ISI 24-1e Determination of Protein by Kjeldahl

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1. Scope	The method is applicable to starch, starch hydrolysate and starch syrups.	LT 24 Jan.. 1967 Rev. LT 03 Sep 1999
2. Principle	Destruction of organic matter by sulphuric acid. N is liberated as ammonia, distilled, collected and titrated.
3. Apparatus	3.1 Analytical balance weighing to the nearest 0.1 mg.
	3.2 Kjeldahl distillation assembly with 500 ml Kjeldahl flask, pear-shaped glass bulb, 50 ml - 200 ml dropping funnel, Splash head, 1000 ml distillation flask, condenser and 500 ml conical flask (collector) 3.3 Electrical heater	Quickfit 280 MC Use an inclined digestion stand.
4. Reagents	4.1 CuSO4, 5H2O p.a. 4.2 Se, p.a. 4.3 K2SO4, p.a. 4.4 Graphite flakes 4.5 Zn, granules p.a. 4.6 H2SO4, conc. p.a. 4.7 NaOH, 33% (w/w) suitable for Nitrogen determination 4.8 HCl 0.02N: Transfer 20.0 ml 0.1N HCl to 100 ml volumetric flask. Add distilled water to mark. Keep for maximum one day. 4.9 H2SO4, 0.1N 4.10 NaOH 0.1N 4.11 H2SO4, 0.5N 4.12 NaOH 0.5N 4.13 Methyl red/Methylene blue indicator: (a) 0.1 g Methyl red C15H15N3O2 is dissolved in 75 ml ethanol. (b) 0.1 g Methylene blue C16H18ClN3S, xH2O is dissolved in 80 ml ethanol. Mix 50 ml of (a) with 25 ml of (b). 4.14 Methyl red/Bromocresol green indicator: 0.6 g Methyl red and 0.3 g Bromocresol green C21H14Br4O5S are dissolved in 100 ml ethanol. Add drop by drop NaOH 1N until a dark red colour is formed. 4.15 Pumice
5. Procedure	Weigh sample according to table one to the nearest 1 mg and transfer to a 500 ml Kjeldhahl flask. Add according to table one the reagents (4.1) CuSO4, 5H2O, (4.2) Se, (4.3) K2SO4, (4.4) Graphite and (4.6) H2SO4, conc. p.a. Close with glass bulb.	Use parchment paper if convenient.
Destruction	Place Kjeldahl flask on heater and heat cautious avoiding vigorously boiling. When foaming ceases heat increases and the destruction continues until the content appears light green for one hour. Cool in air.	Avoid loss of nitrogen by applying heat below liquid level only.
Distillation	Add according to table water or acid to the collecting flask. Place flask so liquid covers the outlet of the condenser. Shield the collecting flask against heat during distillation.
	Connect the Kjeldahl flask to the distillation assembly - flush the glass bulb and the inner neck of the flask with 100 ml distilled water
	Add a small amount of (4.15) pumice and four (4.5) Zn granules. Assemble the apparatus and put on cooling water. Fill 200 ml (4.7) NaOH, 33% (w/w) suitable for Nitrogen determination in the dropping funnel or as much as it takes at a time. Open the valve and add approximately 180 ml (4.7) NaOH to the distillation flask. Heat to boiling. If necessary add more (4.7) NaOH to obtain a brown colour, but avoid excess of (4.7) NaOH. Collect approximately 200 ml distillate.	Ensure the stem of the dropping funnel does not become empty.
Titration	Add immediately indicator according to table one and titrate.
Blank	Carry out a blank test with water, reagents and possible parchment paper.
Cold water soluble protein	Cold water soluble protein in starch: Slurry 200 g starch in distilled water in a 1000 ml volumetric flask. Add distilled water to mark. Transfer to 1000 ml beaker. Stir for 30 minutes at room temperature and filter through filter paper. Discard first 100 ml of filtrate. Transfer 250 ml filtrate to Kjeldahl flask and proceed as above.
Table one	Starch hydro- lysate Glucose syrup, Total Sugar Starch Starch water soluble fraction Various % Protein, approx. 0.02 - 0.5 0.02 - 0.08 0.05 - 0.5 0.01 - 0.04 1-15 15-30 30-80 Sample, g 6.5 DM 8 5   1.5 0.8 1.5 Reagents:               4.1 CuSO4, 5H2O, g 0.5 0.5 0.5 0.5 0.5 0.5 0.5 4.2 Se, g 0.2 0.2 0.2 0.2       4.3 K2SO4, g         15 15 15 4.4 Graphite flakes, g 0.02 0.02 0.02 0.02       4.6 H2SO4, conc. ml 60 60 50 20 25 25 25 Collector pre-filling:               Distilled water, ml 30 30 30 30       4.9 H2SO4, 0.1N ml         30 30   4.11 H2SO4, 0.5N ml             30 Titration               4.8 HCl 0.02N : + + + +       4.10 NaOH 0.1N         + +   4.12 NaOH 0.5N             + Indicator:               4.13 Methyl red/methylene blue + + + +       4.14 Methyl red/bromocresol green         + + + Number of drops 8 8 8 8 4 4 4	As a convenience the method is adapted to "Various" for occasional use. For routine analysis of "Various" dedicated methods should be worked out. Selenium as a catalyst can be replaced by potassium sulphate throughout. DM = Dry Matter
6. Calculation	Calculate protein content of sample by averaging results of two samples with two significant digits:	Protein = N * 6.25
Hydrolysate, Glucose syrup, Total Sugar, Starch	Protein % on dry matter = (a-blankacid) * Nacid*14*100*100*6.25/(g*d*1000)   = (a-blankacid) * Nacid*875/(g*d)
Starch, cold water soluble fraction	Cold water soluble protein % on starch dry matter = (a-blankacid) * Nacid*F
Various	Protein % on dry matter = (blankalkali -t) * Nalkali*875/(g*d)
	Where;
	g = sample weight in g d = sample dry matter in g per 100 g (or Degree Brix)	Degree Brix of hydrolysates and syrups can be used as dry matter
	a = ml acid used to titrate sample blankacid = ml acid to titrated blank Nacid = Normality of acid	blankacid ~ 0 ml
	blankalkali = ml alkali used for titration of blank t = ml alkali used for titration of sample Nalkali = Normality of alkali	blankalkali ~ 30 ml
7. Notes	ISO 3188 are ISO 5378  are simple and recommendable even for routine work.
8. Reference	ISO 3188 (Kjeldahl by Titration); ISO 5378 (Kjeldahl by Spectrophotometric method)
9. Kjeldahl distillation assembly