Permitted Synthetic Food Colours

i) Subsidiary Dyes

The subsidiary dyes are separated from the main dye by ascending paper chromatography and are extracted separately from the paper. The optical densities of the extracts are measured at their wavelengths of maximum absorption in the visible spectrum and are used to calculate the content of subsidiary dyes as a percentage by mass of the sample.

Reagents

(a) Chromatographic solvents

•  Water:ammonia (sp gr. 0.880):trisodium citrate (95 ml:5 ml:2 g)
•  n-Butanol:water:ethanol:ammonia (600:264:135:6)
•  Butan-2-one:acetone:water (7:3:3)
•  Butan-2-one:acetone:water:ammonia (700:300:300:2)
•  Butan-2-one:acetone:water (700:160:300:2)
•  n-Butanol:glacial acetic acid:water (4:1:5)

 (b) Extracting solvent : A mixture of equal volumes of acetone and water.

(c) Sodium bicarbonate (0.05N)

Procedure

Not less than 2 h before carrying out the determination arrange the filter paper drapes in the glass tank and pour over the drapes and into the bottom of the tank sufficient of the atmosphere saturating solvent to cover the bottom of the tank to a depth of 1 cm.

Mark out a sheet of chromatography grade paper. Apply 0.1 ml of a 1 % aqueous solution of the dye a s uniformly as possible within the contents of the 180 mm ×7 mm rectangle, holding the nozzle of the micro-syringe steadily in contact with the paper. Allow the paper to dry at room temperature for 1-2 h or at 50°C for 5 min followed by 15 min at room temperature. Mount the sheet together with a plain sheet to act as a blank. Pour sufficient chromatography solvent into the tray to bring the surface of the solvent about 1 cm below the base line of the sheet of paper. Allow the solvent front to ascent the full height of paper,development being continued for 1 h afterwards, then remove the frame and transfer it to a drying cabinet at 50-60°C for 10-15 min. Remove the sheets from frame.Cut each subsidiary band from the sheet as a strip and cut an equivalent strip from the corresponding position of the plain sheet. Place each strip into different test tubes. Add 5 ml of extracting solvent to each test tube, swirl for 2-3 min, add 15 ml of the sodium carbonate solution and shake the tube to ensure mixing. Filter the coloured extracts and blanks through a filter paper and determine wavelengths of maximum absorption against a filtered mixture of 5 ml of extracting solvent and 15 ml of sodium bicarbonate solution. Measure the optical density odf the extract of the blank strips at the wavelengths at which those of the corresponding coloured extracts were measured.

Subsidiary dye, % by mass = F [(D1+D2+…..)-(b1+b2+… )]

Where,

F= conversion factor (11.4),

D1, D2, etc = optical densities of the subsidiary dye extracts; and

b1, b2, etc. = optical densities of extracts of the corresponding blanks.

ii) Dye intermediates

Apparatus

Chromatography tube

Column preparation: Prepare a slurry of Whatman powdered cellulose in a 25 % ammonium sulphate solution. Prepare the column and pass 200 ml of 25% ammonium sulphate solution through it. The UV absorption of solution shall be sufficiently low to avoid interference with the intended analysis. Use about 75 g of cellulose to 500 ml of liquid. Pour sufficient slurry into the tube to give a column to a height of about 5 cm in the mouth of the tube. Tap the tube occasionally to ensure a well packed column. Wash the column with 200 ml of the eluent.

Procedure

Place 0.2 g of the dye sample in a beaker and dissolve in 20 ml of water. Add 5 g of powder cellulose. Add 50 g of ammonium sulphate to the dye. Transfer the mixture to the column, rinse the beaker with 25 % ammonium sulphate solution and add washings to the tube. Allow the column to drain until flow ceases or nearly so. Add the ammonium sulphate solution to the column at a rate equivalent to the rate of flow through the column. Collect the effluent in 100 ml fractions.Continue until 12 fractions have been collected. Mix each fraction well and obtain the UV absorption spectra of each solution from 220 to 400 nm. The specific spectra may be chosen depending on the nature of the dyes.

iii) Unsulphonated Primary Aromatic Amines

Unsulphonated primary aromatic amines are extracted into toluene from an alkaline solution of the sample, re-extracted into acid and then determined spectrophotometrically after diazotisation and coupling.

Procedure

Weigh about 2 g of the colour sample into a separating funnel containing 100 ml of water, swirl down the sides of the funnel with further 50 ml of water. Swirl to dissolve the sample, add 5 ml of 1N NaOH solution. Extract with two 50 ml portion of 0.1N NaOH solution to remove traces of colour. Extract the washed toluene with three 10 ml portions of 3N HCl solution and dilute the combine extract to 100 ml with water. Mix well. Pipette 10 ml of this solution into a clean,dry test tube, cool for 10 min by immersion in a beaker of ice/water mixture; add 1 ml of 50% KBr solution and 0.05 ml of 0.5N sodium nitrite solution. Mix and allow to stand for 10 min in the ice/water bath and add 1 ml of 0.05N disodium salt of 2-napthol-3,6-disulphonic acid (R-salt). Dilute to 25 ml with water, stopper flask and mix the content well and allow to stand for 15 min in the dark. Measure the absorbance of coupled solution at 510 nm in 1 cm cell using as a reference mixture of 10 ml of 1N HCl, 10 ml of 2N sodium carbonate solution and 2 ml of R-salt solution, diluted to 25 ml with water. Similarly, measure absorbances of 10 ml of aniline standard solutions containing 50 to 250 μg aniline, after diazotization and coupling.

iv. Leuco Base

Air is blown through an aqueous solution containing the chloride and dimethylformamide. Under these conditions the leuco base is oxidized to colouring matters and the increase in absorptivity is a measure of the amount of leuco base originally present.

Reagents

Solution A: Weigh 10 g of CuCl2.2H2O and dissolve in 200 ml of DMF. Transfer to a 1 litre volumetric flask and make up to mark with DMF.

Solution B: Accurately weigh the specified quantity of sample, dissolve in 100 ml of water, transfer quantitatively to a 10 litre volumetric flask and make up to the mark with water.

Procedure

Prepare the following solutions:

Solution A-Puipette 50 ml DMF into a 250 ml volumetric flask. Cover with parafilm and place in the dark.

Solution B-Accurately pipette 10 ml of solution B into a 250 ml volumetric flask. Add 50 ml DMF. Cover with parafilm and place in the dark.

Solution C-Pipette 50 ml of solution A into a 250 ml volumetric flask. Bubble air through this solution for 30 min in the following manner.Insert a 5 ml pipette into a box attached to a bench air flow source. Turn on the air, slowly. Stick the pipette down into the solution in the flask and adjust the air flow to a rapid but controlled rate. After 30 min pull the pipette out of the solution and rinse the sides of the pipette into the flask with water from a wash bottle. Then turn off the air flow.

Solution D-Accurately pipette 10 ml solution B into two separate 250 ml volumetric flasks in the same manner as used for solution B. Add 50 ml solution to each flask. Bubble air through the solutions for 30 min, using the above method.After 30 min of rapid bubbling of air through the solutions, dilute all 5 flasks nearly to volume with water. Heat is evolved when DMF and water are mixed, so place the flask in a water bath of tap water until they have cooled to room temperature.Bring accurately to volume with water. Run the solutions on the spectrophotometer immediately. The entire procedure should be completed as quickly as possible.Draw the following curves from 700-500 nm using an absorbance range of 0.1 and 1 cm cells.

Calculation

Leuco base, % by mass = ([(4 3) (2 1)] 25 *100)/ A M Ratio

Where,

A = absorptivity of 100% colouring matters,

M = mass, in g, of sample taken for test,

Ratio = MW of colouring matter/MW of leuco base

4 = Run curve without zero setting, record absorbance at maximum,

3 = Set zero at 700 nm, record absorbance at Abs std colouring matter,

2 = Run curve without readjusting zero setting , record absorbance at maximum,

1 = Set zero at 700 nm, record absorbance at Abs std for colouring matter.

v) Chlorides (As Sodium Chloride)

Accurately weigh 0.5-1.0 g of dye sample, dissolve n 100 ml of water, and acidify with 5 ml of 1.5N nitric acid solution. Place the silver electrode in the colour solution and connect the calomel electrode to the solution by means of the saturated potassium sulphate bridge. The saturated potassium sulphate bridge may be eliminated by using a glass electrode as the reference electrode. Determine the chloride content of the solution by titration against the 0.1N silver nitrate solution and calculate the result as sodium chloride. 1 ml of 0.1N silver nitrate is equivalent to 0.00585 g of sodium chloride.

vi) Metallic Impurities

The samples are dissolved in acid or digested in a mixture of sulphuric acid, nitric acid and in some cases perchloric acid. The barium, cadmium, lead, copper, chromium and zinc in solution are determined by flame atomic absorption spectroscopy. Antimony and arsenic are determined by using a hydride generation technique.

Procedure

Accurately weigh about 2.5 g of the sample into a 500 ml Kjeldahl flask, add 5 ml of dilute nitric acid. As soon as any initial reaction subsides, heat gently until further vigorous reaction ceases and then cool. Add gradually 4 ml of conc. sulphuric acid. at such a rate as not to cause excessive frothing on heating and then heat until the liquid darkens appreciably in colour, that is, begins to clear.Add conc nitric acid slowly in small portions, heating between additions until darkening again takes place. Do not heat so strongly that charring is excessive; small but not excessive amount of free nitric acid should be present throughout.Continue this treatment until the solution is only pale yellow in colour and fails to darken in colour on prolonged heating. If the solution is still coloured run in 0.5 ml of hydrogen peroxide and heat further for a few minutes longer. Allow to cool somewhat and dilute with 10 ml of water. The solution should be quite colourless.Boil down gently, taking care to avoid bumping, until white fumes appear. Allow to cool, add a further 5 ml of water and again boil down gently to fuming. Finally cool and add 10 ml of 5N HCl and boil gently for a few min. Cool and transfer the solution to a 50 ml volumetric flask washing out the Kjeldahl flask with small portions of water and dilute to the mark with water. If barium is present, add 0.0954 g of KCl before dilution as an ionizing buffer to prevent ionization of barium.

Instrumental conditions

Select the wavelength and gases to be used for the particular element under consideration from the table below

 Set the AAS to the appropriate conditions. Aspirate the strongest standard containing the element to be determined and optimize the instrument settings to give maximum deflection. Plot a calibration curve with 3-4 standard solutions.Aspirate the test solution and the corresponding blank solution.

Calculation

Element (X) in the sample, mg/kg = (Conc of X in test solution Conc of X in blank ( g /ml) 50)/ Mass of sample taken (g)

Arsenic By Hydride Generation Technique

Arsenic is determined after preparation of their volatile hydrides mixing of 10% HCl and 10% sodium borohydride solution with sample solution, which are collected in the generation vessel. The gases are then expelled with argon or nitrogen gas into a hydrogen flame. The arsenic is then determined by using all other steps involved in conventional flame atomic absorption spectroscopy.

Mercury By Atomic Absorption Cold Vapour Technique

The sample is digested by heating under reflux with sulphuric and nitric acids. The oxidation is completed by addition of potassium permanganate solution. After successive additions of hydroxylamine hydrochloride solution and stannous chloride solution, the mercury content is measured by cold vapour atomic absorption spectrometry.

vii) Heavy Metals

Reagents

•  Ammonia solution (Dilute 400 ml of ammonium hydroxide (28%) to 1 litre with water)
•  Hydrochloric acid (10%)
•  Lead nitrate stock solution (Dissolve 159.8 mg of lead nitrate in 100 ml of water containing 1 ml of nitric acid. Dilute with water to 1 litre and mix. Prepare and store the solution in lead free glass containers.
•  Standard lead solution (Dilute 10 ml of lead nitrate stock solution, accurately measured with water to 100 ml. Each ml of the solution contains 10 mg of lead.Prepare the solution on the day of use.
•  Nitric acid (10%)
• Concentrated Sulphuric acid
•  Hydrogen sulphide (A saturated solution of hydrogen sulphide gas made by mixing of iron sulphide with dilute hydrochloric acid solution, which passing through cold water).

 Procedure

Solution A : Take the quantity of standard lead solution of concentration equivalent to the limits specified in the individual Nessler tube and add about 23 ml of water.Adjust the pH to between 3-4 by addition of acetic acid or ammonia solution. Dilute with water to 40 ml and mix.

Solution B: Place 500 mg of the sample, accurately weighed, in a suitable crucible,add sufficient sulphuric acid to wet the sample, carefully ignite at a low temperature until thoroughly charred, covering the crucible loosely with a suitable lid during the ignition. After the substance is thoroughly carbonized, add 2 ml of nitric acid and 5 drops of sulphuric acid, and cautiously heat until white fumes are evolved, then ignite, preferably in muffle furnace at 500-600°C until the carbon is completely burned off. Cool and add 4 ml of dilute HCl, cover and digest on a steam-bath to dryness. Moisten the residue with 1 drop of HCl, add 10 ml of hot water and digest for 2 min. Add dropwise ammonia solution until the solution is just alkaline to litmus paper dilute with water to 25 ml and adjust the pH to 3-4 by the addition of dilute acetic acid. Filter if necessary; wash the crucible and the filter with 10 ml of water. Transfer to a 50 ml Nessler tube. Dilute the combined filtrate and washing with water to 40 ml and mix.

To each tube add 10 ml of freshly prepared hydrogen sulphide, mix and allow to stand for 5 min and view over a white surface. The colour of solution B shall not be darker than of solution A.

viii) Ponceau 4r

Purity

Weigh accurately about 250 mg of the dye sample and dissolve in 0.1N HCl in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 1mg per 100 ml. Find out the optical density of this diluted solution against 0.1N HCl solution as blank at 506 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and

from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass = (OD*100)/ M 440

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

440 = Extinction coefficient for Ponceau 4R in 0.1N HCl solution.

Subsidiary dye

The details of the method given above shall be followed.

Developing solvent No. 3

Height of ascent of solvent front= 17 cm then 1 h for further development.

ix. Carmoisine

Purity

Weigh accurately about 250 mg of the dye sample and dissolve in 0.1N HCl in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 1mg per 100 ml. Find out the optical density of this diluted solution against 0.1N HCl solution as blank at 516 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass =   (OD*100)/ M 520

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

520 = Extinction coefficient for carmoisine in 0.1N HCl solution.

Subsidiary dye

The details of the method given above shall be followed.

Developing solvent No. 4

Height of ascent of solvent front= 17 cm.

x. Erythrosine

Purity

Weigh accurately about 125 mg of the dye sample and dissolve in with 0.1N NaOH in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 0.5 mg per 100 ml. Find out the optical density of this diluted solution against 0.1N NaOH solution as blank at 527 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass = (OD *100)/ M 1080

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

1080 = Extinction coefficient for erythrosine in 0.1N NaOH solution.

Subsidiary dye

The details of the method given above shall be followed.

Developing solvent No. 5

Height of ascent of solvent front= 17 cm.

Fluorescein

Solvent - methanol: water: ammonia = 500 :400:100

Sample – Weigh 1 g sample and dissolve in 50 ml solvent and dilute to 100 ml in a volumetric flask.

Standard- Weigh an amount of fluorescein corresponding to 1 g at the colouring matter content of sample. Dissolve in water and dilute to 100 ml. Make furher sequential dilutions as follows:

1 ml to 100 ml with water, 1 ml to 100 ml with water and 20 ml to 100 ml with solvent.

Chromatography solvent

N-Butanol:water:ammnia:ethanol = 100:44:1:22.5

TLC: Spot 25 ml of sample and standard solutions side by side on a cellulose plate. Develop for 16 h in the chromatography solvent. Allow the plate to dry.View under UV light and compare the fluorescence of the corresponding area on the chromatogram of the sample. The intensity of the latter shall not be greater than that of the former.

Organic compounds other than colouring matter

The method described as dye intermediates shall be followed :

2(2,4-dihydroxy-3,5-diiodobenzoyl)benzoic acid-0.047 mg/L/cm at 348 nm(alkaline)

Tri-iodoresorcinol-0.079 mg/L/cm at 223 nm (acidic)

xi. Tartrazine

Purity

Weigh accurately about 250 mg of the dye sample and dissolve in 0.1N HCl in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 1mg per 100 ml. Find out the optical density of this diluted solution against 0.1N HCl solution as blank at 428 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass = (OD *100)/ M 485

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

485 = Extinction coefficient for tartrazine in 0.1N HCl solution.

Subsidiary dye

The details of the method given above shall be followed.

Developing solvent No. 4

Height of ascent of solvent front= 12 cm.

xii. Sunset Yellow FCF

Purity

Weigh accurately about 250 mg of the dye sample and dissolve in 0.1N HCl in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 1mg per 100 ml. Find out the optical density of this diluted solution against 0.1N HCl solution as blank at 482 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass = (OD* 100)/ M 543

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

543 = Extinction coefficient for sunset yellow in 0.1N HCl solution.

Subsidiary dye

The details of the method given above shall be followed.

Developing solvent No. 4

Height of ascent of solvent front= 17 cm.

xiii. Indigo Carmine

Purity

Weigh accurately about 250 mg of the dye sample and dissolve with ammonium acetate solution in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 1 mg per 100 ml. Find out the optical density of this diluted solution against ammonium acetate solution as blank at 610 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass =( OD* 100)/ M 450

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

450 = Extinction coefficient for indigo carmine in ammonium acetate solution.

Subsidiary Dye

The details of the method given above shall be followed.

Developing solvent No. 3

Height of ascent of solvent front= 17 cm.

xiv. Brilliant Blue FCF

Purity

Weigh accurately about 100 mg of the dye sample and dissolve with ammonium acetate solution in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 0.2 mg per 100 ml. Find out the optical density of this diluted solution against ammonium acetate solution as blank at 630 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass = (OD *100)/ M 1640

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

1640 = Extinction coefficient for brilliant blue FCF in ammonium acetate solution.

Subsidiary Dye

The details of the method given above shall be followed.

Developing solvent No. 4

Develop chromatogram for approx. 20 h.

xv. Fast Green FCF

Purity

Weigh accurately about 100 mg of the dye sample and dissolve with ammonium acetate solution in a 250 ml volumetric flask. Dilute this with the same solvent to make a final concentration of 0.2 mg per 100 ml. Find out the optical density of this diluted solution against ammonium acetate solution as blank at 625 nm in a glass cell with 10 mm path length. Simultaneously weigh accurately about 2 g of the dye sample and dry this in an air oven at 105±1°C for 2 h. Calculate the loss of mass on drying and from this data calculate the dry mass of the sample in the final solution taken for measurement of the optical density.

Total dye, % by mass = (OD *100)/ M 1560

Where,

OD = optical density found,

M = dry mass of sample in 100 ml of solution, and

1560 = Extinction coefficient for Fast green FCF in ammonium acetate solution.

Subsidiary Dye

The details of the method given above shall be followed.

Developing solvent No. 4

Height of ascent of solvent front= 12 cm.