Biomedical subjects

J W Dolan

Publications and source records attributed to J W Dolan.

At least 19 recordsLinked to original sources

PubMed2005

"Orthogonal" separations for reversed-phase liquid chromatography.

A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is "orthogonal" to a pre-existing ("primary") method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the "orthogonal" B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative "orthogonality" of the primary and "orthogonal" RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop "orthogonal" methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here.

Chromatography, Liquid↗

PubMed2005

Column selectivity in reversed-phase liquid chromatography. VIII. Phenylalkyl and fluoro-substituted columns.

As reported previously, five solute-column interactions (hydrophobicity, steric resistance, hydrogen-bond acidity and basicity, ionic interaction) quantitatively describe column selectivity for 163 alkyl-silica, polar-group and cyano columns. In the present study, solute retention and column selectivity for 11 phenyl and 5 fluoro-substituted columns were compared with alkyl-silica columns of similar ligand length. It is concluded that two additional solute-column interactions may be significant in affecting retention and selectivity for the latter columns: (a) dispersion interactions of varying strength as a result of significant differences in bonded-phase polarizability or refractive index and (b) pi-pi interactions in the case of phenyl columns and aromatic solutes. These 16 phenyl and fluoro columns were also characterized in terms of hydrophobicity, steric resistance, hydrogen-bond acidity and basicity, and ionic interaction.

Chromatography, Liquid↗

PubMed2005

Column selectivity in reversed-phase liquid chromatography. VII. Cyanopropyl columns.

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.

Chromatography, Liquid↗

PubMed2004

The hydrophobic-subtraction model of reversed-phase column selectivity.

A recently developed treatment of reversed-phase column selectivity (the hydrophobic-subtraction model) is reviewed and extended, including its characterization of the selectivity of different column types (e.g., C1-C30, cyano, phenyl, etc.). The application of this model to retention data for various solutes and columns has provided new insights into the nature of different solute-column interactions and their relative importance in affecting sample retention and separation. Reversed-phase columns can be characterized by five selectivity parameters (H, S*, A, B and C), values of which are summarized here for more than 300 different columns. The selection of columns of either equivalent or different selectivity is readily achievable on the basis of their values of H, S*, etc. The development of the hydrophobic-subtraction model, its use in characterizing the selectivity of different reversed-phase liquid chromatography (RP-LC) columns, and its application to various practical problems as described here began in 1998. The original inspiration for this project owes much to Jack Kirkland, who also contributed actively to the initial studies that laid the foundation of this model; he has since provided other important support to this project. Jack and one of the authors (LRS) have enjoyed a strong professional relationship and personal friendship for the past 35 years, and it is the privilege of the authors to dedicate this paper and the work that it represents to Jack. His contributions to HPLC column technology have extended from the mid-1960s into the present century, and it is impossible to conceive of present day HPLC practice without Jack's contributions over the years. In this and other ways, his position as a pioneer and key implementer of HPLC is widely recognized. We wish Jack well in the years to come.

Chromatography, Liquid↗

PubMed2004

A fast, convenient and rugged procedure for characterizing the selectivity of alkyl-silica columns.

Previous work has shown that the selectivity of reversed-phase columns for HPLC can be described by means of five column parameters: H (hydrophobicity), S* (steric resistance), A (hydrogen-bond acidity), B (hydrogen-bond basicity) and C (cation-exchange capacity). Values of H, S*, etc. can be determined by carrying out retention measurements for 18 test solutes under standardized conditions. The reproducibility of the latter procedure has been evaluated by comparison testing in four different laboratories and found acceptable. An alternative 10-solute test procedure which is more reproducible and convenient (but somewhat less accurate), requires only 2-3 h per column.

Cation Exchange Resins↗

PubMed2004

Choosing an equivalent replacement column for a reversed-phase liquid chromatographic assay procedure.

The column selectivity parameters (H, S*, A, B and C) described in the preceding paper [L.R. Snyder, A. Maule, A. Heebsch, R. Cuellar, S. Paulson, J. Carrano, L. Wrisley C.C. Chan, N. Pearson, J.W. Dolan, J.J. Gilroy, J. Chromatogr. A 1057 (2004) 49-57] can be used to compare columns in terms of selectivity. A detailed procedure for such column comparisons is presented here, and evaluated by its use in finding suitable replacement columns for 12 different routine separations performed in five different pharmaceutical analysis laboratories.

Chromatography, Liquid↗

PubMed2004

Column selectivity in reversed-phase liquid chromatography. VI. Columns with embedded or end-capping polar groups.

A previous model of column selectivity for reversed-phase liquid chromatography (RP-LC) has been applied to an additional 21 columns with embedded or end-capping polar groups (EPGs). Embedded-polar-group columns exhibit a significantly different selectivity vs. non-EPG, type-B columns, generally showing preferential retention of hydrogen-bond donors, as well as decreased retention for hydrogen-bond acceptors or ionized bases. EPG-columns are also generally less hydrophobic (more polar) than are non-EPG-columns. Interestingly, columns with polar end-capping tend to more closely resemble non-EPG columns, suggesting that the polar group has less effect on column selectivity when used to end-cap the column versus the case of an embedded polar group. Column selectivity data reported here for EPG-columns can be combined with previously reported values for non-EPG columns to provide a database of 154 different columns. This enables a comparison of any two of these columns in terms of selectivity. However, comparisons that involve EPG columns are more approximate.

Acids↗

PubMed2003

Slow equilibration of reversed-phase columns for the separation of ionized solutes.

Reversed-phase columns that have been stored in buffer-free solvents can exhibit pronounced retention-time drift when buffered, low-pH mobile phases are used with ionized solutes. Whereas non-ionized compounds exhibit constant retention times within 20 min of the beginning of mobile phase flow, the retention of ionized compounds can continue to change (by 20% or more) for several hours. If mobile phase pH is changed from low to high and back again, an even longer time may be required before the column reaches equilibration at low pH. The speed of column equilibration for ionized solutes can vary significantly among different reversed-phase columns and is not affected by flow rate.

Chromatography, Liquid↗

PubMed2002

Column selectivity in reversed-phase liquid chromatography III. The physico-chemical basis of selectivity.

Reversed-phase liquid chromatography (RP-LC) retention data for 23 additional solutes have been acquired to further test and evaluate a general relationship from part I: log alpha = log (k/kref) = eta'H(i) + sigma'S(ii) beta'S(iii) + alpha'B(iv) +kappa'C(v) The physico-chemical origin of terms i-v above is examined here by comparing values of (a) the solute parameters of Eq. (1) (eta', sigma', etc.) vs. solute molecular structure, and (b) the column parameters (H, S, etc.) vs. column properties (ligand length and concentration, pore diameter, end-capping). We conclude that terms i-v correspond, respectively, to hydrophobic (i), steric (ii), hydrogen bonding (iii, iv) and ionic (v) interactions between solute and stationary phase. While steric interaction (term ii) is superficially similar to what previously has been defined as "shape selectivity", the role of the solute and column in determining steric selectivity (term ii) appears more complex than previously proposed for "shape selectivity". Similarly, what has previously been called hydrogen bonding between donor solutes and an acceptor group in the stationary phase (term iv) is very likely an oversimplification.

Chromatography, Liquid↗

PubMed2002

Column selectivity in reversed-phase liquid chromatography I. A general quantitative relationship.

Retention factors k have been measured for 67 neutral, acidic and basic solutes of highly diverse molecular structure (size, shape, polarity, hydrogen bonding, pKa, etc.) on 10 different C18 columns (other conditions constant). These data have been combined with k values from a previous study (86 solutes, five different C8 and C18 columns) to develop a six-term equation for the correlation of retention as a function of solute and column. Values of k can be correlated with an accuracy of +/- 1-2% (1 standard deviation). This suggests that all significant contributions to column selectivity have been identified (and can be measured) for individual alkyl-silica columns which do not have an embedded polar group. That is, columns of the latter kind can be quantitatively characterized in terms of selectivity for use in the separation of any sample.

Chromatography, Liquid↗

PubMed2002

Column selectivity in reversed-phase liquid chromatography II. Effect of a change in conditions.

The isocratic retention of 67 widely-different solutes in reversed-phase liquid chromatography (RP-LC) has been investigated as a function of temperature and mobile phase composition (% B) for three different C18 columns. Similar studies were also carried out in a gradient mode, where temperature, gradient time and solvent type were varied. These results show that changes in retention with these conditions are similar for each of these three columns. This suggests that relative column selectivity as defined by experiments for one set of experimental conditions will be approximately applicable for other conditions, with the exception of changes in mobile phase pH-which can affect values of the column parameter C (a measure of silanol ionization). Column selectivity as a function of pH was explored for several columns.

Buffers↗

PubMed2002

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

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.

Chromatography, High Pressure Liquid↗

PubMed2002

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

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.

Acids↗

PubMed2001

Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae.

Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.

Culture Media↗

PubMed2001

Controlled expression in Klebsiella pneumoniae and Shigella flexneri using a bacteriophage P1-derived C1-regulated promoter system.

The utility of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor was examined in Shigella flexneri and Klebsiella pneumoniae. Promoters carrying C1 operator sites driving LacZ expression had induction/repression ratios of up to 240-fold in S. flexneri and up to 50-fold in K. pneumoniae. The promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. This system will provide a new opportunity for controlled gene expression in enteric gram-negative bacteria.

Base Sequence↗

PubMed2000

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

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.

Chromatography, Liquid↗

PubMed2000

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

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.

Chromatography, Liquid↗

PubMed2000

Expediting the method development and quality control of reversed-phase liquid chromatography electrospray ionization mass spectrometry for pharmaceutical analysis by using an LC/MS performance test mix.

Mass spectrometry combined with liquid chromatography (LC/MS) has become an important analytical methodology in both pharmaceutical and biomolecule analyses. LC/MS, especially with reversed-phase HPLC (RP-LC), is extensively used in the separation and structural identification of pharmaceutical samples. However, many parameters have to be considered when a new LC/MS method is developed for either separation and structural analysis of unknown mixtures or quantitative analysis of a set of known compounds in an assay. The optimization of a new LC/MS method can be a time-consuming process. A novel kit-LC/MS performance test mix-composed of aspartame, cortisone, reserpine, and dioctyl phthalate has been developed to accelerate the process of establishing a new RP-LC/MS method. The LC/MS mix makes the evaluation and validation of an LC/MS method more efficient and easier. It also simplifies the quality control procedure for an LC/MS method in use.

Chromatography, High Pressure Liquid↗