1 Determination of Protein in Bread 4

Method 9.1 Determination of Protein in Bread 4

Description of the Method. This quantitative method of analysis for proteins is based on a determination of the %w/w N in the sample. Since different cereal proteins have similar amounts of nitrogen, the experimentally determined %w/w N is multiplied by a factor of 5.7 to give the %w/w protein in the sample (on average there are 5.7 g of cereal protein for every gram of nitrogen). As described here, nitrogen is determined by the Kjeldahl method. The protein in a sample of

bread is oxidized in hot concentrated H 2 SO 4 , converting the nitrogen to NH 4 + . After making the solution alkaline, converting NH 4 + to NH 3 , the ammonia is distilled into

a flask containing a known amount of standard strong acid. Finally, the excess strong acid is determined by a back titration with a standard strong base titrant.

Procedure. Transfer a 2.0-g sample of bread, which has previously been air

Representative Methods dried and ground into a powder, to a suitable digestion flask, along with 0.7 g of

HgO as a catalyst, 10 g of K 2 SO 4 , and 25 mL of concentrated H 2 SO 4 . Bring the

solution to a boil, and continue boiling until the solution turns clear, and for at

The photo in Colorplate 8a shows the

least an additional 30 min. After cooling to below room temperature, add 200 mL

indicator’s color change for this titration.

of H 2 O and 25 mL of 4% w/v K 2 S to remove the Hg 2+ catalyst. Add a few Zn

granules to serve as boiling stones, and 25 g of NaOH. Quickly connect the flask to

a distillation apparatus, and distill the NH 3 into a collecting flask containing a

known amount of standardized HCl. The tip of the condenser should be placed below the surface of the strong acid. After the distillation is complete, titrate the excess strong acid with a standard solution of NaOH, using methyl red as a visual indicator.

Questions

1. Oxidizing the protein converts the nitrogen to NH 4 + . Why is the amount of nitrogen not determined by titrating the NH 4 + with a strong base?

There are two reasons for not titrating the ammonium ion. First, NH 4 + is a very weak acid (K a = 5.7 × 10 –10 ) that yields a poorly defined end point when titrated

with a strong base. Second, even if the end point can be determined with acceptable accuracy and precision, the procedure calls for adding a substantial

amount of H 2 SO 4 . After the oxidation is complete, the amount of excess H 2 SO 4 will be much greater than the amount of NH 4 + that is produced. The presence

of two acids that differ greatly in concentration makes for a difficult analysis. If

the titrant’s concentration is similar to that of H 2 SO 4 , then the equivalence point volume for the titration of NH 4 + may be too small to measure reliably. On the other hand, if the concentration of the titrant is similar to that of NH 4 + , the volume needed to neutralize the H 2 SO 4 will be unreasonably large.

2. Ammonia is a volatile compound as evidenced by the strong smell of even dilute solutions. This volatility presents a possible source of determinate error. Will this determinate error be negative or positive?

The conversion of N to NH 3 follows the following pathway N → NH 4 + NH 4 + → NH 3

Any loss of NH 3 is loss of analyte and a negative determinate error.

—Continued

298 Modern Analytical Chemistry

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3. Discuss the steps taken in this procedure to minimize this determinate error.

Three specific steps are taken to minimize the loss of ammonia: (1) the solution is cooled to below room temperature before adding NaOH; (2) the digestion flask is quickly connected to the distillation apparatus after adding NaOH; and (3) the condenser tip of the distillation apparatus is placed below the surface of the HCl to ensure that the ammonia will react with the HCl before it can be lost through volatilization.

4. How does K 2 S remove Hg 2+ , and why is this important?

Adding sulfide precipitates the Hg 2+ as HgS. This is important because NH 3 forms stable complexes with many metal ions, including Hg 2+ . Any NH 3 that is complexed with Hg 2+ will not be collected by distillation, providing another source of determinate error.