ESTIMATION OF SUGAR BY HAGEDORN-JENSON METHOD
The standard sodium is treated with potassium ferricyanide. A part of ferricyanide is reduced by glucose to ferrocyanide. The remaining ferricyanide is determined from the amount of iodine liberated. The ferrocyanide forms a salt with zinc and is not deoxidized to ferricyanide by atmospheric oxygen. The principle reactions are -
2K3 [Fe (CN) 6] + 2KI ⎯⎯→ 2K4 [Fe (CN) 6] + I2
2K4 [Fe (CN) 6] + ZnSO4 ⎯⎯→ K2Zn [Fe (CN) 6] + 3K2SO4
The liberated iodine is estimated by titrating against sodium thiosulfate (0.005 N) using starch as an indicator. The amount of thiosulfate required is noted. The difference between the blank and the sample is measured. This shows the amount of thiosulfate required for sugar. By plotting a graph the concentration of sugar can be calculated.
1. Iodide sulfate chloride solution: 25 gms of ZnSO4 and 12.5 gms of NaCl2 are dissolved in 500 mL of water. To this 12.5 gms of KI is added on the day of the experiment.
2. 3% acetic acid: 3 mL of acetic acid made up to 100 mL.
3. Potassium ferricyanide solution K3 [Fe (CN) 6]: 0.82 gm of K3 [Fe (CN) 6] and 5.3 gms of anhydrous sodium carbonate is dissolved in water. Store it in a dark bottle.
4. Preparation of standard sugar solution = 100 mg of sugar in 100 mL is prepared. Take 10 mL of stock and make it up to 100 mL.
5. 0.1N Na2S2O3 solution: 12.4 gms of sodium thiosulfate in 500 mL of distilled water.
6. Starch indicator: 1 gm of starch in 100 mL of H2O and 5 gms of NaCl.
7. 0.1N K2Cr2O7: 0.49 gm in 100 mL of H2O.
Standardization of Na2S2O3 solution. Add 10 mL 0.1N K2Cr2O7 solution in a conical flask to 1 gm of KI and half of a test tube. H2SO4 1–2 drops of starch indicator and then titrate it against 0.1 N Na2S2O3 solution. The end point is blue to colorless. Seven clean and dry test tubes are used and sugar solutions ranging from 0.0, 0.4, 0.8, to 2 mL are added it is made up to 2 mL by adding distilled water. It produces 0–200 mg of sugar solution. Add 3 mL of potassium ferricyanide to each of the conical flasks. Boil the contents for 15 minutes and cool to room temperature. Then add 3 mL of iodine sulfate chloride solution and shake well. Add 2 mL of 3% acetic acid just before titrating against 0.005 N Na2S2O3 using the starch indicator. Titrate until the blue color disappears. Note the reading substrate from the first value. The values are plotted against the concentration of sugar solution from this graph. Concentration of unknown sugar is determined.
Result: The unknown solution contains ................ mg of sugar.
ESTIMATION OF BLOOD GLUCOSE
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.
1. 0.45% zinc sulfate: prepared fresh every week by dilution of 45% stock solution.
2. 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. 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.
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, was 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.