Decades of chromatography research done with DryLab®.

LC-GC Europe · 2001

Computer-Assisted Optimization of Reversed-Phase HPLC Isocratic Separations of Neutral Compounds

Tomasz Baczek, Roman Kaliszan, H.A. Claessens, M.A. Straten

Rational selection of optimized experimental conditions for chromatographic separation of analytes is realized nowadays by means of specialized computer programs. Two such programs, DryLab (LC Resources, Walnut Creek, California, USA) and ChromSword (Merck KCaA, Darmstadt, Germany), were compared in this research. The aim of the study was to compare the optimization of chromatographic separations of neutral compounds performed isocratically in reversed-phase high performance liquid chromatography (HPLC) systems. A detailed description and discussion of the differences in predicted and experimental chromatographic retention parameters is reported. The conclusion reached is that the two programs provide identical predictions of retention data, when predictions are based on two initial experimental chromatographic measurements. An additional option of ChromSword, employing the quantitative structure-retention relationships (QSRR), appears to provide satisfactory predictions of the separation when molecular structural data along with retention data from a single initial experimental run are used as inputs. Predictions based on molecular structure alone are not very accurate and are not likely to provide useful separation information.

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.

Journal of Chromatography A · 1996

Initial experiments in high-performance liquid chromatographic method development I. Use of a starting gradient run

L. R. Snyder, J. W. Dolan

A single gradient elution run with acetonitrile (B)-water (A) as mobile phase can be used to estimate preferred conditions for subsequent method development experiments based on reversed-phase high-performance liquid chromatography. For a broad range of sample types,that includes both very hydrophilic and hydrophobic compounds, it was found that isocratic retention is given by log k ≈ log kw −4.2ϕ, where ϕ = 0.01 %B. An initial gradient run allows values of log kw to be estimated for each compound in the sample, which then permits compound retention to be approximated as a function of either isocratic or gradient experimental conditions. The use of an initial gradient run in this way provides a rational basis for the subsequent development of a final HPLC method. Predictions based on this initial run can be used to (a) select between isocratic or gradient elution for further experiments and the final method, (b) choose a value of %B to achieve a desired value of k for the initial or final band in isocratic separation and/or (c) choose values of %B for the initial and final mobile phase in gradient elution. The present approach is based on a wide range of sample types and different reversed-phase columns; for this reason it is expected to be reasonably general and accurate. Errors in subsequent predictions based on an initial gradient run can also be estimated as a function of experimental conditions, allowing the selection of conditions for minimum error.

Analytical Chemistry · 1986

Separation of peptide mixtures by reversed-phase gradient elution. Use of flow rate changes for controlling band spacing and improving resolution

Joseph L. Glajch, M. A. Quarry, J. F. Vasta, Lloyd R. Snyder

World Scientific Publishing Europe Ltd · 2019

Software-assisted method development in high performance liquid chromatography

Imre Molnár, Szabolcs Fekete

"This handbook gives a general overview of the possibilities in recent developments in chromatographic retention modeling. As a result of the latest developments in modeling software, several new features are now accessible, opening a new level in HPLC method development. Many of these current possibilities in software assisted liquid chromatographic method modeling for analytical purposes are presented. Several modes of chromatography, including Reversed-Phase Liquid Chromatography (RPLC), Ion Exchange Chromatography (IEX), Hydrophobic Interaction Chromatography (HIC), and Hydrophilic Interaction Liquid Chromatography (HILIC) are explained in detail. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Beside the industrial and practical benefits of retention modeling, the possibilities in teaching and education are also illustrated. Finally, numerous representative industrial examples are shown, to highlight the benefits, time and cost savings offered by state-of-the-art software assisted HPLC method development."--Publisher's website

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).

Journal of Chromatography A · 2002

Variability of column selectivity for reversed-phase high-performance liquid chromatography: Compensation by adjustment of separation conditions

J. W Dolan, L. R Snyder, T. H Jupille, N. S Wilson

Reversed-phase columns are widely used in assays based on high-performance liquid chromatography (HPLC). When such assays are repeated over time, it is often necessary to replace the column. In such cases, the selectivity of columns from different production batches may prove sufficiently variable to result in a failed separation. It is possible to compensate for differences in column selectivity by making small changes (adjustments) in separation conditions. The present paper describes an efficient procedure for choosing adjusted conditions and discusses its general applicability.

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 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 · 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.

Journal of Chromatography · 1984

Measurement and use of retention data from high-performance gradient elution: Correction for “non-ideal” processes originating within the column

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

Under “ideal” conditions it is possible to model retention in gradient elution so as to be able to calculate retention times, tg, as a function of isocratic retention in corresponding liquid chromatographic systems. In this paper we consider various “non-ideal” processes that lead to errors in calculated values of tg. The more important of these are solvent demixing due to uptake of one mobile phase component by the column packing, non-linear plots of log k′ vs. gradient time or mobile phase composition and changes in column dead-time, t0, due to changes in mobile phase composition and flow-rate. Expressions are derived to correct for these various “non-ideal” effects, including equipment limitations discussed in the preceding paper. Calculated values of tg for reversed-phase gradient elution systems then agree with experimental values to within ± 1 % (1 standard deviation) of the total gradient time, tG. These results should prove useful in (a) improving the precision of retention in gradient elution (which should be comparable to that in isocratic elution), (b) using gradient elution for more efficient method development in isocratic procedures and (c) better understanding gradient separations of macromolecules such as proteins.

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.

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 · 2017

Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass spectrometric peak tracking

Róbert Kormány, Imre Molnár, Jenő Fekete

An older method for terazosin was reworked in order to reduce the analysis time from 90min (2×45min) to below 5min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified impurities separately. The reason of the different methods is that the retention of two impurities is not adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by different method than the other specified impurities. In our new method we could improve the retention with pH elevation using a new type of stationary phases available for high pH applications. Also a detection wavelength could be selected that is appropriate for the detection and quantification of all impurities. The method development is the bottleneck of liquid chromatography even today, when more and more fast chromatographic systems are used. Expert knowledge with intelligent programs is available to reduce the time of method development and offer extra information about the robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good alternative way to identify a certain peak in different chromatograms is the molecular mass of the compound, due to its high specificity. Liquid Chromatography–Mass Spectrometry (LC–MS) is now a routine technique and increasingly available in laboratories. In our experiment for the resolution- and retention modeling the DryLab4 method development software (Version 4.2) was used. In recent versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking technology. The modelled and measured chromatograms showed excellent correlations. The average retention time deviations were ca. 0.5s and there was no difference between the predicted and measured Rs,crit −values.

Journal of Chromatography A · 2000

Selectivity differences for C18 and C8 reversed-phase columns as a function of temperature and gradient steepness: II. Minimizing column reproducibility problems

J. W Dolan, L. R Snyder, T. Blanc

The choice of T and tG as variables for controlling selectivity and resolution during reversed-phase liquid chromatography (RPLC) method development can be used to minimize problems caused by column batch-to-batch irreproducibility. When a new column fails to provide adequate separation of the sample, altered values of T and tG can be predicted that will restore the separation obtained with the previous column. Alternatively, columns from different manufacturers can be tested during method development, in order to find a common set of conditions (T and tG) that provide acceptable separation with two or more of these columns. In this way, any of several columns from different sources become usable for the method. Examples are shown of these different computer-assisted procedures for minimizing problems due to column variability.

Journal of Chromatography A · 1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: II. Two-run assay procedures

J. W Dolan, L. R Snyder, N. M Djordjevic, D. W Hill, T. J Waeghe

By optimizing column temperature T and gradient time tG, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15–20 components are present in the sample. An alternative approach is to carry out two separations with different conditions (T, tG) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and tG are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.

Journal of Chromatography A · 2002

Two-dimensional optimization using different pairs of variables for the reversed-phase high-performance liquid chromatographic separation of a mixture of acidic compounds

Tom H. Jupille, John W. Dolan, Lloyd R. Snyder, Imre Molnár

Computer-facilitated method development has been extended for the simultaneous optimization of any two variables in separations by HPLC and other chromatographic procedures (gas chromatography, supercritical fluid chromatography, capillary electrophoresis, etc.). The application of this approach to HPLC method development is illustrated by the reversed-phase separation of a nine-component mixture of organic acids. Two of four variables (temperature, solvent strength (%B), pH and buffer concentration) were separately optimized in terms of selectivity, and the results are compared in terms of which variables and other conditions are most effective in providing maximum resolution for samples that contain ionizable compounds.

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 Pharmaceutical and Biomedical Analysis · 2017

Separation of antibody drug conjugate species by RPLC: A generic method development approach

Szabolcs Fekete, Imre Molnár, Davy Guillarme

This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

Journal of Chromatography · 1985

Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography: Application to method development and the choice of column configuration

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