Selerity Technologies Inc.
2484 West Custer Road
Salt Lake City, UT, 84104
Website: http://www.selerity.com
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Isocratic Separation Of Proteins Using A Thermal Gradient
Introduction
HPLC analysis at elevated temperatures reduces analysis time and improves resolution. Reduced viscosities at elevated temperatures result in significantly lower backpressure, allowing for higher flow rates and shorter analysis times. It also permits the use of smaller particle size packings to increase efficiency while operating at lower backpressure. Flatter van Deemter curves mean that operating at higher flow rates does not sacrifice efficiency. Solvent properties also change as the temperature is increased. Hydrogen bonding interactions in water are reduced at higher temperatures, making water less polar so that it behaves more like an organic solvent as the temperature is elevated. This means that most solvent gradients can be replaced with temperature gradients so that samples requiring complex solvent gradients can be separated isocratically using a simple temperature program.
Experimental conditions
Conditions are summarized in Table 1. A Milton Roy CM4000 pump (Milton Roy USA, Ivyland, PA), Alltech vacuum degasser (Alltech Associates, Inc., Deerfield, IL), Thermo Separations UV2000 variable wavelength detector (Thermo Electron Corp., Beverly, MA) and Alcott autosampler (Alcott Chromatography, Norcross, GA) were used in conjunction with a Selerity Technologies Series 8000 programmable HTLC oven (Selerity Technologies, Inc., Salt Lake City, UT).
Results
Figure 1 shows the separation of protein standards on a Hamilton PRP-3 (Hamilton Co., Reno, NV) column using an isocratic mobile phase and a temperature gradient. Baseline resolution of five standards is achieved in about six minutes. Reduced viscosity of the mobile phase at higher temperature allows a flow rate of 1.0 ml/min - a high flow rate for a 2.1 mm column with a 3 mm particle size. The backpressure at 50 C was about 3100 psi. A backpressure regulator installed on the system after the detector contributes 250 psi to that total.
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