Carr Research Group

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CONTACT INFORMATION

University of Minnesota
Department of Chemistry
Smith and Kolthoff Halls
207 Pleasant St. SE
Minneapolis, MN 55455

(612) 624-5870 (lab)
(612) 626-7541 (fax)
schellinger@chem.umn.edu

EDUCATION

Graduated from Viterbo University (LaCrosse, WI) with a B.S. in Chemistry, minor in Biology

Thesis Title: Improving the speed and transferability of gradient elution RPLC,

Objectives of Thesis Work

To determine the solubility of common buffers used in RPLC in various aqueous - organic mixtures.
To develop a simple guideline for transferring a gradient elution method between different instruments and / or columns accounting for the dwell volume, column volume and gradient steepness.
To determine the column volumes of initial eluent that must be flushed through the column to obtain excellent run-to-run repeatability in gradient retention time (< ± 0.002 min) and / or to obtain full equilibration (i.e. retention time becomes independent of the re-equilibration time).
To find the appropriate conditions for repeatability and / or full equilibration in gradient elution by flushing less than the standard of 10-15 column volumes of initial eluent through the column. To speed re-equilibration, we will investigate the effect of the eluent composition (i.e. addition of small amounts of n-propanol and n-butanol), column temperature, pore size and flow rate on repeatability and full equilibration. Also, we will develop novel instrumentation that dramatically reduces the flush-out volume to further speed re-equilibration.
To investigate the reproducibility in retention time, peak height and peak area for samples that are adequately separated using either isocratic or gradient elution. Using conditions to speed gradient elution re-equilibration eliminates the main drawback of this technique. Therefore, we will re-evaluate method development guidelines for these sample types that are adequately separated isocratically or using a gradient in situations where the speed of each technique is not significantly different.
To optimize the gradient elution separation of many solute mixtures such as 16 EPA priority PAHs, 10 triazine herbicides and 14 nitroaromatic explosives by simultaneously optimizing the gradient slope, dwell volume, column volume, particle size and column temperature to obtain an adequate separation based on defined goals (i.e. a specific resolution and analysis time) and limitations of the instrumentation (i.e. back-pressure). The ultimate goal is to obtain a faster separation with similar resolution compared to the current "best" separation in the literature.

Accomplished Work (January 2005)

A paper has been published that details the solubility of common RPLC buffers in aqueous-organic mixtures (Schellinger, A. P.; Carr, P.W. (2004). "Solubility of Buffers in Aqueous-Organic Eluents for Reversed-Phase Liquid Chromatography." LC-GC 22(6): 544, 546, 548).
Using the Schoenmakers theory of gradient elution, which includes the effect of the dwell volume on the selectivity, a simple guideline for transferring a gradient elution method between different instruments or different columns packed with identical materials has been developed. This work is summarized in a paper submitted to the Journal of Chromatography A (Schellinger, A. P.; Carr, P.W. (2004). "A Practical Approach to Transferring Gradient Elution Methods Between Instruments" JCA-) Previous method transfer guidelines have not properly accounted for the relationship of dwell volume and column volume on the selectivity in gradient elution.
Using non-ionizable soluted and non-buffered eluents, excellent repeatability in gradient retention time (< 0.002 min) is possible after one column volume of initial eluent has flushed the column. Using the appropriate ternary composition of ancillary solvent (i.e. n-propanol or n-butanol), organic modifier (i.e. acetonitrile) and water, full equilibration is obtained after less than two column volumes of initial eluent have flushed the column. Furthermore, a novel scheme has been developed to dramatically reduce the flush-out volume of typical HPLC instrumentation. The reduced flush-out instrument further improves the speed of gradient elution by reducing the time required for repeatability and / or full equilibration. A paper summarizing this work has been published (Schellinger, A.P.; Stoll; Dwight, R.; Carr, P.W. "High Speed Gradient Elution RPLC." J. Chromatogr. A 1064, 143-156).
A Monte Carlo optimization technique (i.e. "shot-in-the-dark" generation and "weed-whacking" refinement) has been used to locate separation conditions that provide baseline separation of 10 triazine herbicides on a polybutadiene-coated zirconia column in less than 3 minutes. This represents a significant improvement compared to the 10 minute separation of these solutes employing two columns of radically different selectivity in series. Also, this optimization technique has indicated that a separation of the 16 EPA priority PAHs is possible in less than 1 minute which represents a significant improvement from the 4 minute separation reported by Supelco.

Work in Progress

Knowing that the speed of gradient elution is no longer significantly limited by re-equilibration requirements under the appropriate conditions, we will re-evaluate method development guidelines for samples that are adequately separated using either isocratic or gradient elution. Based on preliminary studies, we believe that gradient elution is more ideal for many samples which are separated isocratically mainly to avoid the supposed long re-equilibration time in gradient elution.
Although the speed of gradient elution (i.e. time required for re-equilibration) using non-ionizable solutes and non-buffered eluents can be made very fast, we still need to find ways to improve the speed of gradient elution for ionizable solutes and buffered eluents. Preliminary work has shown that excellent repeatability in gradient retention time for tryptic peptides separated using a 0.1 % TFA mobile phase after 2 column volumes of initial eluent has flushed the column. We intend to collaborate with Dan Marchand at the University of Wisconsin River Falls for the remainder of this study.
Continued work involving goal-based optimization of fast gradient elution separations will be performed.
Importance of This Work to the Field of Analytical Chemistry
The work outlined above will have a significant impact in the field of analytical chemistry, especially in the field of RPLC. All of the work performed is practical and extremely useful for chromatographers of any expertise. The buffer solubility paper alleviates the guess work in creating buffers for RPLC. The gradient elution method transfer paper will simplifying the previously frustrating task of transferring a method from one instrument and / or column to another by properly accounting for the effect of dwell volume on the selectivity. The paper focused on speeding up gradient elution re-equilibration debunks many of the traditional myths regarding the speed of gradient elution for non-ionizable solutes and non-buffered eluents. For example, excellent repeatability in gradient elution retention time is possible after only 1 column volume of initial eluent has flushed the column. Although full equilibration can require much more than 10-15 column volumes of initial eluent be flushed through the column, we have shown that addition of a small amount of n-propanol or n-butanol to eluent allows for full equilibration after less than 2 column volumes of initial eluent has flushed the column. Furthermore, reduction of the flush-out volume of the instrumentation further speeds full equilibration. Thus, we feel we have increased the speed of gradient elution to the point where the re-equilibration time is insignificant compared to the gradient time and / or cycle time of the instrument. Also, by distinguishing between repeatability of full equilibration, chromatographers will have a better understanding of what state of re-equilibration they require. For example, one may desire full equilibration to obtain an absolute value of gradient retention time to improve the accuracy of predicting gradient elution using gradient training runs.

Modified by Hao Luo --- hluo@chem.umn.edu