FOOD ANALYSIS : PROTEIN METHODS
Digest with acid, distil the ammonia, titrate
Burn in oxygen, measure the nitrogen gas
Figure 1: Both methods measure nitrogen, not protein itself. The factor turns the nitrogen into crude protein.
Introduction
The Kjeldahl method and the Dumas method are the two standard ways to find protein in food, and both work by measuring nitrogen and then multiplying by a factor. Kjeldahl is the older reference method: the sample is digested in strong acid, the nitrogen is distilled as ammonia, and a titration measures it, which takes hours. Dumas is the modern fast method: the sample is burned completely in oxygen, its nitrogen is released as nitrogen gas, and the gas is measured, which takes minutes.
Quick comparison
Table 1: Kjeldahl method compared with Dumas method
| Aspect | Kjeldahl method | Dumas method |
|---|---|---|
| What it measures | Nitrogen, after digestion and distillation | Nitrogen, as nitrogen gas after combustion |
| Stages | Three: digestion, distillation, titration | One: combustion, then gas measurement |
| Reagents | Concentrated sulfuric acid, H₂SO₄, catalyst, alkali, standard acid | Oxygen for burning, no acid and no titration |
| Time per test | Hours | Minutes |
| Sample | Grams, weighed into a digestion flask | Small, weighed into a combustion boat |
| Result | Nitrogen percent, times the factor gives crude protein | Total nitrogen, times the same factor gives crude protein |
| Reading | Usually very slightly lower than Dumas | Usually very slightly higher, because burning releases a little nitrogen that digestion misses |
| Role | Reference method that settles disputes | Fast method for routine work with many samples |
| Hazards | Strong acid burns, hot fumes, work in a fume cupboard | High temperature combustion, no strong acid |
| Factor | 6.25 for most foods, 6.38 for milk, 5.70 for wheat | Same factors: 6.25, 6.38 for milk, 5.70 for wheat |
Scientific explanation
Definition
Kjeldahl method: the reference method for protein, in which the sample is digested, distilled, and titrated to find its nitrogen. Dumas method: a fast protein method that burns the sample in oxygen and measures the nitrogen gas released. Crude protein: nitrogen in a food multiplied by a factor, used as the protein value.
Working principle
Kjeldahl: in digestion, the weighed sample is heated with concentrated sulfuric acid, H₂SO₄, with a catalyst, until the organic matter breaks down and all the nitrogen is turned into ammonium sulfate. In distillation, alkali is added, the ammonium is released as ammonia gas, NH₃, and the gas is distilled into a measured volume of standard acid. In titration, the acid left over is titrated with standard alkali, and the volume used tells how much ammonia was trapped, which tells how much nitrogen was in the sample. Dumas: the sample is burned completely in oxygen at high temperature, all its nitrogen is released as nitrogen gas, N₂, and the gas is measured. There is no digestion, no distillation, and no titration.
Apparatus
Kjeldahl: digestion flask and heater, distillation unit, burette, and standard acid and alkali. Dumas: a combustion furnace with an oxygen supply and a gas detector that measures the nitrogen gas released.
Measurement procedure
Kjeldahl: weigh the sample, digest it until the liquid turns clear, distil the ammonia into standard acid, titrate the acid left over, and run a blank test with no sample so the nitrogen in the reagents can be subtracted. Dumas: weigh a small sample, burn it in oxygen, and read the nitrogen gas. Laboratories that change methods check the new method against the old one on the same samples before reporting results from it.
Factors affecting readings
Kjeldahl: the wrong factor gives a wrong protein value even when the nitrogen measurement is perfect, so milk uses 6.38 and wheat uses 5.70 instead of 6.25. Incomplete digestion misses nitrogen, and a missing blank counts reagent nitrogen as sample nitrogen. Dumas: incomplete burning misses nitrogen, and the small sample must represent the whole lot, so sampling and grinding matter more. Both methods count all nitrogen as protein, so a nitrogen-rich chemical that is not protein would count as protein in the calculation.
Formulas and calculations
Nitrogen percent = titration volume in mL times normality times 1.4007, divided by sample weight in grams. Crude protein percent = nitrogen percent times the factor. One millilitre of 0.1N sulfuric acid equals 0.0014 g of nitrogen.
Worked example from the FTN Food Analysis notes: a 1.000 g flour sample needs 14.3 mL of 0.1N acid, and the blank needs 0.3 mL, so the volume is 14.0 mL. Nitrogen percent = 14.0 times 0.1 times 1.4007 divided by 1.000 = 1.961 percent. Crude protein = 1.961 times 6.25 = 12.3 percent.
Industrial applications
- Flour mills check wheat protein by the Kjeldahl method, because buyers pay by protein.
- Milk buyers pay by protein, and the Kjeldahl method settles the protein value in trade disputes.
- Laboratories with many samples a day use the Dumas method for routine protein, checked against Kjeldahl.
- Snack makers print protein on every pack, and the value comes from one of these two nitrogen methods.
Advantages and limitations
Kjeldahl method: it is the accepted reference, it handles large samples, and its chemistry is well understood. Its limits are time, strong acid and fumes, and the fact that it misses a little nitrogen held in some ring compounds.
Dumas method: it is fast, it uses no strong acid, and it needs little analyst time per test. Its limits are the small sample, which must represent the lot, the cost of the instrument, and a reading that runs very slightly higher than Kjeldahl, so a change of method must be checked on the same samples first.
FAQ
Question: Why is the result called crude protein and not protein?
Because the method measures nitrogen, not protein itself. The nitrogen is multiplied by a factor to give the protein value, and any nitrogen that is not protein is counted too.
Question: Why does Dumas usually read slightly higher than Kjeldahl?
Because burning releases a little nitrogen that digestion misses, such as the nitrogen in some ring compounds.
Question: Which factor do I use for milk?
Milk uses 6.38. A milk sample with 0.50 percent nitrogen gives crude protein of 0.50 times 6.38 = 3.19 percent. Using 6.25 would give 3.13 percent, which is wrong for milk.
Question: Why run a blank test in the Kjeldahl method?
To measure what the reagents themselves contribute. The blank shows the nitrogen in the acid, which is subtracted from the sample result.
Question: If a flour is spiked with a nitrogen-rich chemical that is not protein, what would Kjeldahl report?
It would report high protein, because Kjeldahl measures nitrogen, not protein itself. That is why a result that looks too good is checked by other means.
References and further reading
- FTN Food Analysis, Unit 4: Protein and fat analysis (notes on disk): Kjeldahl stages, Dumas method, factors 6.25, 6.38, 5.70, worked examples 12.5 percent and 12.3 percent crude protein.
- Nielsen, S. S., Food Analysis Laboratory Manual, Springer: Kjeldahl nitrogen method and nitrogen combustion method (consulted through Google Books).
- Nielsen, S. S., Food Analysis, Springer: protein analysis, Kjeldahl and Dumas methods (consulted through Google Books).
