers is essential for quality assurance of e.g. nutrition supplements, cosmetics, and pharmaceutical ingredients. High performance liquid chromatography (HPLC) is currently the most suitable analytical method for tackling this challenging separation task. However, the existing methods based on normal phase chromatography (NPC) are poorly reproducible due to the typical disadvantages of NPC, such as long equilibration times and fluctuation in retention factors. A new reversed phase method developed in our labs allows the separation of the isomers applying a chiral stationary phase (CSP). This phase consists of an immobilized polysaccharide which can be used in every chromatographic mode. However, they are not typically used in reversed phase mode. Through the screening of various stationary phases with different polysaccharide based chiral selectors, the choice of the ideal stationary phase could be confirmed, allowing to draw conclusions about the retention mechanism. The CSP Chiralpak IG-3 was found to be the most suitable among the examined. Regarding the separation mechanism, the spatial helical structure of the polysaccharide derivatives was confirmed to be of particular significance. In addition to the stationary phase, the mobile phase was tested for optimization regarding composition, gradient parameters as well as temperature using chromatographic method optimization software for the sake of method robustness.

Journal of Chromatography · 1984

Measurement and use of retention data from high-performance gradient elution: Contributions from “non-ideal” gradient equipment

M. A. Quarry, R. L. Grob, Lloyd R. Snyder

Equipment for high-performance liquid chromatographic (HPLC) gradient elution generally distorts the gradient selected by the operator, which in turn affects the retention of solutes separated by gradient elution. A theoretical analysis describes these gradient distortions as a function of equipment design and operating conditions. Comparisons of theory with experimental data show generally good agreement. As a result, it is now possible to select gradient conditions for minimal gradient distortion, or to correct for the effect of gradient distortion on solute retention. This will be shown in later papers to allow the use of gradient elution in new ways for more efficient method development and optimization of separation by HPLC.

Journal of Pharmaceutical Sciences · 2011

Development of Quality-By-Design Analytical Methods

Frederick G. Vogt, Alireza S. Kord

Quality-by-design (QbD) is a systematic approach to drug development, which begins with predefined objectives, and uses science and risk management approaches to gain product and process understanding and ultimately process control. The concept of QbD can be extended to analytical methods. QbD mandates the definition of a goal for the method, and emphasizes thorough evaluation and scouting of alternative methods in a systematic way to obtain optimal method performance. Candidate methods are then carefully assessed in a structured manner for risks, and are challenged to determine if robustness and ruggedness criteria are satisfied. As a result of these studies, the method performance can be understood and improved if necessary, and a control strategy can be defined to manage risk and ensure the method performs as desired when validated and deployed. In this review, the current state of analytical QbD in the industry is detailed with examples of the application of analytical QbD principles to a range of analytical methods, including high-performance liquid chromatography, Karl Fischer titration for moisture content, vibrational spectroscopy for chemical identification, quantitative color measurement, and trace analysis for genotoxic impurities.

Journal of Chromatography A · 2000

Computer simulation for the convenient optimization of isocratic reversed-phase liquid chromatographic separations by varying temperature and mobile phase strength

R. G. Wolcott, John W. Dolan, Lloyd R. Snyder

Software is described which allows the rapid development of separations by means of isocratic reversed-phase liquid chromatography (RP-LC), based on the optimization of column temperature (T) and mobile phase strength (%B). For a given sample, four initial experiments are carried out at two different temperatures, using either isocratic or (better) gradient elution. If isocratic experiments are chosen for computer simulation, it is necessary to select appropriate values of %B for these initial runs. Literature data for solute retention as a function of T are reviewed, as a basis for estimating suitable values of %B at the two values of T selected. The use of optimized values of T and %B led to acceptable separations for three representative samples. The prediction of isocratic separation on the basis of initial gradient experiments is more convenient than the use of initial isocratic experiments, but less reliable. When gradient experiments are used, one additional isocratic experiment can improve the accuracy of such predictions by a "reflection" procedure. The latter approach was confirmed for predictions of both isocratic and gradient separation from initial gradient experiments.

Journal of Chromatography A · 2006

Retention modelling in ternary solvent-strength gradient elution reversed-phase chromatography using 30mm columns

Melvin R. Euerby, Federico Scannapieco, Hans-Jürgen Rieger, Imre Molnár

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (<1, 2 and 6% error, respectively).

Electrophoresis · 2004

Microemulsion electrokinetic chromatography of drugs varying in charge and hydrophobicity Part II: Strategies for optimization of separation

Valérie Harang, Sven P. Jacobsson, Douglas Westerlund

The separation of anionic, cationic, and neutral drugs in microemulsion electrokinetic chromatography (MEEKC) was studied. The concentration of sodium dodecyl sulfate (SDS; surfactant) and 2-propanol (organic solvent) was varied in a three-level full factorial design. 29 different model substances were chosen with different hydrophobicities and charges (neutral, positive, and negative). The models were calculated by means of multiple linear regression (MLR). The compounds were divided into five different subgroups, and different strategies for optimization of the separation within each group were investigated. The optimization was done by maximizing the selectivity using response surface plots in MODDE, by calculation of different chromatographic functions, and by using the software DryLab. For all the different groups, MODDE, almost all chromatographic functions and DryLab gave approximately the same settings of the factors for optimum separation. Attempts were made to fit descriptors of the compounds to the retention data from the three-level full factorial design by means of partial least squares projection to latent structures (PLS). Between 86 and 89% of all predictions of migration times were acceptable (80-120% of the observed value).

SEPARATION SCIENCE PLUS · 2024

Retention modeling of therapeutic peptides in sub-/supercritical fluid chromatography

Jonas Neumann, Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria K. Parr

Chromatographic modeling software packages are valuable tools during method optimization steps. These are well established for reversed-phase applications utilizing retention time (RT) prediction to optimize separations and receive robust methods, which is of high interest for the analysis of pharmaceuticals. In contrast to liquid chromatography, the knowledge of RT prediction in supercritical fluid chromatography is limited to a manageable number of studies. This study uses the software DryLab to predict the RTs of the peptides bacitracin (Bac), colistin, tyrothricin (Tyro), and insulin analogs. Gradient time, column temperature, and the ternary composition (terC) of carbon dioxide, methanol (MeOH), and acetonitrile (ACN) in the gradient elution are varied in a feasibility approach using a neutral (Viridis BEH) and an amino-derivatized aromatic (Torus 2-PIC) stationary phase. In the second part, chromatographic optimization is performed in silico through gradient adjustments to optimize the separation of the fingerprint of Bac. The final gradient method utilizes a Viridis BEH column (100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of ACN/MeOH/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v). In addition, changes in the retention order of Tyro compounds with the proportion of the terC in combination with a Torus Diol column are investigated.

TrAC Trends in Analytical Chemistry · 2017

Current challenges and future prospects in chromatographic method development for pharmaceutical research

Frederick T. Mattrey, Alexey A. Makarov, Erik L. Regalado, Frank Bernardoni, Margaret Figus, Michael B. Hicks, Jinjian Zheng, Lin Wang, Wes Schafer, Vincent Antonucci, Simon E. Hamilton, Kerstin Zawatzky, Christopher J. Welch

A survey of different strategies for chromatographic method development in pharmaceutical research and development is presented. Owing to the widespread utilization of chromatography within diverse areas of pharmaceutical research, a variety of strategies for method development have arisen. We survey the current state of the art, discuss recent trends and approaches and highlight future prospects and capability gaps.

Analytical and Bioanalytical Chemistry · 2002

Use of DRYLAB to compare octadecylsilane and carbon supports for reversed-phase chromatography of triazine herbicide test solutes

Adam P. Schellinger, Yun Mao, Peter W. Carr

One of the most important aspects of method development in reversed-phase high-performance liquid chromatography (RPLC) is the accurate prediction of the resolution as one or more experimental variables (usually the mobile-phase composition) are changed. By use of commercially available computer simulation programs, e.g. Drylab 2000, data from a few initial experimental runs are used to predict how the separation will vary with composition so that a computer program can then rapidly locate the optimum conditions. Assumption of a linear relationship between log k′ (retention factor) and φ (volume fraction of organic modifier in the element) minimizes the number of initial runs required for a separation and is usually adequate when the range of φ is not large. In this work the linearity of plots of log k′ against φ for octadecylsilane-coated silica (ODS) and carbon-coated zirconia (C-ZrO2) columns were compared and the plots were used to determine whether prediction of the separation of triazine herbicides was adequate. Analysis of the slopes and intercepts of such plots are presented for both types of columns.

Journal of Pharmaceutical and Biomedical Analysis · 2018

Automated UHPLC separation of 10 pharmaceutical compounds using software-modeling

Arnold Zöldhegyi, Hans-Jürgen Rieger, Imre Molnár, Liliya Fekhretdinova

Human mistakes are still one of the main reasons of underlying regulatory affairs that in a compliance with FDA’s Data Integrity and Analytical Quality by Design (AQbD) must be eliminated. To develop smooth, fast and robust methods that are free of human failures, a state-of-the-art automation was presented. For the scope of this study, a commercial software (DryLab) and a model mixture of 10 drugs were subjected to testing. Following AQbD-principles, the best available working point was selected and conformational experimental runs, i.e. the six worst cases of the conducted robustness calculation, were performed. Simulated results were found to be in excellent agreement with the experimental ones, proving the usefulness and effectiveness of an automated, software-assisted analytical method development.

Journal of Chromatography · 1977

Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography

Imre Molnár, Csaba Horváth

Microparticulate non-polar stationary phases, such as octadecyl-silica offer a rapid and efficient means for the separation of peptides and amino acids by high-performance liquid chromatography. Retention is attributed to hydrophobic interaction between the solutes and the hydrocarbonaceous functions covalently bound to the stationary phase surface. Consequently the species are eluted in the order of increasing hydrophobicity. Various peptide mixtures were analyzed by using gradient elution with increasing acetonitrile concentration in the eluent and monitoring the column effluent at 200 or 210 nm with an UV detector. The separation of angiotensins and enzymic digest of polypeptides illustrates the speed of the method which can be used to assay the purity of peptide hormones such as α-melanotropin and gramicidin or to analyze the composition of reaction mixtures involving peptides. The efficiency of the method is superior to that obtained on the conventionally used ion-exchanger columns, except for hydrophilic amino acids and peptides that are poorly retarded. Nevertheless, with a suitable ionic surfactant in the mobile phase, non-polar stationary phases can be used for the separation of these species as well.

Journal of Chromatography A · 2013

Intergroup cross-comparison for the evaluation of data-interchangeability from various chromatographic tests

Chamseddin Chamseddin, Imre Molnár, Thomas Jira

The recently dramatic increase in the available choices of reversed-phase columns could be an advantage of this mode of separation. However, due to the insufficiency of available information in terms of the exact functionality of these phases and the similarities and differences between these newly introduced and conventional reversed-phase columns, it is now somehow problematic to determine which could be the best column for a given analytical problem. There is no single column that will give us a good separation for all applications. As a result, there have been several attempts to develop testing strategies to characterize column chemistries. In this study three of the most widely used and acceptable approaches for the characterization of reversed-phase columns, which are Tanaka, United States Pharmacopeia (USP), and Snyder–Dolan, are systemically applied to investigate the chromatographic properties of calixarene- and resorcinarene-bonded stationary phases, polar-embedded and polar-endcapped stationary phases, phenyl and ether-linked phenyl with the presence of conventional alkyl-bonded phases (octyl- and octadecylsilane). Although all column classification systems aim to evaluate “more or less” the same characteristics, each system uses different test mixtures in different chromatographic conditions. It is therefore very important to evaluate the similarities and differences in the resulted “column parameters” and the possible interchangeability of them. The results of this comparative study show that the used parameters of Tanaka and of Snyder–Dolan have in many cases a good to very good correlation. The USP approach, which is based on single run, is related to Tanaka and Snyder–Dolan only in terms of hydrophobic characters, and no relation could establish in the other parameters. The hydrophobic-subtraction model could be extended to describe the ligand–solute interactions of calixarene- and resorcinarene-bonded stationary phases, which are belonging to reversed phase material. However they show, depending on the analytes, some additional interactions, since their steric, polar and ionic properties are different compared to those of conventional alkyl-bonded phases.

Journal of Chromatography A · 1996

Combined use of temperature and solvent strength in reversed-phase gradient elution II. Comparing selectivity for different samples and systems

P. L. Zhu, John W. Dolan, Lloyd R. Snyder

A change in temperature (T) or gradient steepness (b) can result in changes in reversed-phase selectivity (α). The magnitude of these changes in α will vary with other separation conditions (column, pH, etc.) and with sample type. In this paper, selectivity changes as a function of T and b are discussed and a simple treatment that allows changes in selectivity to be compared quantitatively for different samples and HPLC conditions is developed. Following papers in this series will apply this theory to arrive at conclusions concerning the use of temperature and gradient steepness in HPLC method development. The present treatment assumes that gradient-steepness selectivity (measured by the parameter S) does not change significantly with temperature. Data for a wide range of compound types and conditions are provided in support of this assumption.

Analytical Methods · 2014

Multifactorial design principles applied for the simultaneous separation of local anesthetics using chromatography modeling software

Chamseddin Chamseddin, Thomas Jira

This study describes the development of liquid chromatographic methods for the simultaneous separation of some of the most popular local anesthetics in pharmaceutical preparations and medical praxis (benzocaine, bupivacaine, chloroprocaine lidocaine, oxybuprocaine, prilocaine, procaine, propipocaine and tetracaine) based on a systematic approach using experimental design methodology in which one or more factors are changed at the same time. The strategy employs a chromatography modeling software for the simultaneous optimization of critical chromatographic parameters, which are gradient time tG, temperature T and the ternary composition of the organic eluent B. Three different stationary phases were investigated, which are: Kromasil C18, Prontosil C18-AQ and Luna Phenyl-Hexyl. To build the design space for each column, 12 initial experiments were carried out by systematical variation of the selected critical parameters simultaneously. The chromatographic conditions of these initial runs are based on two different gradient times (tG = 20 and 60 min, linear gradient system from 10–90% organic modifier B) each at two different temperatures (T = 25 and 40 °C) repeated at three different ternary composition of the eluent B (a) 100% acetonitrile, (b) acetonitrile–methanol (50 : 50) (v/v), and (c) 100% methanol. In all experiments the pH value of the eluent A (20 mM phosphate buffer) was kept at 3.0. The mixture of local anesthetics could be well separated on all three stationary phases. Although not demonstrated in this paper, this method should be suitable for the analysis of LAs in pharmaceutical preparations or to detect them in some illegal cosmetics. The results showed that the selectivity and the elution order were similar on Kromasil C18 and Prontosil C18-AQ. On the other hand, a unique selectivity is resulted on Luna Phenyl-Hexyl, which shows, depending on the analytes, some additional interactions, since the separation mechanism on this column is influenced by its different steric and polar properties compared to the separation mechanism of alkyl-bonded phases. The predictions and real experiments were strongly correlated with an average absolute error (ΔtR) of 0.13 min (<8 s).

Decades of chromatography research done with DryLab®.