NTR 325- Food Analysis Dr C.

Bou Mitri

Lab Session 7
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Protein Determination (Kjeldahl Method)

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Introduction
The protein content of foods varies widely: most fresh fruits and vegetables have less than 6%;
white flour and fresh eggs about 12%; and most meats, fish and cheese about 25%. Nitrogen is
the most distinguishing chemical element present in proteins, which are the most common
organic nitrogen compounds among foodstuffs. Protein analysis in foods has been mostly done
by determining Nitrogen content (Kjeldahl method). Food proteins contain on average 16% N by
mass. In order to get protein content, we multiply nitrogen content by the conversion factor 6.25
(conversion factor = 100/16). Since the protein content ranges between 13.4 and 19.1, the
conversion factor varies accordingly. For wheat and cereals it is 5.70, eggs 6.25, and milk 6.38.
The determination of protein content from total nitrogen is only a crude estimate, since many
other non-protein nitrogenous substances are also present in foods. Other methods (Dumas
method, Biuret method, Lowry method, Bradford method) find also extensive use.

Principle of Kjeldahl method

The Kjeldahl method (developed in 1883) is the method that is most universally used, and
probably the most accurate, to determine proteins. It is based on the determination of total
proteins from the total nitrogen content, using the previously mentioned conversion factor (0.25
for many applications).
The Kjeldahl method is carried out in two distinct phases. Firstly, the nitrogen compounds are
digested with concentrated H2SO4 to produce (NH4)2SO4 with the aid of catalysts (copper,
mercury, or selenium) and K2SO4, which raises the boiling point. Secondly, The NH4+ produced

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is neutralized to ammonia with NaOH; ammonia is then evacuated by steam distillation into a
weak acid (boric acid) solution (containing an acid base indicator), where it is converted again to
NH4+. The borate ions produced from the previous neutralization are titrated against standardized
HCl.

Steps of Kjeldahl method:

The Kjeldahl method can be divided into 3 steps: digestion, neutralization, and titration.

1- Digestion
Digestion is performed with boiling sulfuric acid and the following reaction takes place:
Protein + H2SO4  (NH4)2SO4 + CO2 + H2O
Nitrogen is converted to ammonium sulfate and carbon and hydrogen are oxidized to carbon
dioxide and water. It is performed in the presence of potassium sulfate (increases the boiling
point of sulfuric acid), and a metallic catalyst (mercury, copper, or selenium).

2- Neutralization and distillation

During neutralization: the digest is diluted and neutralized with concentrated sodium hydroxide.
The reaction that takes place transforms ammonium ion (NH4+) into ammonia (NH3):
(NH4)2SO4 + 2NaOH  2NH3(g) + 2 H2O + Na2SO4
The produced ammonia is driven off as a gas and distilled into a boric acid solution containing
an acid base indicator. Ammonia reacts with boric acid to form ammonium and borate ion
according to the following equation:
NH3(l) + H3BO3  NH4+ + H2BO3-

3- Titration
The borate ion (H2BO3-) is titrated with standardized HCl (endpoint: change in color from green
to violet):
H2BO3- + H+  H3BO3
The indicators used are methylene blue and methyl red.

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Calculation of results
Finding the amount of nitrogen requires simple skills in stoichiometry. The stoichiometric
relationship between different substances involved in Kjeldahl reactions can be summarized into
the following:
nHCl required = nborate = nNH4+ = nN in the original sample

The following equation is a handy formula that can be used for the direct calculation of the
percentage of total nitrogen in the sample:

%N = Normality HCl x (S – B) x (14 g N/1 mol N) x (100/g sample)

Where:
• S: mL HCl required for sample neutralization
• B: mL HCl required for blank neutralization

The percentage of proteins can be afterward calculated simply using the following equation:

% protein = %nitrogen x conversion factor (6.25)

Kjeldahl method is very accurate, precise & widely used. The problem that might be encountered
in biological systems is due to the fact that such systems contain non-protein nitrogenous
compounds (e.g., DNA). Such problem might be overcome by a prior separation of proteins by
precipitation (e.g., with 12% trichloroacetic acid).

Advantages and disadvantages of Kjeldahl method

Advantages
 Applicable to all foods
 Relatively simple
 Inexpensive
 Accurate (official method)
 Can be modified for micro analysis

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NTR 325- Food Analysis Dr C. Bou Mitri

Disadvantages
 Measures total N not only nitrogen in proteins
 Different proteins contain different amino acid => needs different correction factors
 Time consuming: ~ 2 hrs
 Corrosive chemicals are used

Experimental Procedure
Materials:
- Concentrated acid and concentrated caustic soda (sodium hydroxide)
- Ashless filter paper
- Piece of blank paper
- Kjeldahl flask
- Potassium sulfate (K2SO4)
- Copper sulfate
- Dispenser
- Concentrated sulfuric acid
- Gerhardt apparatus
- 2% boric acid solution
- Gerhardt conical flask
- Distilled water
- Some boiling chips
- 100 ml cylinder
- 0.1M HCl solution

CAUTION: The Kjeldahl analysis uses hot concentrated acid and concentrated caustic soda
(sodium hydroxide). It is imperative that you follow safety directions carefully and wear safety
goggles and gloves.

A. Digestion
If the sample you are provided is not finely ground, crush it with mortar and pestle so that it can
pass through a sieve with 2 mm mesh. Weigh out accurately about 1.5 to 2 g sample on an

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NTR 325- Food Analysis Dr C. Bou Mitri

ashless filter paper. Roll up a piece of blank paper and insert it into the neck of the Kjeldahl
flask. This will keep the neck of the flask clean while you add the sample. Place the sample
inside a labeled digestion flask.
Add to the flask 7 g of potassium sulfate (K2SO4), 0.1 g of copper sulfate (CuSO4). Finally, using
a dispenser (shown below), add to the flask 20 mL of concentrated sulfuric acid (H2SO4; 98%).

CAUTION: 98% sulfuric acid is dangerous. Wear safety goggles and gloves. If you get any on
your skin, wash immediately with copious amounts of cold water, followed by soap and water.

Place the digestion (Kjeldahl) flask on the flask heater in the Gerhardt apparatus. Once all flasks
positioned, the system will be heated till boiling of the digestion solution and white vapor comes
up. After 20 to 30 minutes the digestion solutions should be clear; any sample residue remaining
on the glass walls are washed down into the flask with the condensating sulfuric acid.
During the entire digestion period the scrubber should be on.

B- Suction and cooling

The cooling off period after the lifting of the insert rack (or turning off the heating) is about 30
minutes. During this time the scrubber should be working as well.

Meanwhile, prepare in the Gerhardt conical flask 50 mL of the 2% boric (H3BO3) acid solution.
Add 3 to 4 drops of the indicator mixture.

C- Dilution, neutralization and distillation

Place the Gerhardt conical flask containing the boric acid solution plus indicator in its position
on the Gerhardt apparatus. The tip of the condenser outlet-tube should be immersed in the
solution.

When the Kjeldahl flask is cool, place it in an ice bath (at the same time, prepare 300 mL of
distilled water in an ice bath). After 10 to 15 minutes, add cautiously and slowly the 300 mL of
distilled water (FLASK POINTED TOWARD THE REAR OF THE HOOD) so as to wash down
the neck of the flask, and mix the content thoroughly. Recall the rule “always add acid to water;

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NTR 325- Food Analysis Dr C. Bou Mitri

never add water to acid.” Now we have to break that rule, so use caution (safety goggles and
gloves). Add some boiling chips (about 10). Now, you will add the sodium hydroxide solution
that will transform ammonium ions into volatile ammonia. This operation should be performed
carefully under the hood (safety goggles and gloves worn), since the sodium hydroxide solution
used is highly concentrated:
With the help of a dispenser, place about 70 mL of the 32% caustic soda (NaOH) solution in a
100 mL cylinder. Add the caustic soda to the to the Kjeldahl flask pointed toward the rear of the
hood. Do not wet the top of the neck with the sodium hydroxide solution. The combined volume
of water and NaOH solution is 370 mL. This will allow 150 mL of distillate to be collected just
before irregular boiling (bumping) ensues.

Immediately connect the Kjeldahl flask to the distillation unit of the Gerhardt apparatus. Make
sure that the tip of the condenser outlet-tube is immersed in the boric acid solution.
Swirl the content of the Kjeldahl flask to mix thoroughly and boil, gently at first, to prevent
excessive frothing. When 100 to 125 mL of distillate have been collected, lower each conical
flask until the tip of the condenser outlet-tube is 40 mm above the 200 mL mark. Continue each
distillation, until irregular boiling (bumping) starts and then immediately stop the heating.
Disconnect each Kjeldahl flask and rinse the tip of each condenser outlet tube with a little
distilled water, collecting the rinsing in the conical flask.

The distillation rate shall be such that approximately 150 mL of distillate are collected when
irregular boiling (bumping) starts, the volume content of each conical flask will be
approximately 200 mL, i.e. approximately 150 mL of distillate will have been collected as
required.

D- Titration
Titrate the ammonia collected in the Kjeldahl conical flask with the 0.1 M HCl solution. The end
point is detected when the indicator color changes from green to violet.
Leave a copy of your titration volumes to your lab instructor

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NTR 325- Food Analysis Dr C. Bou Mitri

E- Calculation

• Using your titration volume and the provided blank volume, calculate the percentage of
nitrogen in your sample
• Using the conversion factor, calculate the percentage of nitrogen in your sample
• Looking at the website of the of the United States Department of Agriculture
(http://www.nal.usda.gov/fnic/foodcomp/search/), compare your result to that given in the
USDA nutrient database.

References
AOAC International. (1995). ‘Official Methods of Analysis’. 16th Ed. (AOAC International:
Gaithersburg, MD)

D’Arcy B.R., Hawes G., Bentley I. (2002). “Food Chemistry: A Practical Manual” (University of
Queensland Publications)

Nielsen, S. S. (ed.) (1998) ‘Food Analysis’ 2nd Ed. (Aspen Publishers, Inc.: Gaithersburg,
Maryland, USA).

Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.

Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

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