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Author: Subject: Making biuret reagent
cnidocyte
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[*] posted on 2-2-2011 at 08:00
Making biuret reagent


The wiki page claims that its made with Cu(II)SO4, KOH and potassium sodium tartrate. I thought that the principle behind this test was that Cu2+ cations in an alkaline solution turn violet in the presence of peptide bonds instead of the usual blue colour. Would a mixture of Cu(II)SO4 and NaOH not work?
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[*] posted on 2-2-2011 at 08:20


The tartrate is needed to complex the copper II ions otherwise they would drop out of solution as the hydroxide.
I do not think it matters if you use sodium or potassium hydroxide.
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[*] posted on 2-2-2011 at 15:07


I do not have a recipe for biuret reagent off hand but I think you could adapt one of the published recipes to use off the shelf chemicals.

"Biuret Reagent: Add, with stirring, 300 mL of 10% (w/v) NaOH to 500 mL of a solution containing 0.3% copper sulfate pentahydrate and 1.2% sodium potassium tartrate, then dilute to one liter. The reagent is stable for a few months but not a year. Adding one gram of potassium iodide per liter and storing in the dark makes it stable indefinitely.

The reagent can be used either qualitatively or quantitatively. In a typical reaction, one volume of sample is mixed with two to five volumes of reagent; the optimal ratio depends on the maximum protein concentrations you want to be able to resolve. The presence of protein gives a violet color with maximum absorbance around 550-555 nm; we typically read absorbances at 540 nm."

This one uses sodium potassium tartrate, sodium hydroxide and copper sulphate but you could use sodium hydroxide, potassium hydrogen tartrate aka cream of tartar and copper sulphate with appropriate adjustments.
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cnidocyte
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[*] posted on 4-2-2011 at 10:10


I wonder if citrate can be used as a substitute to tartrate similar to the way its used in Benedicts solution (as opposed to Fehlings solution).

I don't have a spectrophotometer but I find that quantitative analysis method interesting. I'm guessing this can be done with all colour indicating tests like this (i.e. benedicts solution, fehlings solution). Although you'd need a spectrophotometer to get an actual value, I suppose you can get a rough estimation of the concentration with your eyes.

[Edited on 4-2-2011 by cnidocyte]
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cnidocyte
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[*] posted on 20-2-2011 at 06:21


Quote: Originally posted by ScienceSquirrel  

This one uses sodium potassium tartrate, sodium hydroxide and copper sulphate but you could use sodium hydroxide, potassium hydrogen tartrate aka cream of tartar and copper sulphate with appropriate adjustments.

I have plenty of potassium bitartrate but I'm not sure what adjustments need to be made here. My coordination chemistry theories hazy, does potassium sodium tartrate have 2 coordination sites whereas potassium bitartrate only has one? In other words do I need 2 moles of bitartrate to complex one copper(II) ion as opposed to one mole if I was using the di-salt?
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[*] posted on 20-2-2011 at 09:44


bitartrate is just hydrogen tartrate, so you would use the same number of moles of K bitartrate as K Na tartrate, but you would add that many moles of K/Na OH to the bitartrate.
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cnidocyte
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[*] posted on 22-2-2011 at 13:06


I know what it is, I'm just wondering where its coordination sites are. If potassium bitartrate complexes Cu(II) ions as effectively as potassium sodium tartrate then tartaric acid must too. The strong base will deprotonate all the carboxyl groups of tartaric acid so I this is probably the case isn't it.
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[*] posted on 1-3-2011 at 13:32


The stuff I'm making doesn't seem to be working. I used these instructions:

1. Weigh 1.50 g of cupric sulfate pentahydrate (CuSO4 5 H2O) with 6.0 g sodium potassium tartrate tetrahydrate (NaKC4H4O6 4 H2O).

2. Dissolve in 500 ml of H2O.

3. Add 300 ml of 10% NaOH.

4. Make up to total volume of 1 liter. Store in a plastic bottle protected from light.

but replaced the 6g of potassium sodium tartrate with 4g of potassium hydrogen tartrate. The bitartrate complexed the Cu2+ ions fine but the colour changes aren't occuring for some reason. I just added a teaspoon of table sugar to 10mL of the solution and placed the beaker in a water bath but no red precipitate is forming. I did the same thing with a urea containing salt mixture but the solution didn't turn violet. One area I may have swayed from the instructions is the mass of Cu(II)SO4.5H2O used. My copper sulfate is still wet so it weighs far more than it should. Any idea where I may be going wrong?

[Edited on 1-3-2011 by cnidocyte]
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