Determination of Blood Glucose by Hagedorn-Jenson Method

Principle
Blood proteins are precipitated with zinc hydroxide. The reducing sugars in the protein-free filtrate reduce potassium ferricyanide on heating. The amount of unreduced ferricyanide is determined iodimetrically.

Reagents

1. 0.45% zinc sulfate: prepared fresh every week by dilution of 45% stock solution.
2. 0.1 N NaOH: prepared fresh every week by dilution of 2 N NaOH.
3. Potassium ferricyanide solution: 1.65 gms crystallized potassium ferricyanide and 10.6 gms anhydrous sodium carbonate were dissolved in 1 liter of water and stored in a dark bottle, protected from light.
4. 4. Iodide-Sulfate-Chloride Solution: zinc sulfate 10 gms and NaCl 50 gms were dissolved in 200 mL of water. On the day of use, 5 gms potassium iodide is added to the solution.
5. 3% acetic acid.
6. 0.005 N sodium thiosulfate: prepared fresh daily by diluting 0.5 N Na2S2O3. 0.5 N Na2S2O3 was prepared by dissolving 70 gms of salt in 500-mL water. It was better if a solution of slightly higher normality was prepared since sodium thiosulfate decomposes rapidly. The solution should be protected from light and stored in the cold. The normality was checked daily with 0.005 N potassium iodate.
7. 0.005 N potassium iodate: This is a stable solution and should be made accurately. 0.3566 gm of the anhydrous salt was weighed accurately and dissolved in 2 liters of H₂O. That was used to check Na2S2O3 and potassium ferricyanide solution.
8. Starch indicator: 1 gm soluble starch was dissolved in 100 mL saturated NaCl solution.

Procedure

1. One-mL 0.1 N NaOH and 5 mL 0.45% zinc sulfate were pipetted into a test tube. 0.1-mL blood was taken in a dry pipette and introduced into the gelatinous zinc hydroxide in the test tube, rinsing out the pipette twice with the mixture. The tube was kept in boiling water for 3 minutes and cooled without disturbing the precipitate. The mixture was filtered through a Whatman No. 42 filter paper of lightly pressed moisture cotton.
2. The tube was washed twice with 3-mL portions of water and filtered into the same containers.
3. Two-mL potassium ferricyanide was then added to the filtrate and heated in boiling water for 15 minutes. After cooling, 3-mL iodide-sulfate-chloride solution, followed by 2 mL 3% acetic acid, were added.
4. The liberated iodine was then titrated against 0.005 N Na2S2O3 using 2–3 drops of 1% starch as an indicator toward the end of the titration.
5. A blank was run through the entire procedure simultaneously. The blank should yield a liter value of 1.97 to 2.00 mL.

Result:
The concentration of blood glucose in given sample was .............. mg of glucose.