Improved measurement of the rotor temperature in analytical ultracentrifugation

Abstract

Sedimentation velocity is a classical method for measuring the hydrodynamic, translational friction coefficient of biological macromolecules. In a recent study, comparing various analytical ultracentrifuges, we have shown that external calibration of the scan time, radial magnification, and temperature are critically important for accurate measurements (Anal. Biochem., 2013, doi: 10.1016/j.ab.2013.05.011). To achieve accurate temperature calibration, we have introduced the use of an autonomous miniature temperature logging integrated circuit (Maxim Thermochron iButton ™) that can be inserted in an ultracentrifugation cell assembly and spun at low rotor speeds. In the present work, we developed an improved holder for the temperature sensor located in the rotor handle. This has the advantage of not reducing the rotor capacity and allows for a direct temperature measurement of the spinning rotor during high-speed sedimentation velocity experiments up to 60,000 rpm. We demonstrate the sensitivity of this approach by monitoring the adiabatic cooling due to rotor stretching during rotor acceleration, and the reverse process upon rotor deceleration. Based on this, we developed a procedure to approximate isothermal rotor acceleration for better temperature control.

Measurement of the temperature of the resting rotor in analytical ultracentrifugation

“…In this first study, we observed highly reproducible instrument-dependent deviations between the temperature reported on the ultracentrifuge console, as measured by the instrument radiometer, and the values measured by the iButton. More recently, we have developed a modified rotor handle that can hold the iButton in the center of rotation, allowing for temperature measurements at even the highest rotor speeds currently permitted in analytical rotors [14]. In such a manner, the rotor temperature can be monitored independently throughout the actual sedimentation experiment, including the cooling from adiabatic stretching of the rotor during acceleration [14].…”

“…Interestingly, we found that the iButton temperatures were highly consistent and independent of iButton placement [14]. Furthermore, we found that the difference between the apparent temperature reported by the instrument and the iButton temperature to be independent of rotor speed, and, when tested in a single instrument, largely independent of the temperature set-point [14].…”

“…Furthermore, we found that the difference between the apparent temperature reported by the instrument and the iButton temperature to be independent of rotor speed, and, when tested in a single instrument, largely independent of the temperature set-point [14]. Finally, when using the evacuated and temperature equilibrated rotor chamber as a convenient device to compare calibration accuracy of different iButtons, we fortuitously observed that the temperature measured on top of the resting rotor was, within the precision of the iButtons, identical to the temperature of the spinning rotor [14]. This coincidence was unexpected, due to the potentially different heat flows and temperature regulation in the resting or spinning rotor.…”

“…Temperature measurements were carried out using Maxim Thermochron iButtons (DS1922L, Maxim Integrated Products, San Jose, CA, USA) [12,14], with 11-bit resolution corresponding to 0.0625 °C increments. iButtons were either calibrated with a reference thermometer [12], or purchased with a calibration provided by the vendor (Thermodata Corporation, Marblehead, MA, USA) [14].…”

“…iButtons were either calibrated with a reference thermometer [12], or purchased with a calibration provided by the vendor (Thermodata Corporation, Marblehead, MA, USA) [14]. iButton temperature logs were retrieved using a USB connector attached to an office computer and temperature equilibration was verified from the time-course of readings.…”