Thichaphat Nawalohakul,a Pannarat Charoenjiraroj,a Rattikan Chantiwas,b Prapin Wilairatc and Wiboon Praditweangkum*a


aDepartment of Chemistry, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. E-mail:; Fax: +66 2329 8412; Tel: +66 2329 8400 ext. 3604

bDepartment of Chemistry, Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand

cAnalytical Sciences and National Doping Test Institute, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand



This work presents the use of a 96-well plate as headspaces for the determination of ascorbic acid in samples loaded in the 96-well plate. Ascorbic acid in the sample is oxidized to iodide by the addition of excess acidic iodate solution into the well. The iodide is further oxidized by the remaining iodate to molecular iodine. A single sheet of moist starch indicator paper is immediately placed over the 96-well plate after the addition of the iodate with the moisture forming a gas seal. The iodine gas in each well diffuses through the headspace to react with the starch paper producing circular areas of a colored starch–iodine complex. After 15 min the indicator paper is scanned, and the digital images of the complex are analyzed by using ImageJ software to obtain blue intensity values. The precision of the intensity values from 12 wells containing 20 mL of 2.84 mM standard ascorbic acid is 0.998. Samples of fruit juice and dietary supplements were analyzed for their ascorbic acid contents. The results obtained from the headspace reflectance method are not statistically different from values obtained from the titration method using paired t-tests (α = 0.05).


T. Nawalohakul, P. Charoenjiraroj, R. Chantiwas, P. Wilairat, W. Praditweangkum, A ninety-six well plate as headspaces with moist starch indicator paper as a cover for the determination of ascorbic acid by iodate oxidation and formation of volatile iodine, Analytical Methods 14(7) (2022) 741-749. doi:10.1039/D1AY02050A