Science Buddies: "Ask an Expert"

Hi, my science fair project involves testing several different factors involved in the loss of vitamin C during preparation. Vitamin C (ascorbic acid) either becomes permanently destroyed or can be oxidized by various enzymes or substances to dehydroascorbic acid. Dehydroascorbic acid can be converted back to ascorbic acid using a suitable reducing agent and I would like to use this to test how much vitamin C was oxidized versus completely destroyed. The titration analysis only registers the reduced ascorbic acid form so I need to find a reducing agent to reduce the dehydroascorbic acid back in order to measure it. What reducing agent (preferably relatively inexpensive or easily obtainable) would work for this? Thanks.

Hi,

The authors of this paper:

http://www.pl.barc.usda.gov/downloads/jp22.pdf

Used 2,3-dimercapto-1-propanol to reduce dehydroascorbic acid to ascorbic acid. This is stated in on the second page of the paper in the Assays subsection of Experimental Methods, citing reference 10


See also this site:

http://en.wikipedia.org/wiki/Dehydroascorbic_acid

Which states that glutathione and other thiols will reduce dehydroascorbic acid back to ascorbic acid.

A third agent is described in this abstract:

http://cat.inist.fr/?aModele=afficheN&cpsidt=18924125

And yet another:

http://informahealthcare.com/doi/abs/10 ... 9309086629

http://www.ingentaconnect.com/content/a ... 1/art04592

The problem you will have is that if you attempt to assay the ascorbic acid by oxidation (with iodine?) the reducing agent you use on dehydroascorbic acid, whatever it is, will likely also react with the oxidizing agent, interfering with your assay result. You will need to either destroy the reducing agent, separate out of the reaction mixture, or use an ascorbic acid assay that is not sensitive to the reducing agent.

The last link above refers to High Performance Liquid Chromatography (HPLC) using an electrochemical detector as one method that might avoid this problem. I suspect there are also other electrochemical methods that can be used.

This paper, while old, appears very relevant to your question:

http://www.plantphysiol.org/cgi/reprint/26/4/757.pdf

This analysis is also discussed here in a clinical lab context:

http://www.healthomedical.com/Dehydroascorbic_acid

Here is a link to a current standard approach to the analysis:

http://www.currentprotocols.com/protocol/tx0706

This is an older paper describing determination of both ascorbic acid and dehydroascorbic acid by chemical and photometric method:

http://www.jbc.org/content/152/3/511.full.pdf
http://www.clinchem.org/cgi/reprint/41/8/1177.pdf

Here is a Google book review that summarizes analysis approaches:

http://books.google.com/books?id=0pl61o ... cid&f=true

http://www.nowfoods.com/M086887.htm

This is not a very focused answer to your question, but I hope it is an adequate one. To summarize it looks like you can’t just use a reducing agent to convert dehydroascorbic acid back to ascorbic acid and then do the usual iodine oxidation of ascorbic acid to assay it because your residual reducing agent will also react, giving false results. There are three or four techniques discussed above that will do the analysis for you, but they may be beyond your resources (either available lab equipment or knowledge). I suggest you try to find a mentor who can support you appropriately. Here is a Science Buddies resouce to help you find one:

https://www.sciencebuddies.org/science- ... cess.shtml

https://www.sciencebuddies.org/science- ... tors.shtml

This looks like a really fun project. I wish you the best of luck with it!!!

Best regards,

Barrett L Tomlinson

Hi,

Here are some papers on the use of polarography to measure ascorbic acid and dehydroascorbic acid:

http://article.pubs.nrc-cnrc.gc.ca/ppv/ ... tPage=2799

http://www.sciencedirect.com/science?_o ... archtype=a

http://www.tau.ac.il/~advanal/Polarography.htm

http://findarticles.com/p/articles/mi_h ... n55066988/

This document contains a survey of methods for analyzing both forms of vitamin C(on page 9 of 22), and includes cryptic references to the original sources:

http://www.fcf.usp.br/FBA0200/Grupo%208 ... nalysis%20(somente%20vitaminas%20hid.pdf

This is another more complete survey of analytical methods with complete bibliographic references:

http://www.iupac.org/publications/pac/p ... 1x0075.pdf

This set of materials should give you a relatively complete survey of your options to analyze both ascorbic acid and DAA.

Best of luck on the project!

Barrett L Tomlinson