紫外-可见吸收停流系统快速测定2,6-Dichloroindophenol

吸收停止流测量系统旨在测量快速的酶促、催化和氧化还原反应。该系统由FS- 110快速扫描分光光度计和SFS-822停止流附件组成，允许2到4个液体样品快速混合。停流吸收光谱可以在5毫秒间隔内测量，以获得在毫秒时间尺度上发生的反应。Application Note Reduction Reaction of 2，6-Dichloroindophenol using theAbsorption Stopped-Flow System2/2Application Note Introduc t ion The absorption stopped -f low measurement system is designed to meas u re r apid enzymatic， catalytic， and oxidation-reduction r eactions. T h e system consists of the FS-110 fast scan spectrophotometer and the SFS-822 stopped-flow accessory and allows for two to four liqui d samples to be r apidly mixed. Stopped-f l ow absorption spectra can be measured in 5 msec i nterva l s to acquire reactions occurr i ng at millisecond t i mescales . This application n ote i llu st r ates how the absorpt i on stopped-f low system can determine the r eaction rate fo r t he reduc t io n of 2，6-dic h olorindeophenol (DCIP)， whose color in aqueous sol ut io n cha n ges from to blue to color l ess as a r esult of its r eaction with L-ascorbic acid. FS-110 Fast Sc a n Spectrophotometer K e ywords FS-110 f a s t s c an spect r op h ot o m e t e r， U V-Vi s ible/NIR， SFS-852 S to p p e d -f l ow sy s tem ， Stopped -flow Measure m e nt pr ogram ， Reac ti on Rate Calculat i o n p r ogram Measurement Conditions Spectrophotometer Stopped-flow System Optical Pathlength 2 mm Time of solution sending 10 msec Data Interval 1nm Mixing ratio 1：1 Measurement Interval 0.010 sec Volume of solutionsending 50 pL Measurement Time 0-3 seconds The measurement is started when the syringe isstopped. 28600 Ma r y 's C ou r t， Easton， M D 21601 US A L-ascorbic acid was dissolved i n NaOH/Na2HPO4 to acq u ire a f inal concent r ation of 20 mmol/L a n d the pH was adjusted to 7.6. Aqueous solu ti ons of DCIP were u sed at a conce n trat i on of 1 mmol/L. Resul t s Figure 1 shows the 3D spec t ra of the DCIP react i on. When the r eac t ion begi n s， the spect r a i ll ustrates a n absorption maximu m around 600 nm which coinc i des with t he sample exh i bi t ing a blue color . Wi t hi n 1 second afte r starting the measurement， the absorba n ce d r ops to approx i mately zero and the sample changes to colorless. Figure 1. 3D s p e ct ra of t h e re d u c t ion o f DCI P. Figure 2 s h ows the time course absorbance data at the a bsorption maximum (604 nm) a n d the curve f i tted to t h e r eac t ion dat a b etween 0.03 and 2.0 second s. The r eac t ion is assumed t o be a primary reac t i on， a n d the f it results are in excelle n t agreeme n t wi th the measu r ement results. Using t h e Reaction Rate Calc u lat i on progr a m， a reaction rate o f 4.3 sec -1 was calculated. Re a c t i o n r a te c a l cul a ti o n e q ua ti o n： Y(t)=0.615066 x exp(-t /0.230571) Basel i ne e q uation：Y(t)=0.0105329 T i me cons t a nt ： 0.230967 sec Rate consta n t ： 4.32963 sec-l Half -life per i o d ： 0.160094 sec Figure 2. T i me cours e me a su r emen t res u lts a n d t h e fi t t e d c u rv e for s a mp l e a b so r p t io n mea s ur e d a t 604 nm. JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A