Decades of chromatography research done with DryLab®.

Chromatographia · 2005

Searching for Robust HPLC Methods – Csaba Horváth and the Solvophobic Theory

Imre Molnár

This paper is written in remembrance of the work of Csaba Horváth on Reversed Phase Chromatography, (RPC) and the fundamental theory of the mechanism of retention on non-polar stationary phases, the ``Solvophobic Theory''. The paper discusses some steps in the development of this important theory and examines its consequences in developing robust methods for routine RPC. Reliable product quality requires the understanding of selectivity changes, which in RPC govern the development of robust and reliable methods involving continuous changes of liquid chromatographic parameters in aqueous eluents. The application of RPC is still growing in scientific research and in pharmaceutical and chemical production. The impact of the Solvophobic Theory in life sciences has been enormous but it was only a part of Horváth's scientific work. RPC is today one of the most popular, most widely used tools in analytical chemistry and will remain so for many years due to its stability and to its robustness.

Chromatographia · 2011

From Csaba Horváth to Quality by Design: Visualizing Design Space in Selectivity Exploration of HPLC Separations

Imre Molnár, K.E. Monks

The present paper starts by taking a look back at some of the pioneering work in high pressure liquid chromatography (HPLC) that went on in Csaba Horváth’s laboratory in 1970s, through the eyes of I. Molnár. It then goes on to describe a very modern approach to HPLC method development within the Quality by Design framework: the multifactorial optimization of three critical HPLC method parameters, i.e. gradient time (tG), temperature (T), and ternary composition (B1:B2) based on 12 experiments. The effect of these experimental variables on critical resolution and selectivity was carried out in such a way as to systematically vary all three factors simultaneously.

Peptides: Proceedings of the 5th American Peptide Symposium · 1977

Rapid Analysis of Peptide Mixtures by High Performance Liquid Chromatography With Nonpolar Stationary Phases

Murray Goodman, Johannes Meienhofer, Imre Molnár, Csaba Horváth

Journal of Chromatography B: Biomedical Sciences and Applications · 1977

Rapid separation of urinary acids by high-performance liquid chromatography

Imre Molnár, Csaba Horváth

Over a hundred acidic urinary constituents were separated within 30 min by using 5-μm octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.

Journal of Chromatography · 1976

Solvophobic interactions in liquid chromatography with nonpolar stationary phases

Csaba Horváth, Wayne Melander, Imre Molnár

Solute interaction with nonpolar stationary phases in liquid chromatography is examined on the basis of the solvophobic theory. The chromatographic process is viewed as a reversible association of the solute with the hydrocarbonaceous ligands of bonded phases. A detailed analysis of the effect of the solvent on this process yields an expression for the capacity factor with essentially no adjustable constants. The theory satisfactorily accounts for the factors affecting solute retention under a wide range of experimental conditions. It makes possible the characterization of the solvophobic (eluent) strength of mixed solvents having different composition and the evaluation of the various solvophobic forces representing incremental values of the logarithm of the capacity factor. The wide applicability of nonpolar stationary phases (reversed phases) in liquid chromatography is demonstrated by the rapid separation of biogenic acids and bases on octadecylsilica columns with neat aqueous elements.

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.

Analytical Chemistry · 1977

Enhancement of retention by ion-pair formation in liquid chromatography with nonpolar stationary phases

Csaba Horváth, Wayne Melander, Imre Molnár, Petra Molnár

In Ion-pair reversed-phase chromatography, the retention of ionized analytes on a nonpolar bonded stationary phase is enhanced by the presence of a "hydrophobic" counterlon (hetaeron) in the mobile phase. Either ion-pair formation in the mobile phase with relatively strong retention of the complex or the conversion of the stationary phase into an ion-exchanger may explain the phenomenon. Analysis of the pertinent equilibria shows that the observed hyperbolic or parabolic dependence of the capacity factors on the hetaeron concentration cannot shed light on the mechanism. The experimental data obtained for the retention of catecholamlnes by using C4-C10 alkyl sulfates and other similar hetaerons in a wide concentration range, however, could be mechanistically interpreted from the chain length dependence of the parameters for the relationship between the capacity factors and hetaeron concentration. Although the results clearly demonstrate that in the system investigated, ion-pair formation governs retention, ion-exchange mechanism can be operative under certain conditions. Changes in retention upon addition of salt to the eluent are treated both theoretically and experimentally. The effect of organic solvents on the behavior of the chromatographlc system is discussed in view of the proposed theory.

Journal of Chromatography B: Biomedical Sciences and Applications · 1978

Catecholamines and related compounds: Effect of substituents on retention in reversed-phase chromatography

Imre Molnár, Csaba Horváth

The capacity factors of 32 compounds were measured on octadecyl-silica columns by using neat aqueous phosphate buffer, pH 2.1, as the eluent. The tabulated results allow the estimation of the effect of various substituents on the retention of catecholamines and related compounds under similar chromatographic conditions.

Analytical Chemistry · 1977

Liquid chromatography of ionogenic substances with nonpolar stationary phases

Csaba Horváth, Wayne Melander, Imre Molnár

The effect of solute ionization on the retention of weak acids, bases, and ampholytes on octadecylsilica was investigated both theoretically and experimentally. The retention was attributed to a reversible association of the solutes with the hydrocarbonaceous ligand of the stationary phase. A phenomenological treatment of the corresponding equilibria was developed for various types of ionogenic substances. The energetics of the association process was analyzed in a rigorous fashion in the light of the solvophobic theory and a semi-empirical extension of the Debye-Hiickel theory to high ionic strength. The predicted effect of solute ionization on the capacity factors was substantiated by experimental data. The observed dependence of the capacity factors on the ionic strength of the eluent and the hydrophobic surface of the solute molecules showed good agreement with the theory. The advantages of the technique in the separation of biological substances are illustrated.

High-Performance Liquid Chromatography: Advances and Perspectives · 1980

Gradient Elution

Csaba Horváth, Lloyd R. Snyder

High-Performance Liquid Chromatography: Advances and Perspectives, Volume 1 deals with the fundamental aspects of high-performance liquid chromatography, a technique used in chemical analysis. The publication provides accounts, presented by experts in the field, of a variety of topics in high-performance liquid chromatography. Each chapter covers interesting subjects such as the evolution of liquid chromatography; the use of bonded phases in high-performance chromatography; effects of ionization and complex formation on retention and selectivity in reversed-phase chromatography; and gradient elution. Chromatographers, chemists, and researchers in the field of chemical analysis will find this book a valuable reference material.

Chromatographic and Membrane Processes in Biotechnology · 1991

Gradient Elution Chromatography

Firoz D. Antia, Csaba Horváth, Carlos A. Costa, Joaquim S. Cabral

Theoretical principles of gradient elution in both linear and nonlinear chromatography are discussed. In analytical applications, where linear chromatography prevails, the theory of gradient linear elution is well developed. On the other hand, in preparative applications,where the high concentrations encountered force the adsorption isotherms into the nonlinear range, gradient elution theory is as yet in its infancy. In this work, the governing principles of the theory, its limitations, and its application in some special cases are outlined.

High-Performance Liquid Chromatography: Advances and Perspectives · 1986

HPLC Separation of Large Molecules. A General Model

Csaba Horváth, Lloyd R. Snyder, M. A. Stadalius

CRC Press · 2021

Advances in Chromatography: Volume 57

Nelu Grinberg, Peter W. Carr

Chromatography Today · 2013

Chromatography Modelling in High Performance Liquid Chromatography Method Development

Imre Molnár, Róbert Kormány

This article discusses the development of chromatography modelling of the last 30 years from the first software package for calculating resolution and capacity factors to the visual modelling of chromatograms for testing peak movements with altering elution conditions. Different approaches are discussed, such as retention modelling based on measurements, others based on molecular structure or on statistical considerations. The state-of-the-art will be demonstrated with a few applications of industrial importance.

Journal of Pharmaceutical and Biomedical Analysis · 2013

Exploring better column selectivity choices in ultra-high performance liquid chromatography using Quality by Design principles

Róbert Kormány, Imre Molnár, Hans-Jürgen Rieger

An older method for amlodipine was reworked with the goal to reduce the analysis time of 60min below 6min. To select the best column for short and robust analysis, 9 different UHPLC column chemistries were investigated using 3-dimensional resolution spaces based on 12 experiments using modelling software. The main variables used were gradient time (tG), temperature (T) and the pH of eluent A. The best critical resolution was calculated and located in a 3-dimensional space in an automated fashion and the corresponding best experiments were carried out. The work (9×12=108 runs) was finished with an UHPLC instrument in less than 24h. The comparison between predictions and real experiments showed an excellent correlation with differences typically less than 0.04min (<3s) in average, although the set points were located at quite different conditions on gradient times, pH's and temperatures for the individual columns. All columns could perform the required baseline separation at their individual best working points with satisfactory results.

Software-assisted method development in high performance liquid chromatography · 2019

Modeling of HPLC Methods Using QbD Principles in HPLC

Szabolcs Fekete, Imre Molnár, Imre Molnár, Hans-Jürgen Rieger, Róbert Kormány

HPLC method modeling is becoming a powerful tool to be used in the communication about method quality in HPLC between different labs, different companies, and between companies and regulatory agencies. The understanding of simple rules of peak movements will facilitate the development of new drugs, which are badly needed for smaller patient populations. The new features of HPLC modeling software, such as 3D resolution map, the modeled robustness testing, a practicable method transfer, or a method knowledge management offer a closed loop of all information about the birth and practical use of a method, and it further suggests the use of such software solutions in regulated laboratories to make analyst's life easier-especially in the pharmaceutical industry.

Liquid Chromatography · 2017

Chapter 1 - Milestones in the development of liquid chromatography

Lloyd R. Snyder, John W. Dolan, Salvatore Fanali, Paul R. Haddad, Colin F. Poole, Marja-Liisa Riekkola

The evolution of high-performance liquid chromatography is reviewed with an emphasis on the innovations that occurred in the technique in response to the sample needs. The general themes are the development and/or applications of the basic theory as catalyst for change, invention of new chromatographic modes, evolution of column technology, and the development and improvement of instrumentation. Steady progress in these areas rather than sudden change is responsible for the column chemistries, particle technology, instrumentation, and data handling tools available today.

Journal of Pharmaceutical and Biomedical Analysis · 2021

Computer-assisted UHPLC method development and optimization for the determination of albendazole and its related substances

Elek Ferencz, Éva-Katalin Kelemen, Mona Obreja, Emese Sipos, Sára Vida, Melinda Urkon, Zoltán-István Szabó

Computer-aided ultrahigh performance liquid chromatographic (UHPLC) method development and optimization was undertaken in order to replace an underperforming European Pharmacopoeia method for the determination of albendazole and its related substances. In the preliminary screening, a temperature-gradient time bidimensional model was chosen to aid selection of the proper stationary phase. Hereinafter temperature-gradient time-ternary composition and temperature-gradient time-pH tridimensional models were applied for the optimization of critical method parameters. The simulation and in silico robustness testing were realized using DryLab modeling software. The final method was validated for quantification of impurities and assay of the active substance according to the current ICH guidance. The validated methods were tested on a real, commercial tablet formulation. The experimental design-based and software-assisted method development proved to be a fast and reliable way of replacing a method with inadequate selectivity and long runtime with a robust UHPLC-based method, which offers baseline separation for all monitored impurities in 10 min. Results confirm that software-based chromatographic modelling can not only speed up the analytical method development process, but also improve the reliability of the developed method.

Analyst · 2016

Computer assisted liquid chromatographic method development for the separation of therapeutic proteins

Eva Tyteca, Jean-Luc Veuthey, Gert Desmet, Davy Guillarme, Szabolcs Fekete

This review summarizes the use of computer assisted liquid chromatographic method development for the analytical characterization of protein biopharmaceuticals. Several modes of chromatography including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and some perspectives are discussed. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Then, the retention models that were applied in the literature in RPLC, IEX and HIC are described and critically discussed. Finally, some representative examples of separation of therapeutic proteins and mAbs are shown, to illustrate the possibilities offered by the retention modeling approach. At the end, the reliability of the models was excellent, whatever the chromatographic mode, and the retention time prediction errors were systematically below 2%. In addition, a significant amount of time can be saved during method development and robustness testing.

Journal of Chromatography A · 2009

Rapid high performance liquid chromatography method development with high prediction accuracy, using 5cm long narrow bore columns packed with sub-2μm particles and Design Space computer modeling

Szabolcs Fekete, Jenő Fekete, Imre Molnár, Katalin Ganzler

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5cm×2.1mm columns packed with sub-2μm particles and computer simulation (DryLab® package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3–5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p>400bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.