Method of Analysis for Total Coloring Matter

Two methods are used for the determination of total colouring matters:

1. Colouring Matters Content by Spectrophotometry
2. Colouring Matters Content by Titration with Titanous Chloride

When using the spectrophotometric method, all colours present in the sample that absorb in the same region as that of the main colour will contribute to the absorbance figure used to calculate the results; subsidiary colouring matters of markedly different hues will not be accounted for by this method.

The titanous chloride reduction method assumes that isomers and subsidiary colouring matters have the same titanous chloride equivalent as the main colouring matter.

Colouring Matters Content by Spectrophotometry

Three experimental procedures are described. 

1. Procedure 1 is used for water-soluble colouring matters. 
2. Procedure 2 is used for organic solvent-soluble colouring matters. 
3. Procedure 3 is used for lakes. 

Information about the wavelength of maximum absorbance, absorptivity or specific absorbance necessary for determination of percent colouring matters for the lakes of synthetic colours is included in the below Table.

Principle

The absorbance of a solution of the colouring matter is determined at its wavelength of maximum absorption and the total colouring matter content is calculated using the standard absorptivity or specific absorbance value provided in the below Table.

Apparatus used

1. UV-visible spectrophotometer capable of accurate measurement of absorbance in the region of 350 - 700 nm with an effective slit width of 10nm or less
2. Spectrophotometer cells, 1 cm path length

Procedure 1 – Colouring matters content of water-soluble colouring matters

• Accurately weigh 0.25 g of the sample (W). Transfer to a 1 liter volumetric flask. Add the solvent prescribed in the Table and swirl to dissolve. Makeup to volume and mix. Dilute the solution with the same solvent to obtain an absorbance between 0.3 and 0.7. 
• Measure the absorbance (A) at the wavelength of maximum absorption using the prescribed solvent as the blank.

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Calculation

Calculate the total colouring matter content using either of the following equations:

Procedure 2 – Colouring matters content of organic solvent-soluble colouring matters

Reagents

1. Chloroform, reagent grade, acid-free
2. Cyclohexane, reagent grade

• Accurately weigh 0.08 g of the sample (W) into a 100 ml volumetric flask (V1). Add 20 ml of chloroform and dissolve by swirling briefly. Make sure that the solution is clear. Makeup to volume with cyclohexane and mix. 
• Pipet 5.0 ml of the solution (v1) into a second 100 ml volumetric flask (V2) and make up to volume with cyclohexane. Pipet 5.0 ml of this diluted solution (v2) into the final 100 ml volumetric flask (V3) and make up to volume with cyclohexane. 
• Measure the absorbance (A) of the twice diluted solution at the wavelength of maximum absorption in a 1 cm cell, using cyclohexane as the blank.

NOTE: Perform this procedure promptly, avoiding exposure to air insofar as possible and undertaking all operations in the absence of direct sunlight.

Calculation

Calculate the total colouring matter content using either of the following equations:

Procedure 3 – Colouring matters content of lakes

Reagents

Potassium dihydrogen phosphate, reagent grade

Sodium hydroxide, reagent grade

Phosphoric acid, reagent grade

Hydrochloric acid, reagent grade

Prepare 0.1 M phosphate buffer pH 7 

• Weigh 13.61 g of potassium dihydrogen phosphate into a 2000 ml beaker and dissolve in about 900 ml of water. Add about 90 ml of 1 N sodium hydroxide.  
• Measure the pH using a pH meter and adjust the pH to 7.0 using 0.1 N sodium hydroxide or diluted phosphoric acid. Make to volume in a 1 liter volumetric flask.
• Accurately weigh a quantity of lake which will give an absorbance approximately equal to that of the parent colour when the latter is tested according to Procedure 1, above. Transfer to a 250 ml beaker containing 10 ml hydrochloric acids previously diluted with water to approximately 50 ml.

• Heat with stirring to dissolve the lake, and then cool to ambient temperature. Transfer to a 1-litre volumetric flask, makeup to volume with pH 7 phosphate buffer, and mix. 
• Proceed as detailed in Procedure 1, above, and in the specification monograph, using the values for wavelength of maximum absorbance and absorptivity or specific absorbance included in Table 1, and using the phosphate buffer as the spectrophotometric blank.

JECFA Colour	Wavelength of Maximum Absorbance (nm)	Absorptivity	Specific Absorbance	Solvent
Allura Red AC	500 (FDA), 504 (EU), 502 (FCC), 497-501 (Japan)	52.0 (FDA, FCC)	540 (EU)	Purified Water : HCl
Amaranth	520 (EU), 518-522 (Japan)	44.0	440 (EU)	Concentrated HCl
Azorubine	515 (EU)		516 (EU)	Purified Water : HCl
Brilliant Black PN	570 (EU)	53.0	530 (EU)	Concentrated HCl
Brilliant Blue FCF	630 (FDA & EU), 628-632 (Japan)	64.0	1630 (EU)	1N Sodium Hydroxide
Brown HT	460 (EU)	40.3		Purified Water : HCl
Erythrosine	527 (FDA & EU), 524-528 (Japan)	110 (FDA)	1100 (EU)	1N Sodium Hydroxide
Fast Green FCF	625 (FDA), 622-626 (Japan)	156 (FDA)	15605 (FDA)	Purified Water : HCl
Fast Red E		50.5	450	Concentrated HCl
Green S	632 (EU)	172	1720 (EU)	Purified Water : HCl
Indigotine	610 (FDA & EU), 610-614 (Japan)	47.8 (FDA)	480 (EU)	Concentrated HCl
Patent Blue V	638 (EU)	200	2000 (EU)	Purified Water : HCl
Ponceau 4R	505 (EU), 506-510 (Japan)	43.0	430 (EU)	Concentrated HCl
Quinoline Yellow	415 (JECFA)	86.5 (JECFA)		Concentrated HCl
Red 2G	532 (EU)	62.0	620 (EU)	Purified Water : HCl
Sunset Yellow FCF	484 (FDA), 485 (EU), 480-484 (Japan)	54.0	555 (EU)	Purified Water : HCl
Tartrazine	428 (FDA), 426 (EU), 426-430 (Japan)	53.0	530 (EU)	Concentrated HCl

1. Values based on information from the United States Food and Drug Administration (FDA)
2. Values based on information obtained from the European Union (EU)
3. Values based on information obtained from Japan
4. Calculated from specific absorbance
5. Calculated from absorptivity

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