Protonation and deprotonation processes are the key step of acid-base reaction and occur in many biological processes. Study on the deprotonation process of molecules and/or functional groups in confined conditions would help us understand the acid-base theory and confinement effect of biomolecules. In this paper, we use a recently established approach to the study of protonation and deprotonation processes of functional groups in porous anodic alumina array nanochannels by measuring the flux of electrochemical active probes (ferricyanide ions) using an Au film electrochemical detector sputtered at the end of nanochannels. The protonation and deprotonation processes of surface functional groups in nanochannels will change the surface charges and in turn modulate the transportation of charged electroactive probes through nanochannels. The titration curve for the deprotonation of carboxylic groups in nanochannel confined conditions is obtained by measuring the current signal of ferricyanide probe flowing through an carboxylic-anchored PAA nanochannels array at different solution pH. Results show that the deprotonation of carboxylic group in nanochannel occurs in one step with a pK(1/2) = 6.2. The present method provides an effective tool to study the deprotonation processes of various functional groups and biomolecules under confined conditions. (C) 2012 Elsevier Ltd. All rights reserved.