Decades of chromatography research done with DryLab®.

J Chromatogr · 1989

Practical approach for high-performance liquid chromatographic method development: assaying synthetic intermediates of a leukotriene inhibitor

John Fulper

High-performance liquid chromatographic (HPLC) methods for analyzing new drugs and their synthetic intermediates are needed as the synthesis is optimized and scaled up from making milligram amounts for initial evaluation of biological activity to producing kilogram amounts of the drug for thorough testing purposes. The most efficient solution is a single HPLC method that can be used for each step of the synthesis. A practical approach for the development of a single HPLC method is the use of computer-assisted method development to maximize the resolution within a reasonable analysis time. The computer program DryLab I was used in the development of an HPLC assay for the synthetic intermediates of a leukotriene inhibitor. The use of DryLab I with binary mixtures of organic solvents in the organic portion of reversed-phase HPLC systems is reported. With the retention data from two initial analyses, resolution can be optimized as a function of solvent strength.

Journal of Chromatography · 1989

Peak tracking in high-performance liquid chromatography based on normalized band areas: A ribosomal protein sample as an example

I. Molnár, R. Boysen, P. Jekow

Complex chromatograms that result from reversed-pahse gradient elution often exhibit changes in band order when the gradient steepness is changed. This complicates the interpretation of the resulting separation, and prevents the application of computer simulation for method development. A simple procedure based on normalized band areas was used to match bands between runs where the gradient steepness has been changed. In one example involving a Thermus aquaticus ribosomal protein sample, it was possible to find an additional band that was not apparent in tw initial experimental runs with different gradient slopes.

Journal of Chromatography · 1989

Separation of mixtures of o-phthalaldehyde-derivatized amino acids by reversed-phase gradient elution: Accuracy of computer simulation for predicting retention and band width

James D. Stuart, Diana D. Lisi, Lloyd R. Snyder

The separation of seven o-phthalaldehyde-derivatized amino acids by reversed-phase gradient elution was studied as a function of gradient time and mobile phase flow-rate. The resulting separations were compared with those from computer simulation (Drylab G). Predictions of retention time via computer simulation were found to be quite accurate, being about ±0.7% for retention time and about ±7% for retention-time differences (resolution). Predictions of band width were accurate within about ±15% for all but the steepest gradients (b > 1.0). Consequently, the ability of computer simulation to predict chromatograms reliably as a function of gradient conditions and flow-rate was confirmed for a sample that is representative of “real life”. For very steep gradients (b > 1.0), significant errors in band width were observed. The source of these errors could arise from various effects which are discussed.

Journal of Chromatography · 1989

Prediction of retention times in ion-exchange chromatography

Tatsuru Sasagawa, Yasuhiro Sakamoto, Tsukasa Hirose, Tatsunari Yoshida, Yoshimi Kobayashi, Yoshihiro Sato, Kyoko Koizumi

A method for prediction ion-exchange isocratic capacity factors from two initial gradient runs is developed. This does not assume so-called linear solvent strength (LSS) conditions, which cause significant errors in k'(C) vs. C relationships in ion-exchange chromatography. The errors associated with this approach and the LSS model are examined. The present approach allows a more accurate prediction of isocratic capacity factors for ion-exchange chromatography. Experimental application of the method to a variety of compounds, including peptides, polynucleotides and polysaccharides, separated by ion-exchange chromatography is described.

LC GC · 1988

Developing a Gradient Elution Method for Reversed-Phase HPLC

Lloyd R. Snyder, J. W. Dolan

Application of DryLab G method development strategy to a real sample

Journal of Chromatographic Science · 1988

Quantitative Determination of Limonin in Citrus Juices by HPLC Using Computerized Solvent Optimization

Philip E. Shaw, Charles W., III Wilson

The commercially available computer program, Dry lab®, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-μm C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.

American Laboratory · 1988

Computer Simulation in HPLC: Making Multistep Gradients Practical

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

Journal of Chromatography · 1988

Design of optimized high-performance liquid chromatographic gradients for the separation of either small or large molecules: II. Background and theory

B. F. D. Ghrist, Lloyd R. Snyder

The effect of the gradient on high-performance liquid chromatographic separations has been examined from a theoretical standpoint, using computer simulations to visualize the effects of different variables. Samples to be separated by gradient elution can be classified according to their separation characteristics into three groups (referred to here as cases I, II and III). Each of these sample types responds differently to a change in gradient conditions. Case III samples exhibit changes in band spacing when the gradient conditions are varied, and gradients composed of multiple linear segments are especially useful for controlling band spacing and resolution for such samples. The effect of different gradient conditions (starting %B, gradient steepness and gradient shape) on the separation of case III samples is examined in detail.

Journal of Chromatography · 1988

Design of optimized high-performance liquid chromatographic gradients for the separation of either small or large molecules: I. Minimizing errors in computer simulations

B. F. D. Ghrist, B. S. Cooperman, Lloyd R. Snyder

Computer simulations can be used to develop high-performance liquid chromatographic gradient elution methods. However, the usefulness of this approach depends on the accuracy of the resulting predictions. Possible sources of error in computer simulation for the prediction of separation based on gradient elution have been investigated. This has in turn led to recommendations for minimizing such errors. With suitable precautions it appears possible to make adequately reliable predictions of separation by gradient elution. Several examples with protein mixtures as samples are reported.

Journal of Chromatography · 1988

Design of optimized high-performance liquid chromatographic gradients for the separation of either small or large molecules: III. An overall strategy and its application to several examples

B. F. D. Ghrist, L. R. Snyder

Recommendations are presented for an efficient approach to the design of optimized gradients for complex samples using computer simulation. Examples based on the separation of polyaromatic hydrocarbon and ribosomal protein mixtures are shown.

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.

Journal of Liquid Chromatography · 1987

Computer Simulation in HPLC Method Development. Reducing the Error of Predicted Retention Times

L. R. Snyder, M. A. Quarry

Computer simulation uses two experimental HPLC runs to allow prediction of sample retention as a function of mobile phase composition or gradient conditions. The general approach is rigorous, but it is assumed that reversed-phase retention obeys the relationship log k' = log kw - S 0. Small deviations in this relationship can lead to error in predicted retention times. We have studied this error empirically for several different reversed-phase systems. This provides a basis for partially correcting these errors, and suggests recommendations for avoiding significant errors during computer simulation.

Journal of Chromatography · 1987

Predicting bandwidth in the high-performance liquid chromatographic separation of large biomolecules: II. A general model for the four common high-performance liquid chromatography methods

M. A. Stadalius, B. F. D. Ghrist, L. R. Snyder

A general model for describing gradient elution separations of peptides and proteins by reversed-phase high-performance liquid chromatography (HPLC) has been presented previously. This model has now been modified so that it can be applied to any of the four HPLC methods used for separating biological macromolecules: reversed-phase, ion-exchange, hydrophobic-interaction and size-exclusion chromatography, carried out in either an isocratic or gradient elution mode. The role of sample molecule structure and the particular column used has been further studied, so that previous empirical parameters for different column/sample choices can now be estimated from three physical properties of the sample and the column: (a) sample molecular weight, (b) native vs. denatured sample, (c) column packing pore diameter. This eliminates much of the empiricism of our preceding model, and minimizes the number of experimental runs now required in order to apply the model in practice. The final model has been tested for (a) several hundred runs involving peptides and proteins in the molecular weight range 600–162000, (b) all four of these HPLC methods, (c) in both isocratic and gradient elution modes, and (d) using data from several different laboratories (including our own). The model is able to predict bandwidth in HPLC separations of proteins and peptides with an accuracy of ±17% (1 standard deviation), for the case of “well-behaved” separations. Separations that are not “well-behaved” will give wider bands than predicted by the model.

Journal of Chromatography · 1987

Predicting bandwidth in the high-performance liquid chromatographic separation of large biomolecules: I. Size-exclusion studies and the role of solute stokes diameter versus particle pore diameter

B. F. D. Ghrist, M. A. Stadalius, Lloyd R. Snyder

Column plate numbers, N, were measured for 12 different proteins as a function of mobile phase flow-rate in two gel filtration systems (either denaturing or non-denaturing conditions). These data were used to extend a previous model that predicts bandwidths in reversed-phase and ion-exchange chromatography. Restriction of diffusion of large molecules within column packing pores is now defined more precisely, with a single relationship describing this effect for both reversed-phase and size-exclusion chromatography (SEC) (and presumably other high-performance liquid chromatography systems). Separations by gel filtration (SEC) are now included in our general model. A total of 17 flow-rate studies were carried out, involving different proteins, columns and/or mobile phase conditions (denaturing or non-denaturing). Comparisons of plate numbers predicted by the model with experimental values were satisfactory in 15 out of 17 cases. The remaining two cases appear to represent “non-well-behaved” systems, where experimental bandwidths were higher than predicted values by more than 20%. Initial attempts at understanding the origin of these non-ideal effects are described.

American Laboratory · 1987

HPLC Method Development and Column Reproducibility

Lloyd R. Snyder, John W. Dolan, M. A. Quarry

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.

Chromatographia · 1987

Solvent-strength selectivity in reversed-phase HPLC

L. R. Snyder, M. A. Quarry, Joseph L. Glajch

Solvent-strength selectivity refers to the variation of band spacing by changing the %-organic in the mobile phase (ion-pair or reversed-phase HPLC). A review of the literature has been combined with new experimental data to illustrate the general potential of this approach for HPLC optimization. It appears that most samples exhibit significant changes in band spacing method development based on solvent-strength optimization plus computer simulation (DryLab software) are given for illustration. For relatively simple mixtures (10 or fewer components), it appears that solvent-strength optimization compares favorably with other methods such as mapping the organic-solvent selectivity of methanol, acetonitrile, tetrahydrofuran, and water.

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.

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

High-Performance Liquid Chromatography: Advances and Perspectives · 1986

HPLC Separation of Large Molecules. A General Model

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