Decades of chromatography research done with DryLab®.

Chromatographia · 2001

Isocratic liquid chromatographic method for the analysis of roxithromycin and structurally related substances in bulk samples

H. K. Chepkwony, F. N. Kamau, E. Rodriguez, E. Roets, J. Hoogmartens

A selective, repeatable, and robust isocratic liquid chromatographic method has been developed for the analysis of bulk samples of roxithromycin, a semi-synthetic macrohde antibiotic. The method involves the use of XTerra RP18 as stationary phase and acetonitrile-2.0 M (NH4)2HPO+ pH 6.4-water, 25:30:45, (v/v) as mobile phase, delivered at a flow rate of 1.0mL min 1. Detection was by UVabsorbance at 215 nm. Sufficient separation of roxithromycin from its homolog containing one more methyleneoxy group (roxithromycin G) and from other related substances was achieved. The robustnessof the method was evaluated by means of a full-factorial experimental design.

Journal of Chromatography A · 2001

Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments

Laurie Van Heukelem, Crystal S Thomas

We used chromatography modeling software to assist in HPLC method development, with the goal of enhancing separations through the exclusive use of gradient time and column temperature. We surveyed nine stationary phases for their utility in pigment purification and natural sample analysis. For purification, a complex algal matrix was separated on an efficient monomeric column, from which partially purified fractions were collected and purified on polymeric columns that exaggerated resolution between pigments of interest. Additionally, we feature an HPLC method that is simple, fast, demonstrates excellent transferability and is ideal for quantitative analysis of pigments in dilute natural water samples.

LC GC Europe · 2001

Unexpected Results in Chromatography

Imre Molnár

Unusual experiments can provide surprisingly good analytical solutions. When developing Chromatographic methods, analysts must use a combination of experience and instinct to choose initial starting conditions. This is often followed by a period of trial-and-error optimization until the desired methodology is achieved. This article illustrates how the process of Chromatographic method development can be improved using computer modelling and simulation.

Fresenius' Journal of Analytical Chemistry · 2001

Automatization for development of HPLC methods

M. Pfeffer, H. Windt

Within the frame of inprocess analytics of the synthesis of pharmaceutical drugs a lot of HPLC methods are required for checking the quality of intermediates and drug substances. The methods have to be developed in terms of optimal selectivity and low limit of detection, minimum running time and chromatographic robustness. The goal was to shorten the method development process. Therefore, the screening of stationary phases was automated by means of switching modules equipped with 12 HPLC columns. Mobile phase and temperature could be optimized by using Drylab after evaluating chromatograms of gradient elutions performed automatically. The column switching module was applied for more than three dozens of substances, e.g. steroidal intermediates. Resolution (especially of isomeres), peak shape and number of peaks turned out to be the criteria for selection of the appropriate stationary phase. On the basis of the "best" column the composition of the "best" eluent was usually defined rapidly and with less effort. This approach leads to savings in manpower by more than one third. Overnight, impurity profiles of the intermediates were obtained yielding robust HPLC methods with high selectivity and minimized elution time.

Journal of Chromatography A · 2000

Selectivity differences for C18 and C8 reversed-phase columns as a function of temperature and gradient steepness: I. Optimizing selectivity and resolution

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

Four experimental runs where temperature T and gradient time tG are varied allow the computer-prediction of reversed-phase liquid chromatographic (RPLC) separation for different combinations of temperature and gradient time. This in turn can provide significant changes in selectivity and a resulting optimization of separation. If this procedure is repeated for different columns, additional control over selectivity and resolution becomes possible. The simultaneous variation of T and tG for columns from different sources was studied for two samples, as a means of evaluating the general advantage of this approach for RPLC method development. Changes in relative retention with T were found to be approximately constant for different values of tG and for different RPLC columns; similarly, changes in relative retention with tG were roughly independent of changes in temperature or the column. The latter relationships can be useful in matching (“tracking”) peaks between runs during method development based on the present approach, as well as for other applications discussed in here and in Part II.

Encyclopedia of Separation Science · 2000

Appendix 2. Essential Guides to Method Development in Liquid Chromatography

J.W. Dolan, L.R. Snyder

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 · 2000

Determination of albendazole and its main metabolites in ovine plasma by liquid chromatography with dialysis as an integrated sample preparation technique

P Chiap, B Evrard, M. A Bimazubute, P de Tullio, Ph Hubert, L Delattre, J Crommen

Albendazole is a benzimidazole derivative with a broad-spectrum activity against human and animal helminth parasites. In order to determine the main pharmacokinetic parameters in sheep after oral and intravenous administration of a new formulation of albendazole (an aqueous solution), a fully automated method was developed for the determination of this drug and its main metabolites, albendazole sulfoxide (active metabolite) and sulfone in ovine plasma. This method involves dialysis as purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatography (LC). All sample handling operations were executed automatically by means of an ASTED XL system. After conditioning of the trace enrichment column (TEC) packed with octadecyl silica with pH 6.0 phosphate buffer containing sodium azide, the plasma sample, in which a protein releasing reagent (1 M HCl) containing Triton X-100 was automatically added, was loaded in the donor channel and dialysed on a cellulose acetate membrane in the static-pulsed mode. The dialysis liquid consisted of pH 2.5 phosphate buffer. By rotation of a switching valve, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The chromatographic separation was performed at 35°C and the analytes were monitored photometrically at 295 nm. Due to the differences in hydrophobic character between albendazole and its metabolites, a gradient elution was applied. The mobile phase consisted of a mixture of acetonitrile and pH 6.0 phosphate buffer. The proportion of organic modifier was increased from 10.0 to 50.1% in 12.30 min, then from 50.1 to 66.9% in 1.70 min. First, the gradient conditions and the temperature were optimised for the LC separation using the DryLab software. Then, the influence of some parameters of the dialysis process on analyte recovery was investigated. Finally, the method developed was validated. The mean recoveries for albendazole and its metabolites were about 70 and 65%, respectively. The limits of quantification for albendazole and its metabolites were 10 and 7.5 ng/ml, respectively.

Journal of Chromatography A · 2000

Reversed-phase separation of achiral isomers by varying temperature and either gradient time or solvent strength

Lloyd R. Snyder, John W. Dolan

The separation of several isomer pairs of widely varied structure was studied as a function of changes in temperature, gradient time, mobile phase pH, column type (“monomeric” vs. “polymeric” C18-silica) and organic solvent (methanol vs. acetonitrile). General conclusions are drawn which may prove useful in future attempts at the separation of these and other achiral isomers.

Journal of Chromatography A · 2000

Control of column temperature in reversed-phase liquid chromatography

R. G Wolcott, John W. Dolan, Lloyd R. Snyder, Stephen R Bakalyar, Megan A Arnold, Jon A Nichols

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.

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 · 1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: I. Peak capacity limitations

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

The separation of samples that contain more than 15 to 20 analytes (n>15–20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8≤n≤48 as a function of temperature T and gradient time tG. The required peak capacity was determined for each sample, after selecting T and tG for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and tG have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and tG vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

Journal of Chromatography A · 1999

Critical comparison of retention models for optimisation of the separation of anions in ion chromatography: III. Anion chromatography using hydroxide eluents on a Dionex AS11 stationary phase

John E. Madden, Nebojsa Avdalovic, Peter E. Jackson, Paul R. Haddad

Three ion chromatography (IC) retention models, namely the linear solvent strength model (LSSM), empirical end points model (EEPM) and three-point curve fitting using DryLab from LC Resources were evaluated in terms of their ability to predict retention factors for inorganic anions separated on a Dionex AS11 column using electrolytically generated hydroxide eluents. Extensive experimental retention data were gathered for 21 anions (fluoride, acetate, formate, bromate, chloride, nitrite, methanesulfonate, bromide, chlorate, nitrate, iodide, thiocyanate, succinate, sulfate, tartrate, oxalate, tungstate, phthalate, chromate, thiosulfate and phosphate) using hydroxide eluents of varying concentration. Although the purely theoretical LSSM was found to give adequate performance, the EEPM (in which a linear relationship is assumed between the logarithm of retention factor and the logarithm of eluent strength, but the slope is determined empirically) and DryLab performed better, with DryLab giving the best accuracy and precision of the three models. The EEPM and DryLab were also shown to have advantages in terms of their low knowledge requirements and ease of solution. Compared with IC using dual eluent species, the retention behaviour in IC using single eluent species was found to be easier to model by both theoretical and empirical approaches.

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.

Chemia Analityczna · 1998

Systematic approaches to HPLC method development for reversed-phase separation

L. R. Snyder, J. W. Dolan

Journal of Chromatography A · 1998

Maintaining fixed band spacing when changing column dimensions in gradient elution

J. W Dolan, L. R Snyder

In gradient elution separations, it may be required to change either column length (to increase resolution or shorten run time) or column diameter (for an increase in sensitivity or for preparative separations). In either of these changes of column dimensions, it is usually desired to maintain the same relative band spacing (selectivity), so as to increase resolution in proportion to (column plate number)1/2 when increasing column length, or to maintain constant resolution when changing column diameter. A general rule for avoiding changes in band spacing in these situations is to maintain the quantity [(gradient time)×(flow-rate)/(column volume)] constant, while holding the initial and final gradient mobile phase compositions (%B) fixed. This rule is only valid as long as the equipment hold-up volume (dwell volume) is negligible, or if all sample components are strongly retained at the start of the gradient. When neither of the latter conditions apply, then significant changes in band spacing may result when changing column size. Rules are presented for recognizing this potential problem for a given sample/HPLC-equipment combination, and adjustments in separation conditions that can avoid this problem are discussed. Changes in band spacing as a result of change in column size are of special concern when developing procedures for preparative chromatography under gradient conditions.

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 A · 1998

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development: II. The use of further changes in conditions

J. W Dolan, L. R Snyder, D. L Saunders, L Van Heukelem

The preceding paper (Part I) suggests that simply optimizing temperature and gradient steepness will often provide an adequate reversed-phase HPLC separation. In some cases, however, this procedure will prove unsuccessful, and then further method-development experiments (involving change in other separation conditions) will be required. One strategy is to change a variable other than temperature or gradient steepness, followed by re-optimization of the latter two variables. The present paper examines the application of this approach with the aid of computer simulation to several samples.

LC GC · 1998

Reversed-Phase Gradient Elution: How to Get Better Results with Less Work

Imre Molnár, Lloyd R. Snyder, John W. Dolan

LC GC · 1997

Selectivity Control in HPLC Method Development

Lloyd R. Snyder, John W. Dolan, Imre Molnár, N. M Djordjevic