Rapid Kinetic Measurement of 2,6-Dichloroindophenol using a UV-visible Absorption Stopped-Flow System

Download PDF October 6, 2017

Introduction

FS-110 Fast Scan Spectrophotometer

An absorption stopped-flow measurement system is designed to measure rapid enzymatic, catalytic, and oxidation/reduction reactions. The system consists of the FS-110 fast scan spectrophotometer and the SFS-822 stopped-flow accessory and allows from two to four liquid samples to be rapidly mixed. Stopped-flow absorption spectra can be measured in 5 msec intervals to acquire reactions occurring at millisecond timescales.

This application note illustrates how the absorption stopped-flow system can determine the reaction rate for the reduction of 2,6-dicholorindeophenol (DCIP), whose color in aqueous solution changes from to blue to colorless as a result of its reaction with L-ascorbic acid.

Experimental

Measurement Conditions
SpectrophotometerStopped-Flow System
Optical Pathlength2 mmSolution Flow Time10 msec
Data Interval1 nmMixing Ratio1:1
Measurement Interval0.10 secVolume of Solution Flow50 μL
Measurement Time0-3 secondsThe measurement is started when the syringe is stopped.

L-ascorbic acid was dissolved in NaOH/Na2HPO4 to acquire a final concentration of 20 mmol/L and the pH was adjusted to 7.6. Aqueous solutions of DCIP were used at a concentration of 1 mmol/L.

Keywords

UV-0019, FS-110 fast scan spectrophotometer, UV-Visible/NIR, SFS-852 Stopped-flow system, Stopped-flow Measurement program, Reaction Rate Calculation program

Results

Figure 1. 3D spectra of the reduction of DCIP.

Figure 1 shows the 3D spectra of the DCIP reaction. When the reaction begins, the spectra has an absorption maximum around 600 nm which coincides with the sample exhibiting a blue color. Within 1 second after starting the measurement, the absorbance drops to approximately zero and the sample changes to colorless.

Figure 2. Time course measurement results and the fitted curve for sample absorption measured at 604 nm.

Figure 2 shows the time course absorbance data at the absorption maximum (604 nm) and the curve fitted to the reaction data between 30 milliseconds and 2.0 seconds. The reaction is assumed to be a primary reaction, and the fitted results are in excellent agreement with the measured results. Using the Reaction Rate Calculation program, a reaction rate of 4.3 sec-1 was calculated.

About the Author

Toshifumi Uchiyama is a member of the electronic spectroscopy team located at the JASCO main laboratory in Tokyo