Feb 14, 2025

Public workspaceColorimetric determination of urea V.4

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.14egnzmzqg5d/v4
  • 1Utah State University;
  • 2Crop Physiology Laboratory, Utah State University
  • USU Crop Physiology Laboratory
Noah Langenfeld
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DOI: dx.doi.org/10.17504/protocols.io.14egnzmzqg5d/v4
External link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0259760
Protocol Citation: Noah Langenfeld, Laurenpayne, Bruce Bugbee 2025. Colorimetric determination of urea. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnzmzqg5d/v4Version created by Noah Langenfeld
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: October 08, 2021
Last Modified: February 14, 2025
Protocol Integer ID: 120244
Keywords: urea, urea assay, colorimetric urea, urea test, thiosemicarbazide, diacetyl monoxime
Abstract
This protocol measures the absorbance of urea in solution in complexation with diacetyl monoxime at 520 nm and is linearly proportional to concentration up to 5 mM urea.
Materials
thiosemicarbazide, diacetyl monoxime, water, sulfuric acid, phosphoric acid, ferric chloride
Safety warnings
This protocol utilizes strong acids. Ensure proper precautions and safety equipment are used when dealing with these chemicals.
Before start
Ensure a lab coat, lab goggles, and plastic gloves are worn throughout this assay.
Mixed Acid Reagent Preparation
Mixed Acid Reagent Preparation



Dissolve Amount2.5 mg ferric chloride (97% purity) in Amount45 mL deionized water in a 250 mL volumetric flask.

Add Amount80 µL of Concentration14.8 Molarity (M) concentrated phosphoric acid (85 wt. % phosphoric acid in water).


Safety information
Caution: Preparation of Concentration4.7 Molarity (M) sulfuric acid is highly exothermic. Slowly add acid toAmount100 mL water and stir frequently to avoid spattering.

Prepare Concentration4.7 Molarity (M) sulfuric acid by diluting Amount65.25 mL concentrated sulfuric acid (Concentration18 Molarity (M) , 98% purity) up to Amount250 mL with deionized water.

Dilute ferric chloride, deionized water, and phosphoric acid mixture to Amount250 mL with Concentration4.7 Molarity (M) sulfuric acid.

Mix until dissolved.
Mixed Color Reagent Preparation
Mixed Color Reagent Preparation
Add Amount20.9 mg diacetyl monoxime (99% purity) and Amount52.4 mg thiosemicarbazide (99% purity) to a 250 mL volumetric flask.

Dilute to volume (Amount250 mL ) with deionized water.

Mix until dissolved.
Urea Assay
Urea Assay
10m 5s
10m 5s
Prepare known concentrations of urea at 0, 1, 2, 3, 4, and 5 mM for a calibration curve.

Fill a 600 mL beaker with Amount200 mL deionized water.

Place the 600 mL beaker on hot plate and bring to a boil.

To test a sample, aliquot Amount1 mL into a 20 mL glass test tube.

AddAmount2 mL Mixed Acid Reagent.

Add Amount2 mL Mixed Color Reagent.

Ensure contents of test tube are vigorously mixed by vortexing for at least Duration00:00:05 .

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 Duration00:20:00 .

Remove tubes from water bath and let cool for Duration00:10:00 .

10m
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.