[HunkaBunka]

Hi all, I'm doing a final year high school self-designed experiment. It involves adding enzyme to a protein solution at different pH levels. I'm going to use a biuret test and colorimeter to measure results, except I can't seem to find the right volumes/concentrations to make the biuret test work and its driving me insane.

My solution of enzyme/protein is 5ml. We don't have a preprepared biuret reagant, so i have to manually add copper sulphate and sodium hydroxide solutions (both 1mol). When I first tried this, I manually added 4ml of hydroxide and 1ml of sulphate which now seems like way too much lol, but the solution did turn a (VERY dark) purple. I then realised this was way too strong and diluted both to 0.5M, before adding the same quantities but it was similarly very dark.

So, now I'm trying by diluting hydroxide to 0.5M, and adding 1ml of this, then diluting the sulphate to 0.25M and adding a few drops with a pipette. Except this time was the opposite issue, the test didn't indicate any protein, and it just looked like suspended drops of the sulphate which eventually turned the solution a milky blue. I doubt that this is a result of absence of protein though, but where did i go wrong with the chemicals?

Is it that the copper sulphate was very diluted and i only added a few drops? Should i have added more than 1ml of hydroxide?

I'd greatly appreciate some advice, as this is the only thing preventing my experiment from running perfectly. Thanks in advance

[Vidya]

Try this site

https://microbenotes.com/biuret-test-for-protein/