Spatial pH jump measures chemical kinetics in a steady-state system.
We measure chemical kinetics in a steady-state solution where we create a microscopic open region with conditions different from the bulk. Individual reactant molecules spontaneously diffuse through this "reaction volume". We measure the changes which take place within their short residence time in the volume. The advantage of this approach is that the time resolution is limited only by the residence time tau(D) of the molecules in the reaction volume (which can easily be <50 mus), while the time taken to average the data can be arbitrarily long. In addition, if the chemical changes are reversible, the system is always in a steady state, and no replenishment of the reactants is necessary. Also, the total specimen volume required can be very small (<20 muL). We demonstrate the scheme by measuring the protonation induced changes of the fluorescence properties of fluorescein. We first show that a pH jump of >1 unit can be achieved by multiphoton excitation of ortho-nitro benzaldehyde (o-NBA). We then perform fluorescence correlation spectroscopy (FCS) to show that the residence time tau(D) of fluorescein in this low-pH region is approximately 30 mus. Subsequently, we use time correlated single photon counting (a widely used probing technique with an inherently long averaging time), and show that the data can be averaged for an arbitrarily long time, yet it captures the fluorescence lifetime of the low-pH species which exists only for the short time tau(D). Finally, we show that the time resolution can be tuned by over 3 orders of magnitude, by changing the focal volume and by changing the viscosity of the solution. The latter experiment also shows that small chemically induced changes in the fluorescence lifetime can be resolved by our technique.
Nag
Suman
S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Bandyopadhyay
Arkarup
A
Maiti
S
S
D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017785:Photons; D014783:Viscosity