Estimation of sugar by HAGEDORN-JENSON method &

estimation of blood sugar level by HJ method

ESTIMATION OF SUGAR BY HAGEDORN-JENSON METHOD
Principle
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.
Reagents Required
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.
Procedure

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 12 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 0200 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

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

2.
3.
4.
5.
6.

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.
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.
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.
3% acetic acid.
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-sulfatechloride solution, followed by 2 mL 3% acetic acid, was added.
4. The liberated iodine was then titrated against 0.005 N Na2S2O3 using
23 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.