Proximate analysis: Crude protein

• Principle and Scope
• Sample preparation
• Reagents
• Procedure
• Calculation
• Possible errors

Principle and Scope

Estimated by a process developed by a Danish chemist/brewer, Johan Kjeldahl. He discovered that "all protein" contains about the same amount of nitrogen (16%). He analyzed for nitrogen, which is relatively easy, and calculated crude protein on the basis: 100/16 = 6.25, therefore: NITROGEN x 6.25 = CRUDE PROTEIN
In the presence of sulfuric acid, sodium sulphate and a catalyst, the amino nitrogen of many organic materials is converted to ammonium sulphate. The ammonia is distilled from an alkaline medium and absorbed in standardized mineral acid. The ammonia is determined by back titration with a standardized mineral base.
This method is applicable to fish, fish products, and fish by-products. Certain species of fish such as dogfish contain non-protein nitrogen; therefore, when analysing these species use AOAC procedure 7.024 (12th Edition) for non-protein nitrogen to correct the results.

Sample preparation

1. Sample preparation should take into account the type of product and how it is used and prepared by the consumer:
   • for fish and fish products that contain no free liquid: comminute the sample until homogeneous.
   • for products that are packed in water, brine or similar medium that is normally discarded by the consumer: open the package and drain the product on an appropriate size sieve for 1 to 1½ minutes. Comminute the part of the sample retained by the screen until a homogeneous blend is obtained.
   • for products that are packed in a medium that may be or is normally used by the consumer, e.g., fish canned in its own juice or oil: transfer the entire contents of the package into a homogenizer and blend for one minute or until a homogeneous mix is obtained.
   • for fish meal: grind the sample in a mill or other suitable apparatus until it will pass through a no. 20 sieve.
2. Collect the homogenized sample into a thoroughly cleaned, sealable plastic cup or glass bottle.
3. Store the sample in a refrigerator or freezer until required.
4. Ensure that the prepared sample is still homogeneous prior to weighing. If liquid separates from the sample, thoroughly reblend before use.

Reagents

• Sulfuric acid (H2SO4), nitrogen-free.
• Cupric Sulphate (CuSO4), nitrogen-free, anhydrous.
• Sodium Sulphate (Na2SO4), nitrogen-free, anhydrous.
• Sodium Hydroxide (NaOH).
  • NaOH solution (50% w/v).
  • NaOH standard solution (0.1 or 0.2 N).
• Boiling granules, selenized. Hengar granules are suitable.
• Hydrochloric acid (HCl).
• HCl standard solution (0.1 N). Standardize against 0.1 or 0.2 N NaOH standard solution.
• Conway indicator.
  • Stock solution. Mix 200 mL of 0.1% Methyl Red solution (in 50% ethanol) with 50 mL of 0.1% Methylene Blue solution (in 50% ethanol).
  • Working solution. Dilute 1 volume of stock with 1 volume of absolute ethanol and 2 volumes of distilled water. (pH change 5.4: Acid - Purple, Alakline - Green).

Procedure

1. Accurately weigh a suitable quantity of fine-grained material (ca 1.2 g for fishmeal, ca 2.5 g for solubles or homogenized fish) and place in digestion flask.
2. Add sequentially 15 g Na2SO4, 1 g CuSO4, one or two selenized boiling granules and 25 mL of conc H2SO4 to the flask.
3. Digest until solution is almost colourless or light green (2 hrs for inorganic material) and then at least a further 30 minutes. Do not heat any part of the Kjeldahl flask above the level of the digestion mixture.
4. Cool (do not allow to solidify), and cautiously add 200 mL water. Add additional boiling granules (if necessary) to prevent bumping.
5. Pipette 100 mL 0.1 N HCl into a 500 mL erlenmeyer flask, add 1 mL Conway's indicator and place the flask under the condenser ensuring that the condenser tip is immersed in the acid solution. (volume of standardized HCl used in distillation may be varied according to the expected nitrogen content of the sample).
6. Tilt the Kjeldahl flask containing the digested sample and add 100 mL of 50% NaOH solution slowly down the side of the Kjeldahl flask so that it forms a layer underneath the digestion mixture.Immediately connect the flask to the distilling bulb of the distillation apparatus. Rotate flask to thoroughly mix contents.
7. Heat until all ammonia has passed over into the standard acid. Collect approximately 150 mL. Caution, flask will bump. Remove immediately (prolonged boiling and too rapid distillation of acid during digestion should be avoided as loss of ammonia may occur).
8. Wash tip of condenser and titrate excess standard HCl in distillate with NaOH standard solution (detailed titration procedure) .

Figure: Soxhlet apparatus and diagram

Provide adequate ventilation for the removal of fumes during digestion.

Calculation

Calculate the percentage nitrogen (wet weight basis) as follows:

where:

• A = vol. (mL) std. HCl x normality of std. HCl
• B = vol. (mL) std. NaOH x normality of std. NaOH

Calculate nitrogen content on dry basis (when moisture content is known) as follows:

Calculate the percentage protein (wet or dry basis) as follows:

• % PROTEIN = % nitrogen x 6.25

where 6.25 is the protein-nitrogen conversion factor for fish and fish by-products.

Possible errors & disadvantages

• This procedure assumes ALL nitrogen present in the sample are in PROTEIN form. This assumption is NOT necessarily true. Nitrogen could be in nucleic acid (RNA, DNA), urea...
• Different proteins need different correction factors because they have different amino acid sequences
• The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts
• The technique is time consuming to carry-out.