Method for Determination of Iodine in Double Fortified Salt

(Quantitative)

Method No.
FSSAI.FS.16.011.2023 Revision No. & Date 0.0

Scope The iodine content can be measured by conventional iodometric titration using
sulphuric acid, but H2SO4 interferes with the estimation of iodine leading to
erroneous results. Hence a modified method with orthophosphoric acid has been
validated for the estimation of iodine in DFS.
Caution Caution should be taken while preparing the solutions and also while analyzing
the samples.
Principle Iodine estimation by Titration Method.
The Iodine content in DFS is measured by a modified iodometric titration.
Apparatus/Instruments Weighing balance

Materials and Reagents Materials

1. Burette
2. Erlenmeyer flask with stopper, 250 mL
3. Beakers, 250mL and 500 mL
4. Pipettes
Reagents
1. 1. Potassium Iodide
2. 2. Orthophosphoric acid
3. 3. Sodium thiosulphate
4. 4. Starch
5. 5. Sodium chloride
6. 6. Potassium iodate
7. 7. Double distilled water
Preparation of Reagents 1. Potassium Iodide, KI (1% solution):
Dissolve 1 g of KI (LR grade) in 100 mL water. Store in a cool, dark place.
The solution is stable for at least 3 months if stored properly.
2. Orthophosphoric acid(H3P04), 4 N:
Slowly add 75.4 mL of AR grade orthophosphoric acid to 900 mL of
ice-cold distilled water. Dilute and make to 1000 mL with water. The volume
is sufficient for 200 samples. The solution is stable.
Note: Always add acid to water dropwise, not water to acid. Stir the solution
while adding acid.
3. Sodium thiosulphate (Na2S2O3), 0.0005M:
Dissolve 1.24 g Na2S2O3.5H2O (AR grade) in 1000 mL water. Store in a cool
place. This volume is sufficient for nearly 200 samples. The solution is stable
at least for 1 month, if stored properly.
4. Starch indicator solution:
4a. Preparation of saturated NaCl solution
Make 100 mL of a saturated NaCl solution, by adding NaCl in small
quantities at a time, to approximately 80 mL water in a beaker, with stirring
and heating, until no further Nacl dissolves. This solution is stable for one
year.

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 5. Preparation of Starch:
Weigh one gram soluble starch (potato, extra pure/LR grade) into a
100 mL beaker, add 10 mL water and make a paste, heat to dissolve. Add
saturated NaCl solution to the hot starch solution to make to 100 mL. Store in
a cool, dark place. This volume is sufficient for 200 samples. The solution is
stable for up to one month, and should be heated (not boiled) each day before
use to resuspend any solids.
6. Standard KIO3 :
Weigh accurately 0.167 g of KIO3 (AR grade) and dissolve in water in a
standard measuring flask (100 mL) and make up the volume to 100 mL. This
will give a concentration of 1 mg of iodine/mL.
Sample Preparation A. DFS Sample Preparation:
a. Weigh accurately 10 g DFS into a 250 mL Erlenmeyer flask with stopper
b. Add 0.5 mL of 1% KI (CAUTION: Do not pipette by mouth)
c. Add 46 mL of water. Swirl the flask to dissolve the salt.
d. Add 5 mL of 4 N H3PO4. The solution will turn yellow if iodine is present.
e. Stopper the flask and put it in the dark (cup board) for 10 min.
(Caution: The reaction mixture should be kept in the dark before
titration because a side reaction can occur when exposed to light that causes
iodide ions to be oxidized to iodine)
B. Standard KIO3:
Run standard KIO3 (1 mg iodine/mL) with 10 g of non-iodized salt as part of
quality control. Take 46 mL of water into a 250 mL Erlenmeyer flask with
stopper. Add 1 mL of standard KIO3 (1mg of iodine/mL) and 10 g of non-
iodized salt. Add 0.5 mL 1% KI. Add 5 mL 4 N H3PO4. Stopper the flask and
put in the dark for 10min.
Precautions: Inaccurate results may occur if starch solution is used while still
warm. If starch indicator is added too early, a strong iodine-starch complex is
formed which reacts slowly and gives falsely elevated results. The reaction
should be performed at room temperature (< 30 ° C), as iodine is volatile and the
indicator solution will lose sensititvity when exposed to high temperature.
Method of analysis Sample Analysis
a. Rinse and fill the burette with 0.005 M Sodium thiosulphate and adjust the
level to zero.
b. Remove the flask from the dark and titrate against Na2S2O3 from the burette
until the solution turns pale yellow (straw yellow)
c. Add 0.5 mL of starch indicator solution and continue titration until the
solution becomes colorless.
d. Record the volume of thiosulfate in the burette and convert to ppm using the
“conversion table”. Refer to conversion table for iodine content.
Standard KIO3 Analysis
i. Rinse and fill the burette with 0.005 M Sodium thiosulphate and adjust the
level to zero.
ii. Then titrate the standard KIO3 solution against 0.005 M Na2S2O3 (repeat
steps b to d as indicated above) to calculate the iodine content. This will give
an iodine value of 100 ppm (100 µg/g).
Calculation with units of The unit of expression is µg/g (ppm)
expression
Inference NA, Quantitative Analysis

2
(Qualitative Analysis)
Reference S. Ranganathan & M. G. Karmarkar, Indian Journal of Medical Research 123,
April 2006, p,531-540; Estimation of Iodine in salt fortified with Iodine & Iron
Approved by Scientific Panel on Methods of Sampling and Analysis

3
 Method for Determination of Iron in Double Fortified Salt
(Quantitative)

Method No.
FSSAI.FS.16.012.2023 Revision No. & Date 0.0

Scope This method is used for the estimation of Iron calorimetrically in Double Fortified Salt
(DFS).
Caution Caution should be taken while preparing the solutions and also while analyzing the
samples.
Principle Iron is determined calorimetrically by the principle that ferric ion (Fe3+) gives a blood
red color with potassium thiocyanate (KCNS).
Apparatus/Instruments 1. Weighing Balance
2. Colorimeter
Materials and Reagents 1. Sulphuric Acid
2. Potassium Persulphate
3. Potassium thiocyanate
4. Standard Iron solution
5. Working standard solution
Preparation of Reagents 1. Sulphuric Acid, H2SO4 (30%):
Take 60 mL distilled water in a beaker. Keep in an ice bath and add slowly drop-wise
30 mL of concentrated H2SO4 with constant stirring. Make the volume to 100 mL
with distilled water.
2. Potassium persulphate, K2S2O8 (7%):
Dissolve seven grams of K2S2O8 in distilled water and make up the volume to 100
mL with distilled water.
3. Potassium thiocyanate, KCNS (40%):
Dissolve 40 g of KCNS in 90 mL distilled water. Add four mL acetone and make up
the volume to 100 mL.
4. Standard Iron Solution:
Dissolve 702.2 mg ferrous ammonium sulphate in 100 mL distilled water. Add five
mL of 1:1 hydrochloric acid (HCL) and make up the volume to 100 mL (0.1 mg/mL).
The standard solution is prepared fresh and can be kept for 6 months. From this
prepare the working standard.
4a. Working Standard (10 µg iron/mL):
Dilute 10 mL of the standard iron solution (0.1 mg/mL) to 100 mL with distilled
water. This will give 1010 µg iron/mL concentration.
Sample Preparation Take one gram of DFS in a 100 mL standard measuring flask using a glass funnel. Add
2.5 mL of concentrated HCL and make up the volume to 100 mL with distilled water.
Mix and use 1 mL – 2 mL aliquots for the estimation of iron as given below.
Reagent Test Test Test Test Test Test Test Test
Tube Tube Tube Tube Tube Tube Tube Tube
1 2 3 4 5 6 7 8
Distilled water (mL) 6.5 5.5 4.5 6.0 5.5 4.5 3.5 2.5
Iron working
0 0 0 0.5 1.0 2.0 3.0 4.0
standard(mL)
DFS Solution(mL) 0 1.0 2.0 0 0 0 0 0
30% H2SO4(mL) 1 1 1 1 1 1 1 1

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 7% K2S2O8(mL) 1 1 1 1 1 1 1 1
40% KCNS(mL) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Method of analysis Prepare the test tubes as above adding all other solutions except 40% KCNS solution.
Add 40% KCNS solution just before taking the readings. Measure the red color
developed within 20 min of addition of 40% KCNS at 540 nm.
Calculation with units of Draw a standard graph of the iron standards by taking iron concentration (µg) on the X-
expression axis and the OD on the Y-axis and calculate the iron content from the standard graph.
Inference NA, Quantitative Analysis
(Qualitative Analysis)
Reference Wong, SY, Hawk’ s. Physiological Chemistry,14th Edition, New York: McGraw Hill,
1965, page 1094
Approved by Scientific Panel on Methods of Sampling and Analysis

2
 Method for Determination of
Phosphorus as (P2O5) in Fortified Salt

Method No. FSSAI.FS.16.013.2023 Revision No. & Date 0.0

Scope Method for Determination of Phosphorous as (P2O5) in Fortified Salt

Caution 1. Sodium molybdate: It May cause eye, skin, and respiratory tract
irritation. May be harmful if swallowed, inhaled, or absorbed through the
skin.
2. Hydrazine sulfate: Hydrazine sulfate is a hazardous chemical. It May
irritate and burn the eyes and skin. Breathing Hydrazine Sulfate can irritate
the nose, throat and lungs causing coughing and shortness of breath.
Exposure can cause dizzy and lightheaded. Higher levels can cause
trembling, a feeling of excitement, and even convulsions.
Principle The method determines Phosphorous as (P2O5) in Fortified Salt in the
presence of Hydrazine sulfate and Sodium molybdate followed by
spectrophotometer measurement of phosphorous as blue phosphomolybdic
acid.
Apparatus/Instruments 1. General glassware and apparatus
2. Volumetric flasks – 50 mL, 100 mL, 250 mL and 500 mL with glass
stoppers
3. Pipette – Mohr ‘s type 10 mL with 0.1 mL subdivision.
4. Spectrophotometer with 1.0 cm cuvettes. For use in the visible region
Materials and Reagents 1. Sodium molybdate, reagent grade
2. Hydrazine sulphate, reagent grade
3. Potassium dihydrogen phosphate, reagent grade dried for 2 h at 101°C
4. Distilled Water
Preparation of Reagents 1. Sodium molybdate - Carefully add 140 mL of concentrated sulphuric acid
to 300 mL distilled water. Cool to room temperature and add 12.5 g of
Sodium molybdate. Dilute to 500 mL with distilled water. Mix thoroughly
and allow to stand for 24 h before use.
2. Hydrazine sulphate – 0.015% Dissolve 0.150 g hydrazine sulphate in 1 L
water.
4. Standard Phosphate solution: Stock solution(A) – Dissolve 1.0967 g of
dry Potassium dihydrogen phosphate in distilled water and make up to 250
mL in a volumetric flask The solution contains 1 mg phosphorous per Ml
5. Working Solution (B) – Dilute 5 mL of standard stock solution A with
distilled water to 500 mL in a volumetric flask. This solution contains 0.01
mg phosphorous per mL.
Preparation of the standard curve: (0, 1, 2, 4, 8 & 10 µg/mL):
Pipette 0.0, 1.0, 2.0, 4.0, 8.0 and 10.0 mL of standard working solution into
50 mL volumetric Flasks & dilute to 10mL with water.
Sample Preparation 1.Weigh accurately 3 – 4 gm of sample in 100mL Volumetric Flask.
2.Dilute to volume with water and mix thoroughly.
Method of analysis 1.Take 10 mL sample solution in clean 50 mL volumetric flask.

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 2.Add 8 mL of hydrazine sulphate solution and 2 mL of sodium molybdate
solution in this order in standard solution and sample solution.
3.Stopper and invert 3 – 4 times. Loosen the stopper and heat for 10 ± 0.5
minutes in a vigorously boiling water bath.
4.Remove from water bath and cool at room temperature.
5.Make the volume upto 50 ml with distilled water and mix thoroughly.
6.Transfer the solution to a clean dry cuvette and measure the absorbance
at 650 nm in a spectrophotometer adjusted to read 0 % absorbance (100 %
transmittance) for distilled water.
7.Plot curve the absorbance of each standard against its phosphorous
content on a linear graph paper.
8.Measure the phosphorus content of the sample and the blank by
comparison with the standard curve.

Calculation with units of Concentration from calibration curve (µg/mL) X Volume

expression made X 141.94
Phosphorous as P2O5 = ------------------------------------------------------------------
(mg/Kg) Sample Weight (g) X 30.97

Inference *****
(Qualitative Analysis)
Reference FSSAI 02.038:2021 of FSSAI Manual of methods of analysis of Food (oil and
Fat)2021
Approved by Scientific Panel on Methods of Sampling and Analysis