Literature
Browse through a complete list of over 280 publications that have been published using DryLab®.
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When carrying out HPLC method development, it is often necessary to vary the relative retention of the sample (values of α) by changing some experimental variable, e.g., solvent type, pH, etc. The choice of which variable will be most suitable for a change in selectivity depends on two conflicting goals: (a) the attainment of maximum changes in α for the better control of resolution and (b) the avoidance of practical problems associated with the use of a given variable to optimize selectivity. This study provides a quantitative evaluation of different variables for their effect on selectivity (α). Various practical problems which must be balanced against this ability of a variable to change values of α are also discussed. The selection of any two variables for their simultaneous use in controlling α is also examined.
The DryLab G+ software (LC Resources, Lafayette, CA, USA) was applied for optimization of HPLC resolution of extract from Archangelica officinalis, a rich source of furanocoumarins. Two preliminary runs based on linear gradient range of methanol from 5% to 100% in gradient times 30 min and 60 min are shown to be satisfactory for optimization of the resolution. A Varian Polychrom 9065 photodiode array detector (DAD) coupled to an IBM personal computer with PolyView (Varian) applications software was used to acquire, manipulate and store spectral data. For quantitative determination of selected furanocoumarins the Multicomponent Analysis (MCA) was applied.
A method is proposed for the sensitive chiral analysis of amino acid enantiomers by high-performance liquid chromatography (HPLC). Thus the enantiomers of a mixture of seven racemic amino acids were resolved as their DNP derivatives from each other and from the peak of the hydrolyzed reagent, employing a quinine carbamate-based chiral anion exchange-type chiral stationary phase (CSP) and aqueous buffered mobile phases. However, the initial isocratic chromatogram yielded many peak overlaps although the corresponding enantiomers were well resolved. Therefore, the separation of the complex mixture had to be optimized; we utilized the commercial computer method development software drylab. First, the influence of the manifold mobile phase parameters and chromatographic conditions (pH, type and content of organic modifier, buffer concentration, temperature, type of co-ion, etc.) on retention and resolution was studied by isocratic elution. Furthermore, with such optimized conditions linear and multi-segmented organic modifier and buffer salt gradients, respectively, were simulated with the computer program and experimentally verified. Average errors of prediction of retention times lay between 2 and 8%. Finally, a highly improved HPLC gradient method resulted in almost all components being baseline separated and equally spaced and accelerated by a factor of more than 3 compared to the initial run.
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.
The DrylabG software (LC Resources, Lafayette, CA, USA) was applied for optimization of HPLC resolution of some nucleosides in the reversed-phase systems. Two preliminary runs based on linear gradient range of acetonitrile (ACN) from 0% (pure buffer) to 20% and of methanol (MeOH) from 0% to 50% are shown to be satisfactory for optimization of the resolution. A good agreement between simulated and experimental chromatograms was observed.
It has been shown previously that computer simulation based on two initial experiments can predict separation in reversed-phase gradient elution as a function of gradient conditions (gradient steepness, gradient range and gradient shape) and column conditions (column length, flow-rate and particle size). The present study extends this capability for changes in temperature. Four initial experiments (two different gradient times, two different temperatures) provide input data that allow predictions of separation as a function of temperature as well as gradient and column conditions. A semi-empirical relationship, tR=a+bT, is able to relate gradient retention time tR to column temperature T (other conditions constant). The accuracy of this approach has been evaluated for 102 solutes and a variety of experimental conditions, including the use of five different HPLC instruments (four different models).
The separation of nine un-ionized samples was studied as a function of temperature (T) and gradient steepness (b). Selectivity values Δlog α∗ were obtained for 160 compounds, ranging from nonpolar hydrocarbons to very polar drugs. Selectivity varied markedly with sample type: nonpolar compounds such as aromatic hydrocarbons and fatty acid methyl esters generally showed only modest changes in band spacing as temperature or gradient steepness was varied. More polar samples exhibited larger changes in α (Δlog α∗) when temperature and/or gradient steepness wee changed, but the largest values of Δlog α∗ for these non-ionized samples are less than the average value of Δlog α∗ for the ionized samples of Part III [1]. Poly-functional silane (“polymeric”) columns exhibit slightly increased b- and/or T-selectivity for some samples.
Initial method development experiments for both neutral and ionic samples are best carried out with reversed-phase high-performance liquid chromatography (HPLC) using acetonitrile-methanol-buffer mobile phases. The preceding paper (Part I) suggests the use of an acetonitrile-buffer gradient to start method development. In this paper, optimum conditions for this first separation are discussed. Sequential method development experiments plus computer simulations are then used to obtain a final HPLC method. In this connection, we have examined how many experiments are required for reliable predictions of ternary-solvent retention. Three experiments are sufficient to predict isoeluotropic retention for methanol-acetonitrile-buffer ternaries where solvent strength does not vary, but five experiments are required for ternaries that contain tetrahydrofuran.
A complex pharmaceutical raw material was characterized by means of reversed-phase gradient elution. By varying gradient steepness and mobile-phase pH, it was possible to optimize band spacing so as to separate 16 impurities or degradation products from the drug substance. Computer simulation was useful in interpreting these complex chromatograms and determining the maximum number of peaks that could be separated in this way. A marginal separation of all 17 sample components could be obtained, but the resulting method was quite pH-sensitive and therefore not very rugged. As an alternative, a rugged method was developed that separates the drug substance from all other sample components. The present study also describes how present computer simulation software for isocratic separation can be used to predict resolution for gradient elution runs as a function of pH.
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.
The ability of temperature and gradient steepness to change band spacing has been investigated for several ionizable samples that include 8 substituted benzoic acids, 9 substituted anilines, 22 basic drugs, 9 structurally-related herbicide impurities, 7 chlorophylls and 72 peptides and proteins. Mobile phase pH was also varied to determine the effect of sample ionization on temperature and gradient-steepness selectivity.
This paper describes the results of a new method for the determination of 1-,-hydroxyanthranoids in senna. This will be illustrated by examples from a study of the occurrence of 17 different 1,8-dihydroxyanthranoids (anthraquinones and their bianthrys) in fruits leaves of PSenna angustifolia amd Senna acutifolia. The anthranoids are extracted using a mixture of acetonitrile and a solution of sodium hydrogencarbonate. The different compounds are separated and detected by HPLC using an Rp-8 column and a photodiode array detector. The method was optimized by means of computer-assisted method development techniques using the DryLab software.
A complex mixture of arachidonic acid metabolites was separated by reversed-phase HPLC using a multi-step gradient, which was modelled by computer-assisted HPLC method development. The metabolites were extracted on-line on a precolumn connected to the analytical column in the same HPLC system. The predictions of the resolution and also the retention times calculated by computer simulation were very accurate when compared with the corresponding experimental run (maximum deviation 0.86%). An appropriate HPLC method and additionally an on-line extraction procedure could be developed with just three experimental HPLC runs. This method could be useful for evaluating the concentrations of arachidonic acid metabolites involved in inflammatory diseases.
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.
Peptide and protein samples are often complex mixtures that contain a number of individual compounds. The initial HPLC separation of such samples typically results in the poor resolution of one or more band pairs. Various means have been suggested tor varying separation selectivity so as to minimize this problem. In this study of a tryptic digest of recombinant human growth hormone, the simultaneous variation of temperature and gradient steepness was found to be a convenient and effective means of varying selectivity and optimizing the separation. The use of computer simulation greatly facilitated this investigation.
Computer simulation by DryLab G/plus has proved to be an invaluable tool in the rapid development of analytical HPLC methods for antibiotics. A glycopeptide antibiotic under study at this Research Center was taken into consideration as a case study. Retention data from two preliminary experiments have allowed us to perform several simulations which greatly shortened the time normally required for the identification of the optimum gradient conditions. The “key steps” in the simulation process have been experimentally verified and a more than satisfactory agreement between calculated and experimental retention times was consistently found. This article presents the preliminary results obtained and proposes an HPLC method for the analysis of a glycopeptide antibiotic. Because of its general approach, the DryLab concept can be extended to any class of chemical compounds not only shortening impressively the HPLC method development time, but also making it possible to adjust/develop a method which takes into account the typical features of the instrument to be used. This last aspect means, among other things, that: (1) HPLC methods already optimized can be transferred from one laboratory to another, differently equipped, without any “local” further adjustment or development work and (2) HPLC methods can be optimized “at a distance” for other laboratories once a few instrumental parameters have been determined and two exploratory runs have been carried out.