Amino Acid Help

[nellermann]

Hi,

I'm doing amino acids for the first time using the HPLC-FID Performic Acid Oxidation method.

So far, I have added my performic acid solution and allowed it to sit overnight for 16 hours. The method states to add sodium metabisulfite to liberate the SO4, stir for 15 minutes and then add 6M HCl-phenol solution. Well, I missed the sodium metabisulfite step until after adding the HCl-phenol solution. As soon as I realized my mistake (it was within 5 minutes), I added it and stirred the sample for 15 minutes.

My question for those who have done this method. Do I need to start over?

Thanks for your help!
Natalie

[Coffo]

Hi Natalie,

I've never performed this method myself but I guess you are following an AOAC or pharmacopeia method to quantify cysteine/cystine as cysteic acid (and methionine as methionine oxide) and the method says neutralize the excess performic acid with sodium metabisulfite before adding the HCl-phenol for hydrolysis, therefore that is what you are going to have to do!!

The metabisulfite is there to neutralize the excess performic acid (preventing any further oxidation). Adding the HCl-phenol for the hydrolysis reaction also neutralizes any unreacted metabisulfite. By adding the HCl-phenol first the metabisulfite may have been decomposed before it has been able to neutralize the performic acid. Personally I don't think I'd take the risk of performing the hydrolysis reaction (refluxing at 110°C) when you may well have un-neutralized performic acid remaining in your sample.