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Determination of Total Coloring Matter

Two methods are used for 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 hue 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 pertaining to 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 below Table.

The absorbance of a solution of the colouring matter is determined at its wavelength of maximum absorption and the total colouring matters content is calculated using the standard absorptivity or specific absorbance value provided in 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 1Colouring 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 Table and swirl to dissolve. Make up to volume and mix. Dilute the solution with the same solvent in order 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.

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

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

  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. Make up 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.

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

Procedure 3Colouring matters content of lakes

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 10ml hydrochloric acid 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 liter volumetric flask, make up 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.

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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