The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

Principle Of Kjeldahl Method

The Kjeldahl procedure measures the nitrogen content of a sample. The protein content then, can be calculated assuming a ratio of protein to nitrogen for the specific food being analyzed. The Kjeldahl procedure can be basically divided into three parts:

(1) Digestion

(2) Distillation

(3) Titration

In the digestion step, organic nitrogen is converted to an ammonium in the presence of a catalyst at approximately 370°C. In the distillation step, the digested sample is made alkaline with NaOH and the nitrogen is distilled off as NH3 . This NH3 is “trapped” in a boric acid solution. The amount of ammonia nitrogen in this solution is quantified by titration with a standard HCl solution. A reagent blank is carried through the analysis and the volume of HCl titrant required for this blank is subtracted from each determination.

Reagents

Sulfuric Acid (concentrated, N-Free)

Catalyst/Salt Mixture (Kjeldahl digestion tablets) Contains potassium sulfate, cupric sulfate, and titanium dioxide. Note: There are several types of Kjeldahl digestion tablets that contain somewhat different chemicals.

Sodium Hydroxide Solution, 50%, w/v, NaOH in deionized distilled (dd) water. Dissolve 2000 g sodium hydroxide (NaOH) pellets in ~3.5 L dd water. Cool. Add dd water to make up to 4.0 L

Boric Acid Solution- In a 4-L flask, dissolve 160 g boric acid in ca. 2 L boiled, and still very hot, distilled water. Mix and then add an additional 1.5 L of boiled, hot distilled water. Cool to room temperature under tap water (caution: glassware may break due to sudden cooling) or leave overnight. When using the rapid procedure, the flask must be shaken occasionally to prevent recrystallization of the boric acid. Add 40 ml of bromocresol green solution (100 mg bromocresol green/100 ml ethanol) and 28 ml of methyl red solution (100 mg methyl red/100 ml ethanol). Dilute to 4 L with water and mix carefully. Transfer 25 ml of the boric acid solution to a receiver flask and distill a digested blank (a digested catalyst/salt/acid mixture). The contents of the flask should then be a neutral gray. If not, titrate with 0.1 N NaOH solution until this color is obtained. Calculate the amount of