Low temperature stopped-flow spectrometry
Hanahan, D.; Auld, D.S.
Analytical Biochemistry 108(1): 86-95
1980
ISSN/ISBN: 0003-2697 PMID: 7457862 Document Number: 168019
A stopped-flow instrument operating over the temperature range from +55 to as low as -55.degree. C features a Teflon stopped-flow module with a quartz flow-through cell, which is placed in a heavy, double-walled, air-insulated, Plexiglas environmental chamber. N2 gas is the temperature control fluid. The combination of the Teflon components, quad x-rings and fluorinated lubricant results in a flow system which is inert, metal free and is leak free at temperatures as low as -55.degree. C. Quartz fiber optics connect the observation cell with the external light supply and detection systems which can monitor fluorescence or absorbance of the sample solution. The performance of the instrument at subzero temperatures was evaluated by measuring the rate of the sodium hydroxide catalyzed hydrolysis of the ester, o-nitrophenylchloroacetate, at 430 nm. At -20.degree. C, in 70% methanol-water the 1st-order rate constant increases linearly from 2.7 to 81 s-1 over the hydroxide concentration range 5-150 mM. Even at -55.degree. C the flow process is artifact free and complete in a few milliseconds. A linear Arrenhius plot is obtained for ester hydrolysis by 5 mM hydroxide over the temperature range from +20 to -55.degree. C.