General Provisions Standard for Manufacturing and
Preparation General Standards for Food
Additive use in Foods Specification and Standards General Test Methods Reagent, Solution, Standard Solution
for Quantitative Analysis and
Standard Solution Appendix
Name
Content
Definition
Description
Identification
Purity
Assay
Permitted Use Level
All
1. Gas Chromatography
2. Residue on Ignition
3. Loss on Drying and Loss on Ignition
4. Refractive Index
5. Lead Limit Test (Dithizone Method)
6. Methoxyl Determination
7. Thin Layer Chromatography
8. Quantitative Test for Generated Gas
9. Arsenic Limit Test
10. Boiling Point and Amount of Distillate
11. Specific Gravity
12. Optical specific rotation
13. Water Determination (Karl Fischer Method)
14. Paper Chromatography
15. Softening Point Measurement
16. Flame Coloration Test
17. Test Methods for Chloride and Sulfate Slats
18. Thermometers
19. Atomic Absorption Spectrophotometry
20. Assay for Alkali Salt of Organic Acid
21. Melting Point
22. Congealing Point
23. Infrared Spectrophotometry
24. Viscosity
25. Heavy Metal Limit Test
26. Nitrogen Determination
27. pH Determination
28. Identification
29. Readily carbonizable substances test
30. Ash and Acid-Insoluble Ash Limit Test
31. Spectrophotometry
32. Pigment Test Methods
33. Coloring Matter Aluminum Lake Test
34. Flavoring Substances Test
35. Oils Test
General Test Methods > Lead Limit Test (Dithizone Method)

5. Lead Limit Test (Dithizone Method)

Unless otherwise specified, the lead limit test by dithizone is carried out by one of the following methods. In this test, water with lowest possible lead content is used. Glass apparatus should be washed with diluted warm nitric acid (1→2) followed by water.

A. Solution
  • Amonium citrate solution : 45 g of ammonium citrate (II) are dissolved in 100 ml of water, where 2~3 drops of phenol red solution are added. Ammonia water is drop-wise added to this solution until the solution becomes red. To remove lead from this solution, it is extracted with 20 ml each of dithizone solution until the color of dithizone solution becomes its characteristic green.
  • Potassium cyanide solution : 50 g of potassium cyanide are dissolved in 100 ml of water. This solution is processed with dithizone solution by following the same procedure as in the previous ammonium citrate solution section. The residual dithizone is extracted out from the solution with chloroform. Water is added to the resulting solution to make the total volume to 500 ml.
    Ammonia potassium cyanide solution : To 20 ml of potassium cyanide solution in the previous section, 15 ml of ammonia water and water are added to make the total volume to 100 ml.
  • hydroxylamine hydrochloridesolution : Hydroxylamine hydrochloride (20 g) is dissolved in water (total volume = approximately 65 ml), where 2~3 drops of thymolblue solution are added. Ammonia water is added to the resulting solution until the color turns yellow, where 10 ml of 4% sodium diethyldithio carbamate solution is added and set aside for 5 minutes. It is then extracted with 10~15 ml each of chloroform. At the end point, a mixture of 5 ml of the extract and 5 drops of cupric sulfate solution (1→100) does not becomes yellow color. To the aqueous phase, 2~3 drops of thymol blue solution, where dilute nitric acid is added until the solution turns red. The total volume of the solution is make to 100 ml with water.
  • Dthizone solution for extraction : Dithizone (30 mg) is dissolved in 1,000 ml of chloroform, where 5 ml of alcohol is added. The solution is stored in a cold place. Before use, necessary amount of the solution is mixed with approximately a half the volume of 1% nitric acid. Water layer is removed from the mixture.
  • Dthizone standard solution : Dithizone (10 mg) is dissolved in 1,000 ml of chloroform. It is then stored in a light shielded lead free bottle in a cold place.
  • Lead standard solution (for dithizone) : Lead standard solution (10 ml) is diluted to 100 ml with 1% nitric acid. This solution is prepared just before use. It contains 1 μg of Pb per 1 ml.

    B. Procedure
    Unless otherwise specified, a specified amount of test solution is taken into a separatory funnel, where 6 ml ammonium citrate solution (10 ml for lead limit test in iron salt), 2 ml of potassium cyanide solution, 2 ml of hydroxylamine hydrochloride solution, and 2 drops of phenol red solution are added. Ammonia water is added until the solution turns red. To this solution, 5 ml of dithizone solution for extraction is added and the solution is shaken for approximately 15 seconds. The chloroform phase is transferred into another separatory funnel. This extraction is repeated three times. The collected chloroform extracts are mixed well by shaking for 30 seconds with 20 ml of 1% nitric acid and the chloroform layer is discarded. With small amount of chloroform, acid solution is washed several times (if necessary) to completely remove dithizone. Small drops of chloroform are removed from the surface of acid solution. To this acid solution,  5 ml of dithizone standard solution and 4 ml of potassium cyanide solution are added and mixed for 30 seconds by shaking. The violet color in the chloroform layer should not be deeper than that of the solution, which is prepared by the same procedure with 8 ml of lead standard solution (for dithizone) instead of sample.