Decades of chromatography research done with DryLab®.

Analytica Chimica Acta · 2021

Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column

Ryan Karongo, Min Ge, Christian Geibel, Jeannie Horak, Michael Lämmerhofer

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient “smart peak parking” algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Journal of Pharmaceutical and Biomedical Analysis · 2015

Establishing column batch repeatability according to Quality by Design (QbD) principles using modeling software

Norbert Rácz, Róbert Kormány, Jenő Fekete, Imre Molnár

Column technology needs further improvement even today. To get information of batch-to-batch repeatability, intelligent modeling software was applied. Twelve columns from the same production process, but from different batches were compared in this work. In this paper, the retention parameters of these columns with real life sample solutes were studied. The following parameters were selected for measurements: gradient time, temperature and pH. Based on calculated results, batch-to-batch repeatability of BEH columns was evaluated. Two parallel measurements on two columns from the same batch were performed to obtain information about the quality of packing. Calculating the average of individual working points at the highest critical resolution (Rs,crit) it was found that the robustness, calculated with a newly released robustness module, had a success rate >98% among the predicted 36=729 experiments for all 12 columns. With the help of retention modeling all substances could be separated independently from the batch and/or packing, using the same conditions, having high robustness of the experiments.

Journal of Chromatography A · 2020

Improving selectivity and performing online on-column fractioning in liquid chromatography for the separation of therapeutic biopharmaceutical products

Szabolcs Fekete, Harald Ritchie, Jason Lawhorn, Jean-Luc Veuthey, Davy Guillarme

A novel column-coupling approach is suggested to improve both the selectivity and efficiency of protein separations in liquid chromatography. Protein separations often suffer from limited selectivity or not appropriate resolving power. For a new biopharmaceutical product, the identification of the main and minor variant species is required. For that purpose, often offline collection fractioning is applied which is time consuming and regularly dilute the samples to an unacceptable extent. By serially coupling columns in the order of their increasing retentivity and applying “multi-isocratic” elution mode, indeed any (arbitrary) selectivity can be attained. Moreover, if a protein peak is trapped at the inlet of a later column segment – of a coupled system -, its band will be refocused and elute in unprecedented sharp peak. Furthermore, it becomes possible to perform online on-column fractioning of protein species within a very short analysis time (∼ 1 min) and without sample dilution. Two-, three- or multiple column systems can be developed and applied for complex sample separations (such as antibody mixtures). This new methodology can be particularly useful to improve the analysis (and therefore, safety and quality) of therapeutic mAbs and related products and offers benefits compared to offline fractionating. It is also demonstrated in this proof of concept study, that methyl (C1) modified RP phase has a great potential for protein separations despite it is not commercially available in state-of-the-art wide pore superficially porous particle format..

Journal of Pharmaceutical and Biomedical Analysis · 2024

Updating the European Pharmacopoeia impurity profiling method for cetirizine and suggesting alternative column, using design space comparison

Róbert Kormány, Barnabás Soós, Krisztián Horváth

The goal of the present study was to develop a generic workflow to evaluate the chromatographic resolution in a design space and find replacement column for the new method. To attain this objective, a limited number of initial experiments have been performed, and a modeling tool was employed to study and compare design spaces obtained with different columns. By overlaying the different individual resolution maps (design spaces), it is possible to quickly identify a robust zone where the different columns meet a given resolution criterion. This new feature of the modeling tool is very useful for finding alternative columns for a given separation, rather than the usual column tests. It was also demonstrated that two different columns can be used as equivalents (replacement columns), providing sufficient resolution at the same working point and with a high degree of robustness.

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 · 2002

Computerized design of separation strategies by reversed-phase liquid chromatography: development of DryLab software

Imre Molnár

The development of DryLab software is a special achievement in analytical HPLC which took place in the last 16 years. This paper tries to collect some of the historical mile stones and concepts. DryLab, being always subject to change according to the needs of the user, never stopped being developed. Under the influence of an ever changing science market, the DryLab development team had to consider not just scientific improvements, but also new technological achievements, such as the introduction of Windows 1.0 and 3.1, and later Windows NT and 2000. The recent availability of new 32-bit programming tools allowed calculations of chromatograms to be completed more quickly so as to show peak movements which result for example from slight changes in eluent pH. DryLab is a great success of interdisciplinary and intercontinental cooperation by many scientists.

Analytical Chemistry · 1983

Gradient elution in reversed-phase HPLC-separation of macromolecules

L. R. Snyder, M. A. Stadalius, M. A. Quarry

Journal of Chromatography · 1989

Drylab® computer simulation for high-performance liquid chromatographic method development: II. Gradient Elution

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

Computer simulation (DryLab software) as an aid for the development of gradient high-performance liquid chromatographic methods is reviewed. Several examples of its application are presented and the accuracy of such predictions is discussed.

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.

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

Chromatographia · 2012

Enhancing the Quality of Separation in One-Dimensional Peptide Mapping Using Mathematical Transformation

Róbert Berky, Szabolcs Fekete, Jenő Fekete

In this study, some practical examples are presented that show the quality of separations using very efficient columns packed with the latest generation of core shell sub-3 μm and fully porous sub-2 μm particles in one-dimensional peptide separations. This paper shows an approach for the analysis of proteins, such as high-resolution separations, and a data transformation process to improve peak recognition and analysis. Applying power functions on raw chromatographic data can be a neat tool in the field of biosimilar analysis, especially in comparability studies regarding the quality (primary structure) of proteins. Based on the results presented here, it can be stated that the use of power functions is beneficial for the comparison of chromatograms when peak areas are considered but has no effect when using peak heights. In this study, the new Acquity CSH columns (C18 and phenyl-hexyl) and the core–shell type wide pore Ascentis Express Peptide ES C18 material were applied with great success in peptide mapping. Finally, using phenyl-hexyl stationary phase in peptide separation seems to be a good alternative to the generally applied C18 or C4 phases.

Journal of Chromatography A · 2013

Optimizing selectivity during reversed-phase high performance liquid chromatography method development: Prioritizing experimental conditions

Lloyd R. Snyder, John W. Dolan

Several different conditions can be varied to improve selectivity for reversed-phase chromatography (RPC). A reexamination of literature data suggests that changes in selectivity due to a change of column or mobile phase pH are largely replicated by changes in temperature or mobile phase composition (concentrations of acetonitrile and/or methanol). This suggests a reconsideration of the role of mobile phase pH and the column during method development.

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.

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.

Journal of Chromatography Open · 2025

Weak to strong ion-pair gradients to expand the selectivity of oligonucleotide separations in reversed phase liquid chromatography - A proof of concept

Szabolcs Fekete, Mateusz Imiolek, Matthew Lauber

This study introduces a novel ion-pair reversed-phase liquid chromatography (IP-RPLC) method using a dual gradient approach to enhance the separation of oligonucleotides. By combining a weak ion-pairing (IP) agent in the starting (weaker) mobile phase with a strong IP agent in the final (stronger) mobile phase, this method widens the elution window, improving the resolution and selectivity for complex ON mixtures. The approach outperforms traditional single IP systems, particularly in separating sequence and size variants of ONs. Experimental results demonstrate that the weak-to-strong gradient yields better peak separation and higher selectivity compared to conventional methods. This innovative gradient strategy, combined with concave gradients, offers significant potential for optimizing ON separations in the development of oligonucleotide-based therapeutics.

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.

TrAC Trends in Analytical Chemistry · 1987

High-performance liquid chromatographic method-development using computer simulation

L. R. Snyder, J. W. Dolan, M. A. Quarry

Computer-assisted method-development schemes for high-performance liquid chromatography have taken another step forward with the introduction of 'computer simulation'. Software for the application of this technique is described and illustrated.

Chromatographia · 1987

Computer simulation as a means of developing an optimized reversed-phase gradient-elution separation

J. W. Dolan, L. R. Snyder, M. A. Quarry

A personal-computer program (DryLab G) is described for the simulation of reversed-phase gradient-elution separations. After two experimental gradient runs are carried out initially, this program allows the user to develop a final separation by varying gradient conditions (gradient time, initial and final %-organic in the mobile phase, gradient shape), column dimensions, flowrate and particle size. This approach takes advantage of “solvent-strength selectivity”, as reported recently [28] for isocratic separations. Method development using this procedure can result in better separations with much less effort. Examples of its validation and application are presented.

TrAC Trends in Analytical Chemistry · 2018

Critical review of reports on impurity and degradation product profiling in the last decade

Sándor Görög

Drug impurity and degradation profiling mean the detection, structure elucidation and quantitative determination of impurities and degradation products in bulk drug materials and pharmaceutical formulations. This is today one of the most important fields of activities in pharmaceutical analysis. The reason for this is that unidentified, potentially toxic impurities are health hazards, and in order to increase the safety of drug therapy, impurities should be identified and determined by selective methods. The aim of this review is to characterise the state-of-art in the field of impurity and degradation profiling of drugs based on papers published in the last decade. The separation and determination of impurities and degradants with a known structure are discussed, but emphasis is placed on the structure elucidation and determination of new (unknown) impurities and degradation products by off-line and on-line chromatographic–spectroscopic methods. The analytical aspects of enantiomeric purity of chiral drugs are also discussed.

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.