INDIGOTINE
Prepared at the 28th JECFA (1984), published in FNP 31/1 (1984) and in
FNP 52 (1992). Metals and arsenic specifications revised at the 59th
JECFA (2002). An ADI of 0-5 mg/kg bw was established at the 18th
JECFA (1974)
SYNONYMS CI Food Blue 1, FD&C Blue No. 2, Indigo Carmine, CI (1975) No. 73015,
INS No. 132
DEFINITION Consists essentially of a mixture of disodium 3,3' -dioxo-[delta2,2'-
biindoline]-5,5'-disulfonate, and disodium 3,3'-dioxo-[delta2,2'-biindoline]-
5,7'-disulfonate and subsidiary colouring matters together with sodium
chloride and/or sodium sulfate as the principal uncoloured components.
May be converted to the corresponding aluminium lake in which case only
the General Specifications for Aluminium Lakes of Colouring Matters
apply.
Chemical names Disodium 3,3'-dioxo-[delta2,2'-biindoline]-5,5'-disulfonate
C.A.S. number 860-22-0 (5,5' isomer)
Chemical formula C16H8N2Na2O8S2
Structural formula
Formula weight 466.36
Assay Not less than 85% total colouring matters.
Not more than 18% of disodium 3,3'-dioxo-[delta2,2'-biindoline]-5,7'-
disulfonate
DESCRIPTION Blue powder or granules
FUNCTIONAL USES Colour
CHARACTERISTICS
IDENTIFICATION
Solubility (Vol. 4) Soluble in water; sparingly soluble in ethanol
Identification of colouring Passes test
matters (Vol. 4)
PURITY
Loss on drying at 135o Not more than 15% together with chloride and sulfate calculated as
sodium salts
Water insoluble matter Not more than 0.2%
(Vol. 4)
Lead (Vol. 4) Not more than 2 mg/kg
Determine using an atomic absorption technique appropriate to the
specified level. The selection of sample size and method of sample
preparation may be based on the principles of the method described in
Volume 4, “Instrumental Methods.”
Subsidiary colouring Not more than 1% (except disodium 3,3'-dioxo-[delta2,2'-biindoline]- 5,7'-
matters (Vol. 4) disulfonate)
Use the following conditions:
Developing solvent: No. 3
Height of ascent of solvent front: approximately 17 cm
Note 1. The 5,7' isomer is separated as a wide blue zone just in front of
the main blue band. Do not include this zone in the subsidiary colouring
matter zones which are cut out and measured.
Note 2. The 15 ml sodium hydrogen carbonate solution used in the
general procedure is replaced by 15 ml 0.05 N hydrochloric acid in order
to avoid the decomposition which the sulfonated indigo undergoes in
alkaline solution.
Organic compounds other Not more than 0.5% of sum of isatin-5-sulfonic acid, 5-sulfoanthranilic acid
than colouring matters and anthranilic acid
(Vol. 4) Use liquid chromatography under the following conditions:
HPLC elution gradient: 2 to 100% gradient followed by elution at 100%
Unsulfonated primary Not more than 0.01% calculated as aniline
aromatic amines (Vol. 4)
Ether extractable matter Not more than 0.2%
(Vol. 4) Weigh accurately about 2 g sample instead of the 5 g stated in the general
methods
METHOD OF Proceed as directed under Total Content by Titration with Titanous
ASSAY Chloride in Volume 4, using the following:
Weight of sample: 1.0-1.1 g
Buffer: 15 g sodium hydrogen tartrate
Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3:
0.02332 g
Isomer content by paper chromatography
Refer to the conditions for the determination of subsidiary colouring
matters (above). Cut the isomer band from the chromatogram in the
manner detailed for the subsidiary bands, extract into solvent and
measure the absorbance at its Smax. Measure the absorbance of the
corresponding blank at the same wavelength. As a standard use 0.1 ml of
an 0.20% solution of the sample applied to the 18 cm x 0.7 cm rectangle.
Isomer expressed as a percentage of the sample =
[A/As] x 20% x [D/100]
where A and As are the net absorbances of the isomer and standard,
respectively, and D is the total colouring matters content of the sample.
Isomer content by HPLC
The 5,7' isomer separates under the HPLC conditions detailed above for
the separation of subsidiary colouring matters, and the amount present
can be quantified.
