I am conducting an experiment to quantification of total anthocyanin in Tea (Camellia sinensis) using spectrophotometric method. I took absorbance at 520nm and 700nm with two buffers (pH=1 and pH=4.5) as formula mentioned in Journal of AOAC International vol. 88:5 1269-1278 (Lee et al, 2005) in determination of total Anthocyanin.
According to formula Absorbance (A)= (A520nm – A700nm)at pH 1.0 - (A520nm – A700nm)at pH 4.5
Literature suggested to take absorbance within 20-50 min of preparation. But absorbance is increasing continuously with the time (even less than 20min) at both 520 nm & 700nm only in pH=4.5 buffer. As a result of higher absorbance values, final value become negative (difference between Abs at pH=1 and pH=4.5). Please anybody explain this incident or suggest any other spectrophotometric method for quantification of Anthocyanin in Tea.
Just after 40 seconds time, you may get maximum binding and reactions that may produce some good color.
Thank you sir. I'll try.
Is it incorrect, If I take absorbance (both 520nm & 700nm) only in pH=1 other than taking measurements in both pH (pH= 1 & 4.5)? Because pH=1 gives coloured oxonium form. pH=4.5 gives colourless hemiketal form. According to literature, monomeric anthocyanin undergo a reversible reaction as a function of pH.
Maybe this is useful for you: http://www.bio-protocol.org/e1098 (a detailed protocol on Quantification of Anthocyanin Content)
I am confusing. Somebody says 30 minutes, others mentioned immediately. Can somebody give me an expert comment about colour development in different pH.
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