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Separation Reproducibility With the BioLogic Chromatography Systems

Kevin Thornton, PhD, Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA


Introduction
In chromatographic separations, sample component elution times are often used both for identification and to program automated chromatography steps like fraction collection. Therefore, the ability to obtain reproducible chromatographic separations is essential. In order to achieve reproducible sample runs, a chromatography system must maintain a precise flow rate, inject a consistent sample volume at a precise time, and detect sample components in a consistent manner. Additionally, the column used must maintain its function and flow characteristics over time. In this report we provide data that shows the highly reproducible separations that can be obtained with both BioLogic DuoFlow and BioLogic LP systems. These results were obtained on a medium-pressure BioLogic DuoFlow QuadTec basic system and a low-pressure BioLogic LP system equipped with a BioFrac fraction collector.


Methods
A test mixture that contained the proteins horse skeletal muscle myoglobin, chicken egg white conalbumin, and soybean trypsin inhibitor type II (all obtained from Sigma) in a ratio of 2:5:5 was dissolved in an appropriate volume of 20 mM Tris buffer, pH 8.1 (buffer A), filtered through a 0.22 m syringe filter, and kept on ice until injected onto a column.

The test mixture was purified in consecutive anion exchange runs using an UNO Q1 column or an Econo-Pac High Q cartridge connected to the DuoFlow or BioLogic LP system, respectively. Proteins were eluted with a linear gradient of 050% buffer B (buffer A
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