The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.
This article would be throwing light on Protein Estimation By Kjeldahl Method.
Principle Of Kjeldahl Method
The Kjeldahl procedure measures the nitrogen content of a sample. The protein content then, can be calculated assuming a ratio of protein to nitrogen for the specific food being analyzed. The Kjeldahl procedure can be basically divided into three parts:
In the digestion step, organic nitrogen is converted to an ammonium in the presence of a catalyst at approximately 370°C. In the distillation step, the digested sample is made alkaline with NaOH and the nitrogen is distilled off as NH3 . This NH3 is “trapped” in a boric acid solution. The amount of ammonia nitrogen in this solution is quantified by titration with a standard HCl solution. A reagent blank is carried through the analysis and the volume of HCl titrant required for this blank is subtracted from each determination.
- Sulfuric Acid (concentrated, N-Free)
- Catalyst/Salt Mixture (Kjeldahl digestion tablets) Contains potassium sulfate, cupric sulfate, and titanium dioxide. Note: There are several types of Kjeldahl digestion tablets that contain somewhat different chemicals.
- Sodium Hydroxide Solution, 50%, w/v, NaOH in deionized distilled (dd) water. Dissolve 2000 g sodium hydroxide (NaOH) pellets in ~3.5 L dd water. Cool. Add dd water to make up to 4.0 L
- Boric Acid Solution- In a 4-L flask, dissolve 160 g boric acid in ca. 2 L boiled, and still very hot, distilled water. Mix and then add an additional 1.5 L of boiled, hot distilled water. Cool to room temperature under tap water (caution: glassware may break due to sudden cooling) or leave overnight. When using the rapid procedure, the flask must be shaken occasionally to prevent recrystallization of the boric acid. Add 40 ml of bromocresol green solution (100 mg bromocresol green/100 ml ethanol) and 28 ml of methyl red solution (100 mg methyl red/100 ml ethanol). Dilute to 4 L with water and mix carefully. Transfer 25 ml of the boric acid solution to a receiver flask and distill a digested blank (a digested catalyst/salt/acid mixture). The contents of the flask should then be a neutral gray. If not, titrate with 0.1 N NaOH solution until this color is obtained. Calculate the amount of NaOH solution necessary to adjust the boric acid solution in the 4-L flask with the formula:
Add the calculated amount of 0.1 N NaOH solution to the boric acid solution. Mix well. Verify the adjustment results by distilling a new blank sample. Place adjusted solution into a bottle equipped with a 50-ml repipettor
Standardized HCl solution** Dilute 3.33 ml conc. HCl to 4 L with dd water. Empty old HCl solution from the titrator reservoir and rinse three times with a small portion of the new HCl solution. Fill the titrator with the new HCl solution to be standardized. Using a volumetric pipet, dispense 10 ml aliquots of the THAM solution prepared as described below into three Erlenmeyer flasks (50 ml). Add 3–5 drops indicator (3 parts 0.1% bromocresol green in ethanol to 1 part of 0.2% methyl red in ethanol) to each flask and swirl. Titrate each solution with the HCl solution to a light pink endpoint. Note the acid volume used and calculates the normality as described below.
Calculation to standardize HCl solution
Tris (hydroxymethyl) aminomethane (THAM) Solution – (0.01 N) ** Place 2 g of THAM in a crucible. Leave in a drying oven (95°C) overnight. Let cool in a desiccator. In a 1-L volumetric flask, dissolve 1.2114 g of oven dried THAM in distilled water. Dilute to volume.
- Turn on digestion block and heat to appropriate temperature.
- Accurately weigh approximately 0.1 g corn flour. Record the weight. Place corn flour in digestion tube. Repeat for two more samples.
- Add one catalyst tablet and appropriate volume (e.g., 7 ml) of concentrated sulfuric acid to each tube with corn flour. Prepare duplicate blanks: one catalyst tablet+volume of sulfuric acid used in the sample+weigh paper (if weigh paper was added with the corn flour samples).
- Place rack of digestion tubes on digestion block. Cover digestion block with exhaust system turned on.
- Let samples digest until digestion is complete. The samples should be clear (but neon green), with no charred material remaining.
- Take samples off the digestion block and allow to cool with the exhaust system still turned on.
- Carefully dilute digest with an appropriate volume of dd water. Swirl each tube.
- Follow appropriate procedure to start up distillation system.
- Dispense appropriate volume of boric acid solution into the receiving flask. Place receiving flask on distillation system. Make sure that the tube coming from the distillation of the sample is submerged in the boric acid solution.
- Put sample tube in place, making sure it is seated securely, and proceed with the distillation until completed. In this distillation process, a set volume of NaOH solution will be delivered to the tube and a steam generator will distill the sample for a set period of time.
- Upon completing distillation of one sample, proceed with a new sample tube and receiving flask.
- After completing distillation of all samples, follow manufacturer’s instructions to shut down the distillation unit.
- Record the normality of the standardized HCl solution as determined by the teaching assistant. 2. If using an automated pH meter titration system, follow manufacturer’s instructions to calibrate the instrument. Put a magnetic stir bar in the receiver flask and place it on a stir plate. Keep the solution stirring briskly while titrating, but do not let the stir bar hit the electrode. Titrate each sample and blank to an endpoint pH of 4.
- Record volume of HCl titrant used.
- If using a colorimetric endpoint, put a magnetic stir bar in the receiver flask, place it on a stir plate, and keep the solution stirring briskly while titrating. Titrate each sample and blank with the standardized HCl solution to the first faint gray color. Record volume of HCl titrant used.
Moles of HCl = moles of NH3 = moles of N in the sample
A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen.
% N = N HCl × Corrected acid volume g of sample ×14 g N mol ×100
A factor is used to convert percent N to percent crude protein. Most proteins contain 16% N, so the conversion factor is 6.25 (100/16 = 6.25).
% N/0.16 = % protein
Kjeldahl Method Video
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