Biomedical subjects

Adam P Schellinger

Publications and source records attributed to Adam P Schellinger.

6 recordsLinked to original sources

PubMed2006

Peak capacity optimization of peptide separations in reversed-phase gradient elution chromatography: fixed column format.

The optimization of peak capacity in gradient elution RPLC is essential for the separation of multicomponent samples such as those encountered in proteomic research. In this work, we study the effect of gradient time (tG), flow rate (F), temperature (T), and final eluent strength (phi(final)) on the peak capacity of separations of peptides that are representative of the range in peptides found in a tryptic digest. We find that there are very strong interactions between the individual variables (e.g., flow rate and gradient time) which make the optimization quite complicated. On a given column, one should first set the gradient time to the longest tolerable and then set the temperature to the highest achievable with the instrument. Next, the flow rate should be optimized using a reasonable but arbitrary value of phi(final). Last, the final eluent strength should be adjusted so that the last solute elutes as close as possible to the gradient time. We also develop an easily implemented, highly efficient, and effective Monte Carlo search strategy to simultaneously optimize all the variables. We find that gradient steepness is an important parameter that influences peak capacity and an optimum range of gradient steepness exists in which the peak capacity is maximized.

Animals↗

PubMed2006

Isocratic and gradient elution chromatography: a comparison in terms of speed, retention reproducibility and quantitation.

Chromatographers are cautioned to avoid gradient elution when isocratic elution will do. In this work, we compared the analytical properties of gradient and isocratic separations of a sample which can be done quite readily under isocratic conditions. We found that gradient elution gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution without sacrificing repeatability in retention time, peak area and peak height or linearity of the calibration curve. We also obtained acceptable repeatability in peak area/height and linearity of calibrations curves for a sample that required gradient elution using a practical baseline subtraction technique. Based on these results and related work which show that columns can be reequilibrated by flushing with less than two column volumes of the initial eluent, we conclude that many of the reasons given to avoid gradient elution deserve serious reconsideration, especially for those samples which are easily separated isocratically. However, we believe isocratic elution will remain preferable when: (1) the sample contains less than 10 weakly retained components (i.e. the last peak elutes with k' < 5) or (2) the gradient baseline impedes trace analysis.

Calibration↗

PubMed2005

A practical approach to transferring linear gradient elution methods.

Attempts to theoretically address the problems involved in transferring linear gradient elution methods have been somewhat ad hoc due to the simplifying assumptions usually made in conventional gradient elution theory. Until now, all equations based on the k* parameter of linear gradient elution theory used as the basis for predicting the separation selectivity have not explicitly included the effect of the dwell volume (VD). Using an exact equation for predicting k*, that is, one which fully accounts in an a priori fashion for VD, we find a set of simple yet exact equations which unequivocally must be satisfied to transfer an optimized linear gradient elution method from one system (column or instrument or both) to another. These relationships absolutely mandate that a change in the instrument dwell volume requires a proportional change in the column volume; in turn, a change in the column volume requires a proportional change in the flow rate and/or gradient time to maintain a constant gradient steepness. Although we are not the first to suggest these guidelines, this work provides a complete theoretical foundation for these exact guidelines for the maintenance of gradient selectivity for the case of transferring a linear gradient elution method between different columns packed with the same particles and/or between different instruments.

Chromatography, Liquid↗

PubMed2005

High speed gradient elution reversed-phase liquid chromatography.

A major disadvantage of gradient elution in terms of speed results from the need to adequately re-equilibrate the column. This work distinguishes two states of re-equilibration: (1) run-to-run repeatability and (2) full equilibration. We find that excellent repeatability (+/-0.002 min in retention time) is achieved with at most 2 column volumes of re-equilibration whereas full equilibration can require considerably more than 20 column volumes. We have investigated the effects of adding ancillary solvents (e.g. n-propanol, n-butanol) to the eluent and changing the particle pore size, initial eluent composition and type, column temperature and flow rate on the speed of full equilibration. Full equilibration seems to be more thermodynamically limited than kinetically controlled. Also, we show that the main limitation to reducing the full equilibration time is related to instrument design issues; a novel approach to overcome these instrumental issues is described.

Chromatography, Liquid↗

PubMed2004

Field screening of waterborne petroleum hydrocarbons by thickness shear-mode resonator measurements.

An inexpensive, field-portable sensor for direct, aggregate determination of aqueous petroleum hydrocarbons (PH) down to sub-ppm levels was developed. The basis of this sensor was an unusual, highly nongravimetric frequency response of 10 MHz (series fundamental) AT-cut quartz crystals when coated with rubbery silicone films. The response depended linearly and reliably on the total concentration of dissolved hydrocarbons over a range of 0.01-100 mg x L(-1) or up to aqueous solubility limits. Calibration sensitivities were measured individually for laboratory-prepared solutions of BTEX (benzene, toluene, ethylbenzene, and xylene isomers) and C6-C8 aliphatic components. Each component demonstrated a method detection limit (MDL) in the low-to sub-ppm range (benzene 10 mg x L(-1), n-hexane 0.54 mg x L(-1)) for light coatings of a commercially available poly-(dimethylsiloxane) gum (OV-1, > 10(6) g x mol(-1)) and lower MDLs for heavier coatings. Pairwise responses for the aliphatic and benzenoid standards were additive, indicating that aggregate determinations of mixtures (especially light fuels) were possible. Natural matrix interferences caused by sample turbidity and ionic strength were overcome by simple preparative methods. Fuel-spiked natural waters were determined with respect to standards and verified by gas chromatography. A 0.19 mg x L(-1) MDL for gasoline was obtained for heavy OV-1 films. Field determinations of groundwater surrounding a leaking underground fuel tank demonstrated that the sensor and method were useful for on-site PH screening. Large differences between the equilibration times of aliphatic and benzenoid components also indicated one avenue for BTEX speciation with the device.

Acoustics↗

PubMed2002

Use of DRYLAB to compare octadecylsilane and carbon supports for reversed-phase chromatography of triazine herbicide test solutes.

One of the most important aspects of method development in reversed-phase high-performance liquid chromatography (RPLC) is the accurate prediction of the resolution as one or more experimental variables (usually the mobile-phase composition) are changed. By use of commercially available computer simulation programs, e.g. Drylab 2000, data from a few initial experimental runs are used to predict how the separation will vary with composition so that a computer program can then rapidly locate the optimum conditions. Assumption of a linear relationship between log k' (retention factor) and phi (volume fraction of organic modifier in the element) minimizes the number of initial runs required for a separation and is usually adequate when the range of phi is not large. In this work the linearity of plots of log k' against phi for octadecylsilane-coated silica (ODS) and carbon-coated zirconia (C-ZrO(2)) columns were compared and the plots were used to determine whether prediction of the separation of triazine herbicides was adequate. Analysis of the slopes and intercepts of such plots are presented for both types of columns.

Chromatography, High Pressure Liquid↗