Colouring matters

Both types of colouring matter natural as well as synthetic are used in dairy products. Natural colours like b-carotene and annatto are preferred or synthetic colors. Methods, for testing of both types of colours are given below.

A. Natural Coloring Matters

i) â-Carotene

Carotene is widely distributed in nature. It is an unsaturated hydrocarbon. It occurs in nature in three isomeric forms, a, â and g, out of which â-carotene is available in pure form and is also manufactured synthetically.

Assaying purity of â-carotene : Weigh accurately about 0.1 g of the dried sample and dissolve it in 100 ml of cyclohexane. Dilute the solution, stem-wise, to a final concentration of approx. 0.2 mg per 100 ml of cyclohexane. Measure the extinction of the resulting solution in a 1 cm cell at 456 nm.

Calculation

Purity, % by mass = (E*1000*100)/ 2441 W

Where

E = observed extinction, and

W= exact concentration of â-carotene in mg per 100 ml of cyclohexane in the final solution for extinction reading.

ii) Annatto Colour

Annatto colour is obtained from the aril (seed coat) of the plant Bixa orellana.Its solution in oil is used for colouring butter while its solution in water with a little alkali is used for colouring cheese and other similar products.

Assaying purity of Annatto

Annatto Extract in oil : Transfer 0.1 to 1 g of the sample, accurately weighed into a 100 ml volumetric flask. Dissolve in chloroform, dilute to volume with chloroform, and mix. Transfer a 1 ml portion of the solution into another 100 ml volumetric flask and dilute to 100 ml. Measure the absorbance A of this solution at 470 nm.

Total carotenoids, % by mass(expressed as bixin) = (A*1000*100)/  2.826 mass of sample (mg) 100

Water Soluble Annatto : Weigh accurately 0.1 to 1 g of the sample, add 0.01N sodium hydroxide to 100 ml, and shake thoroughly. Transfer 1 ml portion of the solution into a separating funnel, add 10 ml of sodium hydroxide solution ( 1 in 10) and water to 50 ml, and add 2 ml of diluted sulphuric acid. Continue to extract the solution with each 10 ml of benzene until the benzene extract is not coloured. Combine benzene extract, wash three times with each 5 ml of water, and allow to stand to separate the water layer. Transfer the benzene extract into another separating funnel containing the water layer three times with 2 ml of benzene, and combine the washings to the benzene extract. To the benzene extract, add an equal volume of petroleum benzene mix, and continue to extract with each 5 ml of 0.01N sodium hydroxide until sodium hydroxide solution is not coloured. Combine extracts, and add 0.01N sodium hydroxide to 100 ml. Measure the absorbance A at 453 nm.

Total carotenoids, % by mass = (A*1000*100)/ 3.473 mass of sample 100

iii) Saffron (Filaments And Powder Form)

Picrocrocine, Safranal & crocine

Weigh exactly 500 mg of sample and transfer quantitatively the test portion into the 1000 ml volumetric flask. Add about 900 ml of distilled water. Stir with a magnetic stirrer (1000 rpm) for 1 h, away from light. Remove the magnetic bar.Make up to the mark with distilled water. Close with a glass stopper and homogenize. Filter the solution, rapidly and away from light through the membrane so as to obtain a clear solution. Adjust the spectrophotometer and read the absorbance between 220 nm and 480 nm using distilled water as the reference liquid. The results are obtained by direct reading of the absorbance at 3 wavelengths, corresponding to the maximum absorbances of picrocrocine, safranal and crocine as follows:

Picrocrocine: absorbance at 257 nm

Safranal : absorbance at 330 nm

Crocine : absorbance at 440 nm

Identification of Saffron pigments

Reagents

a) Methanol

b) Ethanol

c) Chloroform

d) Sulphuric acid (95-97% m/m)

e) 4-Methoxybenzaldehyde (Anisaldehyde)

f) Naphthol yellow (Sodium salt of 2,4-dinitronaphthol)

g) Sudan red G

h) Reference solution prepared by dissolving 5 mg of Naphthol yellow in 5 ml of (expressed as norbixin) 3.473 mass of sample 100 methanol and adding a solution of 5 mg of Sudan red G in 5 ml of chloroform.

i) Elution solvent consisting of the organic phase of a mixture containing ethyl acetate : propan-2-ol : water (65:25:10)

j) Revealing solution prepared by mixing in the following order : 10 ml of anisaldehyde, 90 ml of ethanol and 10 ml sulphuric acid.

Apparatus

a) Chromatography chamber

b) Silica gel plates with indicator of fluorescence GF 254

Procedure

Weighing 0.05 g of sample into a test tube and moisten it with a drop of water. Wait for 2-3 min, and then add 1 ml of methanol. Allow the solution to settle for 29 min away from light. Filter it on a small glass wool plug. Using micropipette load 5 μl of solution on silica gel plate in 2 cm interval and develop in the chromatography chamber with the elution solvent until the solvent front has progressed 10 cm. Allow the solvent to evaporate. Examine the chromatogram in UV light at 254 nm, then in day light. Then spray onto the plate about 10 ml of the revealing solution. Heat for 5-10 min at a temperature of 105-110°C while observing the chromatogram.

Interpretation

1. In daylight the lower third of the chromatogram shows three yellow spots. The spot on the bottom strip is of more intense colour, and corresponds in colour and in size to the Naphthol yellow spot,it characterizes crocine.

2. The chromatogram observed in UV light at 254 nm shows 4 main fluorescent spots, 3 corresponding to the spots observed in daylight and another with a higher Rf value (»0.55), which characterizes picrocrocine. One or two rather faint spots of fluorescence are visible at the level of Sudan red G, characterizing â-hydroxycyclocitral and safranal.

3. After spraying with the revealing solution, the crocine becomes grayish green in colour, and the picrocrocine becomes violet in colour. The chromatogram shall not show any other colour spots before spraying, particularly at the starting point. These would correspond to a deterioration of the crocine and/ or the presence of foreign colouring matter.

iv. Caramel

Solid Content

The solid content of caramel colour is determined by drying a sample with acid washed sand (40-60 mesh). Mix 30 g of prepared sand accurately weighed with 1.5-2.0 g caramel colour accurately weighed and dry to constant weight at 60°C under reduced pressure 50 mm Hg. Record the final weight of the sand plus caramel. Calculate the percent solids as follows:

Soilds content, % by mass =  ((WF −Wi)×100)/ We

Where,

Wf = final weight of sand plus caramel,

Ws= weight of sand, and

Wc= weight of caramel initially added.

Colour Intensity

Prepare a 0.1 percent m/v solution of the sample in freshly boiled and cooled distilled water. If the solution is not clear clarify it by centrifuging, not by filtering.Determine the absorbance of the clear solution in a 1 cm cell at 610 nm with a suitable spectrophotometer previously standardized using water as the reference.

Calculate the colour intensity of the caramel colour as follows :

Colour intensity = (A610 100 )/Percent solids

Ammoniacal Nitrogen

Reagents

a) Sulphuric acid (0.1N)

b) Magnesium oxide (carbonate free)

c) Methyl red indicator solution (0.5% in ethanol)

d) Sodium hydroxide (0.1N)

Procedure

Add 25 ml of sulphuric acid to a 500 ml receiving flask and connect it to the distillation apparatus. Transfer about 20 g of the sample accurately weighed into a 800 ml long neck Kjeldahl digestion flask, and to the flask add 2 g of magnesium oxide, 200 ml of water and several boiling chips. Swirl the digestion flask to mix the contents, and quickly connect it to the distillation apparatus. Heat the digestion flask to boiling and collect about 100 ml of distillate in the receiving flask. Wash the tip of the delivery tube with a few ml of water, collecting the washings in the receiving flask. Then add 4 to 5 drops of methyl red indicator and titrate with the sodium hydroxide, recording the volume (in ml) required as S. Conduct a blank determination and record volume (in ml) of the sodium hydroxide required as B.

Ammoniacal N2, % by mass =   ((S − B)× 0.0014×100)/ Mass of sample in g

v. 4-Methylimidazole

Caramel colour is added to a basic Celite column and eluted with mixture of chloroform and methanol. The eluent is extracted with dilute sulphuric acid and the aqueous extract concentrated, brought to a known volume and neutralized to a slightly alkaline pH. The alkaline extract obtained is analyzed by paper chromatography. The developed spots are visualized by spraying with diazosulfanilic acid reagent, and sodium carbonate solution separately.

Reagents

a) Celite 545

b) Sodium hydroxide solution (2N)

c) Elution solvent (Chloroform:Ethanol=80:20, v/v)

d) Sulphuric acid solution (0.05N)

e) Developing solvent-1 containing diethyl ether, chloroform, methanol and ammonium hydroxide= 80:20:20:4(v/v)

f) Developing solvent-2 containing 3 parts of n-propyl alcohol and 1 part of 0.2N ammonia solution.

g) Spraying reagent-1 : (a) Prepare freshly a 5.0% m/m solution of sodium nitrite, cool to 0°C.

(b) Add 0.9 g of sulphanilic acid to 9.0 ml of conc. HCl taken in a 100 ml flask and dilute to 100 ml with distilled water. Cool to 0°C.

h) Spraying reagent-2 : 5 % m/v sodium carbonate solution.

i) Stock solution of 4MeI : Dissolve 100 mg of 4-methylimidazole in 100 ml of 0.1N sulphuric acid. Store the solution in a refrigerator.

j) Standard solution of 4-MeI: Pipette out 1 ml of 4-MeI stock solution in a 10 ml flask and dilute to volume with distilled water. Bring the pH of the solution to 9 using solid sodium carbonate.

The solution corresponds to 100 mg/kg.

Procedure

Extraction: Prepare a basic column packing by mixing well Celite 545 and 2N NaOH in the proportion of 2 ml of 2N NaOH to 3 g Celite 545. Place a fine glass wool plug at the base of the chromatographic column, followed by 5.0 g of basic column packing. Tap the packing firmly to a uniform mass.

Take 10 g of sample of caramel in 150 ml beaker. To this add 6 g of 20 % Na2CO3 solution. Mix well and add 12.0 g Celite 545. After thoroughly mixing add the mixture to the prepared column. Dry, wash the beaker with about 2.0 g Celite 545 and add this wash to column. Place a plug a glass wool at the top of the dry wash and tap the contents of the column.

Elute the column with elution mixture until 125 ml has been collected. The elution rate should be 5 ml/min and collected into 250 ml separating funnel. Extract the elute with 20 ml portion of 0.05N sulphuric acid and then with second 10 ml portion of 0.05N sulphuric acid. Transfer the combined aqueous layers to 100 ml vacuum flask and concentrate to 5 ml by drying under vacuum at a temperature not exceeding 55°C (the volume should not be reduced below 4 ml)

Transfer this aqueous solution into a 10 ml volumetric flask, which also receives rinsing with 1 ml portion of distilled water until the mark is reached. After mixing transfer the aqueous concentrate to a suitable sample vial and treat with small portion of sodium carbonate anhydrous powder until CO2 evolution ceases and pH meter indicates pH 9.

Paper chromatography: Spot 10 ml of this solution on the chromatographic paper 3 cm from the bottom of the paper, and develop the chromatogram at 25-28°C by ascending paper chromatography using either of the developing solvent.Allow the solvent to travel up to 15 cm.

Remove the paper from the tank and dry it by air drier. Spray the paper with diazosulfanilic acid reagent until the paper is just wet and then develop the colour by a light spray of 5% m/v sodium carbonate solution. Cut the maroon colour spots and elute in 20 % ethanol (5-10 ml). Keep aside for 30 min and immediately note the absorbance of solution at 505 nm. Side by side prepare the calibrated graph using various concentrations of 4-MeI and the amount of 4-MeI in the unknown is the red off directly from the above calibrated graph.

Equivalent colour basis=    (Concentration on solids basis×0.1)/ Colour int ensity

This gives content expressed in terms of a product having a colour intensity of 0.1 absorbance units.

2-Acetyl-4-Tetrahydroxy Butylimidazole (Thi)

THI is converted into its 2,4-dinitrophenyl hydrazone. This derivative is separated from excess reagent and carbonylic contaminants by HPLC on RP-8, then determined its absorbance at 385 nm.

Reagents

Cation-exchange resin (strong): Dowex 50 AG×8, H+, 100-200 mesh

Cation-exchange resin (weak): Amberlite CG AG, H+, 100-200 mesh

Methanol (carbonyl free)

Dimethoxyethane

Procedure

Caramel colour (200-250 mg) is weighed accurately then dissolved in water (3 ml). The solution is passed through a combined column (upper column : weakly acidic cation exchanger, 50-60 mm bed height/ 12.5 mm i.d. or 80-90 mm bed height / 10 mm i.d. and lower column : strongly acidic cation exchanger, 60 mm bed height) with 100 ml of water. The upper column is disconnected. The lower column is eluted with 0.5M HCl. The first 10 ml of elute are discarded then a volume of 35 ml is collected. The solution is concentrated to dryness at 40°C and 15 torr. The syrup residue is dissolved in methanol (250 ml) and 2,4-dinitrophenyl hydrazine reagent (250 ml) is added. The reaction mixture is transferred to a septum-capped vial and stored for 5 h at room temperature. A volume of 1-25 μl is injected on to a Lichrosorb RP-8 (10 mm) HPLC column.

The mobile phase is methanol/0.1M H3PO4 : 50:50 (v/v). At a mobile phase flow rate of 2 ml/min and column dimensions of 250 mm × 4.6 mm, THI-DNPH is eluted at about 6.3±0.1 min. It is detected at 385 nm and the peak height is measured. The amount is calculated from a calibration curve prepared with THIDNPH in methanol.

Total Sulphur

Take a 100 ml crucible, place 1-3 g MgO, 1 g sucrose powder and 50 ml HNO3. Add 5-10 g caramel colour. Place same quantity of reagents in another crucible as blank. Evaporate on steam bath to paste. Place in cold muffle furnace and gradually heat (525°C) until all NO2 fumes are driven off. Cool, dissolve and neutralize with dilute HCL, adding excess of 5 ml. Filter, heat to boiling and add 5 ml of 10 % BaCl2.2H2O solution dropwise. Evaporate to 100 ml, let stand overnight, filter, wash, ignite and weigh the BaSO4. Correct result for BaSO4 obtained in blank and report as mg S/100 g.

Sulphur Dioxide

Reagents

a) Hydrogen peroxide (3% free from sulphate)

b) Barium chloride (10% m/v)

c) Hydrochloric acid (2N)

d) Conc. Hydrochloric acid

e) Carbon dioxide gas

f) Sodium hydroxide (0.1N)

g) Bromophenol blue indicator (Dissolve 0.5 g of bromophenol blue in 7.5 ml of 0.1N sodium hydroxide and dilute to 1 litre.

Procedure

Measure 25 ml of hydrogen peroxide into the conical flask and add 25 ml of distilled water. Measure 10 ml of hydrogen peroxide and add into the first Wohler tube (U tube). In the second tube (as a guard) place 5 ml of a mixture of equal volumes of hydrogen peroxide and barium chloride solutions which has been slightly acidified with 2N HCl solution.

Introduce 500 ml of distilled water and 20 ml of conc. HCl into the round bottom flask. Connect the CO2 tube to the flask and boil the water for a short time in a current of CO2 gas. Introduce about 20 g of sample accurately weighed into the round bottom flask, through the funnel, continuing the flow of CO2 gas. While adding the sample, see that there shall not be any back pressure. Boil it for 2h passing a slow current of CO2 gas throughout the test. Before the end of the distillation stop the flow of water through the condenser to allow any sulphur dioxide which is retained by the condensed moisture in the tube of the condenser to be driven over into the receiver.As soon as the delivery tube, condenser is hot, disconnect the straight tube first, wash it with water. Combine the contents of first Wohler tube in the receiver or conical flask. Cool the conical flask and titrate with the NaOH solution using bromophenol blue indicator. Calculate the percentage of sulphur dioxide. 1 ml of 0.1N NaOH is equivalent to 3.203 mg of sulphur dioxide (Figure ).