Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution.

The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

DryLab G/plus and DryLab I/plus (LC Resources) are shown to be effective aids in the development and optimization of gradient and isocratic HPLC conditions for the assay of drug substances and related compounds. Data obtained after two experimental runs in the laboratory are entered into the appropriate program where HPLC conditions can be altered (e.g. flow-rate, column dimensions, mobile phase composition, gradient steepness and shape, etc.) to arrive at optimum separation conditions with less analyst time required. The computer simulations from DryLab G/plus are shown to be suitably accurate under “real life” conditions in the development of gradient purity methods for two drug substances (Zalospirone and WY-47 384) and two synthetic intermediates (cyclooctatetraene and 2-methylcarboxybenzaldehyde). Moreover, DryLab I/plus was shown to be accurate in predicting isocratic retention for the separation of impurities in cyclooctatetraene, both in scaling down to small columns for speed and scaling up to a semi-preparative separation for isolation of impurities.

The pharmaceutically important conjugated estrogens of the type excreted by pregnant mares were baseline-resolved by gas chromatography (GC) on a SE-30 fused-silica open tubular column after acid hydrolysis and conversion to their tert.-butyldimethylsilyl derivatives. The temperature-programmed conditions were optimized with the aid of DryLab GC software with excellent agreement between the predicted and experimental results. The composition of conjugated estrogens in Premarin tablets is described as an application of the method.

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 × 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10–100 mg.

Description of the general principles underlying DryLab I/mp.

Computer simulation was used to optimize the separation of a tryptic digest of recombinant human growth hormone using reversed-phase high-performance liquid chromatography in a gradient mode. DryLab G/plus software modelled the retention behavior of the complex tryptic digest mixture as a function of gradient conditions, based on data from two experimental gradient runs. The theoretical optimum separation conditions were rapidly obtained and reproduced experimentally. Resolution did not simply increase as gradient steepness was decreased, rather, an intermediate gradient time provided maximum sample resolution. The simulation results also indicate that the method is reasonably rugged, with little change in the separation expected for different high-performance liquid chromatography systems, and changes in the separation can be compensated by a change in the gradient steepness. Computer simulation can also be useful to quickly reoptimize conditions for a new column, if it fails to provide the same separation.