Measuring Food Protein Content

While reviewing one of the laboratory analytical procedures, I am jotting down some important notes. I am trying to make it shorter and easier for me to understand.

The popular method for measuring food protein content is by Kjeldahl procedure. The analytical method developed by Johan Kjeldahl. Most modern and current procedures are also based on his work.

The procedure is not 100 percent accurate. That is why the specific method name is Crude Protein. The procedure specifically targets the element N. Proteins contain N but the food may contain other nitrogen containing compounds. So all N collected might be expressed as protein.

As I said, the following are written specifically as my quick reference. Anyone wanting to learn the protein analysis process should consult the complete procedure approved by Association of Official Analytical Chemist (AOAC) or any reputable institution.

Kjeldahl Method. The shortened procedure


95-98% sulfuric acid, nitrogen free
Copper catalyst – CuSO4-5H2O
Potassium sulfate – nitrogen free
Sodium hydroxide solution – 50% w/w
Boiling chips – high purity amphoteric alundum granules
zinc granules/chips
Methyl red/bromocresol green indicator solution
Ammonium sulfate – 99.9%
Tryptophan or lysine hydrochloride
Sucrose, nitrogen free


Indicator: Dissolve 0.2 g methyl red and dilute to 100 ml 95% ethanol. Dissolve 1 g bromocresol green and dilute to 500ml 95% ethanol. Mix one part methyl red solution with 5 parts bromocresol green.

Boric acid solution: 4% boric acid with 3 ml methyl red/bromocresol green.

1 M HCl … Seed standard preparation.


Add 15 grams potassium sulfate, 8-10 boiling chips, 1 ml copper sulfate pentahydrate, 1 g sample recorded to nearest 0.0001 g and 25 ml sulfuric acid to kjeldahl flask

Heat requirement – 250 ml water rolling boil within 5-6 minutes. Pre-heat the the heating equipment, place the flask containing water then record time. Adjust and repeat.

Place digestion flask on heating apparatus in inclined position. Set to low. Digest for 20 minutes or until white fumes appear in flasks.

Increase heat half-way to maximum. Heat for another 15 minutes.

Set to maximum then continue boiling for 1 to 1.5 hours.

Cool approximately 25 minutes.  Large amount of crystallization should not be observed. Refer to full reference if so.

Add 300 ml distilled water and swirl. Cool to room temperature.


Assemble the distillation apparatus.

Add 50 ml Boric acid solution to 500 ml Erlenmeyer flask. Place it under condenser tip. The tip should be submerged in solution.

To kjeldahl flask, carefully add 75ml 50% sodium hydroxide solution with no agitation. Add few zinc chips – it will prevent bumping during distillation. Connect it to system immediately.

Distill until sufficient volume has been collected. 150ml to 200ml.


Titrate the boric acid solution with 0.1000M HCl to first trace of pink.


Nitrogen loss. Use 0.12 g ammonium sulfate and 0.85 g sucrose. Subject to kjeldahl test. Recovery should be at least 99%.

Digestion efficiency. Use 0.16 g lysine hydrochloride or 0.18 g tryptophan and 0.67 g sucrose.  Subject to kjeldahl test. Recovery should be 98%.


% Nitrogen = (1.4007x[Vs -Vb]xM)/W

where Vs and Vb = ml HCl titrant used for test portion and blank, respectively; M = molarity of HCl solution; and W = test portion weight, g.

Then the percent N is multiplied by factor appropriate for the food analyzed.

coffee bean for protein analysis

Four Reasons Why Compare Different Recipes and Methods

I am going to conduct an array of Kjeldahl Nitrogen  and Moisture Determination activities. I am downloading and reading methods recommended by different organizations. I have gotten more than five procedures each of the same type but still looking for more. I am comparing every procedures, for the following reasons:

1) All of the downloaded files are basically the same but they are uploaded at different times. Methods may changed through the work of different experts. I might find a significant improvement. The use of less chemicals, cheaper reagents or more affordable equipment.

2) Some files are easier to interpret than others, rewritten for better understanding. A recipe with very specific ingredient listing and more elaborate step-by-step instructions. An analytical procedure in translated in less technical manner.

3) Additional information. Safety precautions such as careful handling of sulfuric acid. Proper waste disposal. Other uses – the same Kjeldahl method for determining chlorophyll content and caffeine. A general nata recipe for coconut, kalabasa, mango and banana.

4) Merging of data. It happened to me several times when I cannot made a food product perfectly. Several tries produced the same failed results. Eventually, I managed getting what I want by reading methods from other sources.