Inorganic Constituents AACC Method 40-20Page 1 of 2

Calcium in Bread and FlourFinal approval May 5, 1960; Reapproval November 3, 1999

ObjectiveThis method determines the calcium content of flour and bread by a titrimetric

procedure.

Apparatus1. Platinum, silica, or porcelain ashing dishes, about 60-mm diameter, 35-ml

capacity. Porcelain evaporating dishes of about 25-ml capacity are satisfactory.Do not use flat-bottom dishes of greater diameter than 60 mm.

2. Muffle furnace capable of operating at 550°.3. Filter (choose one of the following):

a. Quantitative filter paper, medium speed; Whatman No. 40 or equivalent.b. Fritted glass crucible, fine porosity.c. Microporous porcelain filter crucible, medium porosity.

4. Glassware as required.5. Acid-washed ashless filter paper.

Reagents1. Ashing aid.

a. Magnesium nitrate solution. Dissolve 50 g Mg(NO3)2·6H2O in water anddilute to 100 ml. Or:

b. Redistilled HNO3.2. Oxalic acid solution, 3%.3. Potassium permanganate, 0.05N. Prepare and standardize as directed in

Method 70-50A.4. Bromcresol green indicator, 1%.5. Sodium acetate solution, 20%.6. Ammonium hydroxide (1 + 50).7. Hydrochloric acid (HCl), concentrated, ACS grade.8. Sulfuric acid (H2SO4), concentrated, ACS grade.

Procedure1. Weigh 10.0 g prepared sample (for bread, Method 62-05) into ashing dish

that has been ignited, cooled in desiccator, and weighed soon after attainingroom temperature. Begin preparation of reagent blank at this point.

2. Incinerate in furnace at about 550° (dull red) until light-gray ash results, orto constant weight. See Note.

3. Cool in desiccator and weigh soon after room temperature is attained, ifpercent ash is desired.

4. Add 5 ml HCl, allowing acid to rinse upper portion of dish; evaporate todryness on steam bath.

Inorganic Constituents AACC Method 40-20Page 2 of 2

Calcium in Bread and Flour (continued)

5. Dissolve residue by adding, accurately measured, 2.0 ml HCl; heat 5 min onsteam bath with watch glass on dish.

6. Wash off watch glass with water; then filter through fast acid-washedashless filter paper into 400-ml beaker. Dilute to about 150 ml.

7. Add 8–10 drops bromcresol green indicator and sufficient 20% sodiumacetate solution to change pH to 4.8–5.0 (blue). Cover with watch glass and heatto boiling.

8. Precipitate calcium slowly by adding 3% oxalic acid solution, 1 drop every3–5 sec, until pH is changed back to 4.4–4.6 (optimum for calcium oxalate pre-cipitation) as indicated by appearance of distinct green shade. (Change of colorindicates excess of oxalic acid—more would develop yellow tints, showingundesirable displacement of pH.)

9. Boil 1–2 min and let mixture settle until clear, or overnight.10. Filter supernatant liquid through quantitative filter paper, fritted glass

filter, or microporous crucible.11. Rinse beaker and transfer precipitate to filter with about 50 ml NH4OH (1

+ 50) in small portions, using wash-bottle delivering very small stream. Discardfiltrate.

12. Dissolve precipitate by washing filter or crucible with mixture of 125 mlwater and 5 ml H2SO4 at 80–90°.

13. Titrate filtrate at 70–90° with 0.05N KMnO4 until slight pink color isobtained, add filter paper or filter, and continue titration if necessary.

14. Correct for blank.

CalculationCalculate calcium as mg/lb; 1 ml 0.05N KMnO4 = 1 mg calcium.

NoteTo diminish ashing time, or for samples that do not burn practically carbon-

free, use one of the following ashing aids: Moisten ash with a) 0.5–1.0 ml mag-nesium nitrate solution or b) redistilled HNO3. Dry contents and carefully ignitein muffle to prevent spattering. (A white ash with no carbon results in mostcases.) Do not add these ashing aids to self-rising flour (products containingNaCl) in platinum dish, because of vigorous action on dish.

ReferenceAOAC International. 1995. Official Methods of Analysis of AOAC International, 16th ed. Methods

944.03 and 945.41. The Association, Arlington, VA.