HPLC Determination of 2,3-Butanedione (Diacetyl) Using 4-Nitro-o-phenylenediamine as the Derivatization Reagent

Separation type: Liquid Chromatography Reverse Phase.

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High Performance Liquid Chromatography (HPLC) Method for Diacetyl.

Diacetyl is a natural by-product of fermentation and is known to be an important flavor compound in many food products. Diacetyl is a reactive diketone in artificial butter flavors. The principal types of flavorings that use diacetyl are dairy flavors, particularly butter flavorings but also cheese, milk, and yogurt. Diacetyl is also sometimes an ingredient in the so-called brown flavors such as caramel, butterscotch, and coffee flavors. The product of the derivatization of diacetyl with NPDA can be retained in HPLC on Newcrom R1 reverse-phase column with the simple isocratic mobile phase consisting of acetonitrile (MeCN), water and phosphoric acid (H3PO4). The analysis method can be UV detected at 260 nm.

Chemicals and Reagents.

  • The stock solutions of 0.1 mg/mL 2,3-Butanedione (Diacetyl) were prepared in distilled water.
  • The stock solutions of 1.0 mg/mL 4-nitro-o-phenylenediamine (NPDA) were prepared in methanol.

Derivatization Procedure.

  • 0.10 mL of diacetyl stock solution,
  • 0.10 mL of HCl (0.1 M),
  • 0.60 mL of methanol, and
  • 0.20 mL of NPDA stock solution were added to a test vial.

The total solution was sonicated for 20 minutes. The resulting solution was filtered through a 0.22 μm filter membrane and injected into the chromatographic system.


Column Newcrom R1, 2.1x100 mm, 3 µm, 100A
Mobile Phase MeCN - 40%
Buffer H2SO4 - 0.2%
Flow Rate 0.2 ml/min
Detection 260 nm


Class of Compounds Ketone
Analyzing Compounds 2,3-Butanedione (Diacetyl), 4-nitro-o-phenylenediamine (NPDA)
Application Analytes: