Colorimetric determination of urea
Noah Langenfeld, Laurenpayne, Bruce Bugbee
Abstract
This protocol measures the absorbance of urea in solution in complexation with diacetyl monoxomie at 520 nm and is linearly proportional to concentration up to 5 mM urea.
Before start
Ensure a lab coat, lab goggles, and plastic gloves are worn throughout this assay.
Steps
Mixed Acid Reagent Preparation
Dissolve 2.5mg ferric chloride in 45mL deionized water in a 250 mL volumetric flask.
Add 80µL phosphoric acid.
18Molarity (M) sulfuric acid by diluting 65.25mL concentrated sulfuric acid up to 250mL with deionized water. Dilute ferric chloride, deionized water, and phosphoric acid mixture to 250mL with 18Molarity (M) sulfuric acid.
Mix until dissolved.
Mixed Color Reagent Preparation
Add 20.9mg diacetyl monoxime and 52.4mg thiosemicarbazide to a 250 mL volumetric flask.
Dilute to volume (250mL) with deionized water.
Mix until dissolved.
Urea Assay
Prepare known concentrations of urea at 0millimolar (mM), 1millimolar (mM), 2millimolar (mM),3millimolar (mM), 4millimolar (mM), and 5millimolar (mM) for a calibration curve.
Fill a 600 mL beaker with 200mL deionized water.
Place the 600 mL beaker on hot plate and bring to a boil.
To test a sample, aliquot 1mL into a 20 mL glass test tube.
Add2mL Mixed Acid Reagent.
Add 2mL Mixed Color Reagent.
Ensure contents of test tube are vigorously mixed by vortexing for at least 0h 0m 5s .
Repeat steps 12-15 for each sample and standard to be analyzed.
Ensure test tubes are labeled and place in boiling water bath for 0h 20m 0s .
Remove tubes from water bath and let cool for 0h 10m 0s .
Fill plastic cuvette to line with cooled solution.
Place sample cuvette in spectrophotometer and record absorbance at 520 nm. Blank against the standard solution with both reagents, but no urea.
Use the calibration curve to determine urea concentrations of unknown solutions.
