My reagent blank (1mL 10^‐2M CuCl2, 1mL 7.5 10^-3Mneocuproine and 1mL ammoniumacetate, the volume was made up with 1.1 ml distilled water)was darker than sample reaction mixtures. Can someone please help!
Mahmoud Elshahawy
Cu(II) chloride solution (10 mM): Prepare by dissolving copper(II) chloride dihydrate in distilled water. Neocuproine solution (7.5 mM): Dissolve neocuproine in ethanol or methanol. Ammonium acetate buffer (1 M, pH 7.0): Dissolve ammonium acetate in distilled water and adjust the pH to 7.0. Combine Reagents: Mix 1 mL of the Cu(II) chloride solution, 1 mL of neocuproine solution, and 1 mL of the ammonium acetate buffer solution in a test tube or cuvette. Add Solvent: Add 1 mL of distilled water (or the solvent used for sample dilution) to bring the final volume to 4 mL. This step replaces the antioxidant solution that would be present in the sample assay. Mix Thoroughly: Vortex or gently shake the mixture to ensure complete mixing. Measure Absorbance: Let the mixture sit for a few minutes to stabilize, and then measure the absorbance at 450 nm. This absorbance value represents the baseline due to the reagent system alone and should be subtracted from the absorbance values of the samples containing antioxidants.
Here’s a step-by-step guide on how to prepare the reagent blank for the CUPRAC assay:
Reagents Needed
Steps to Prepare the Reagent Blank
Notes:
- Neocuproine forms a colored complex with Cu(II) ions, even in the absence of antioxidants. If the concentration of neocuproine or Cu(II) is relatively high (as in your blank), it can yield a darker color due to the formation of the Cu(II)-neocuproine complex. In the sample mixtures, antioxidants reduce Cu(II) to Cu(I), which forms a different, often lighter-colored complex with neocuproine.
- The blank solution should be freshly prepared each time the assay is run. If your CuCl₂ or neocuproine solutions are not freshly prepared, slight degradation or oxidation could lead to increased color in the blank. Neocuproine, especially, is light-sensitive and can oxidize over time, which might darken the blank solution.
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Goddati Nikhil
Make sure to check concentrations of sample mixture and reagent blank to be same. I hope this will help!! Poovamma B C
Thank you.
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