5% Nitric Acid Solution

Preparation of 5% (v/v) nitric acid for acid preservation of biological samples and urea standards.

5% Nitric Acid Solution, v1.0 Kathryn E. Caruso · 0009-0003-2436-1791 Foreman Lab · Center for Biofilm Engineering, Montana State University Updated March 2026

How to cite this protocol Caruso, K.E. (2026). 5% Nitric Acid Solution, v1.0. Foreman Lab, Center for Biofilm Engineering, Montana State University. https://kathryncaruso.github.io/methods/5pct-nitric-acid/

Purpose

Preparation of 5% (v/v) nitric acid for use in acid preservation of biological samples. Used to immediately stop enzymatic activity (e.g., urease) upon sampling and to serve as the matrix for matrix-matched urea standards in colorimetric assays.

Safety

⚠️ WARNING: Concentrated nitric acid is a strong oxidizer and is highly corrosive.

PPE:

  • Lab coat
  • Nitrile gloves
  • Safety glasses or face shield
  • Work in a fume hood

Chemical hazards:

  • Concentrated nitric acid (HNO3, 65–70%): Oxidizer, corrosive, causes severe burns
  • Exothermic reaction when acid contacts water — always add acid to water

Waste:

  • Dispose of excess acid solution in liquid waste container in fume hood

Equipment and materials

  • 1 L volumetric flask
  • Graduated cylinder
  • Concentrated nitric acid (HNO3, 65–70%)
  • Deionized (DI) water
  • Labeled storage bottle with appropriate hazard warnings
  • Fume hood

Volume options

Target volume DI water Concentrated HNO3 Vessel
100 mL 95 mL 5 mL 100 mL volumetric flask or graduated cylinder
200 mL 190 mL 10 mL 200 mL volumetric flask
250 mL 237.5 mL 12.5 mL 250 mL volumetric flask
500 mL 475 mL 25 mL 500 mL volumetric flask
1 L 950 mL 50 mL 1 L volumetric flask

Procedure

  1. Add the appropriate volume of deionized water (see Volume options section) to a clean volumetric flask
  2. In a fume hood, carefully add the corresponding volume of concentrated HNO3
    • ⚠️ ALWAYS add acid to water, never water to acid
    • Pour slowly down the side of the flask
  3. Mix thoroughly
  4. Allow to cool
  5. Transfer to a labeled bottle with appropriate hazard warnings
  6. Store at room temperature