Using Temperature Controlled Water Circulators for Regulating Temperatures in Reagents and Stopped-Flow Spectrometers

With an operating temperature range that measures from -20° C to 60° C, the Applied Photophysics Standard SX20 Stopped-Flow Spectrometer does not require any additional accessories to function. Additionally, when the standard SX20 is fitted with high-temperature drive syringe SX/HT, the operating temperature limit can be as high as 80° C. Applied Photophysics, as well as a number of local suppliers, offer temperature controlled water circulators for regulating temperatures in both the reagents and stopped-flow. While instrument reconfiguration is not necessary when operating stopped-flow systems at lower temperatures, its use at higher temperatures may benefit from utilizing the sequential mixing option on the SX20.

The SX20 software can be used to control a number of circulators, such as those offered in the Thermo Scientific Artic series. The SX20 software’s functionality includes:

  • Setting the instrument through ProData, the stopped-flow control software
  • Initiating the instrument to carry out automatic processes of stopped-flow drives at certain pre-set temperatures
    • Can be used to obtain the kinetics at varying temperatures to obtain an Arrhenius plot
    • Includes a ‘pause’ feature to equilibrate each temperature set
    • Includes a ‘repeat’ feature at each temperature if needed
    • Allow for unattended operation throughout the duration of the experiment

The following experiment was conducted by using a Thermo Neslab Actic SC520-A25 circulator in conjunction with the automated temperature-dependent kinetics feature on the SX20 instrument. The kinetic data shown in Figure 1 is from the base catalyzed hydrolysis reaction of 2,4-dinitrophenylacetate (2,4-DPNA) in methanol. Following the mixing of both 0.3 mM sodium methoxide (NaOMe) and 30 µM 2,4-DPNA, an automated series of stopped-flow kinetic acquisitions were recorded by cooling the temperature from 25 °C to -15° C at intervals of 5° C.

At a path length of 10 mm, 20 µL cells were used and measured at a wavelength of 260 nm, triplicate samples were tested and averaged for each temperature to fit a single exponential.

Figure 1. Base catalyzed hydrolysis reaction of 2,4-DPNA in methanol.

Figure 2. Fitted data plotted as ln(kobs) versus 1/(temperature). The linear relationship visualized here (R2=0.9978) is expected when using the Eyring equation.

This information has been sourced, reviewed and adapted from materials provided by Applied Photophysics.

For more information on this source, please visit Applied Photophysics.

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