Testing of Colouring Matters in Food Ingredients

Food colour is one of the first things a consumer notices – before the smell, before the taste. It signals freshness, flavour identity, and even safety. But the vibrant orange of a cheese rind, the golden hue of a saffron rice dish, or the bright yellow of a bottled soft drink don’t just happen. Every colouring matter used in food production – whether sourced from nature or synthesised in a laboratory – must pass rigorous purity testing before it can legally enter the food supply. Understanding how these tests work, and why they matter, is essential knowledge for anyone working in food quality assurance.

Why testing colouring matters is non-negotiable 🔗

Food colour standards set by the FAO require colouring matters to be tested both for identity and purity content using defined analytical methods. The reason is straightforward: colour additives are used in small quantities, yet even minor contamination or degradation in a single batch can adulterate large volumes of product. According to the US FDA, synthetic colour additives are subject to batch certification, with analysts checking for total colour content, moisture, residual salts, unreacted intermediates, subsidiary colours, and heavy metals including lead, arsenic, and mercury. Using a colour additive that does not conform to purity specifications can render a product adulterated under food law, with serious commercial and legal consequences.

Spectrophotometry is the backbone of food colour testing. The technique works on the principle that molecules absorb light at specific wavelengths, and the degree of absorption is directly proportional to concentration – a relationship formalised as Beer-Lambert’s Law: A = εlc, where A is absorbance, ε is molar absorptivity, l is path length, and c is concentration. A spectrophotometer exposes the sample to a polychromatic light source, splits the reflected or transmitted light into its component wavelengths, and generates a measurable spectral curve. This data can be compared against established reference standards to confirm both identity and the precise concentration of the active colouring compound.

Testing natural colouring matters 🔗

Natural food colours are derived from plant, mineral, and biological sources. Although they are generally perceived as safer than synthetic alternatives, their complex chemical composition means matrix-specific testing methods are required, with careful attention to solvent selection, pH adjustment, and wavelength optimisation for each individual colorant.

β-Carotene 🔗

β-Carotene is the orange-red pigment found in carrots, palm oil, and various algae. As a food colour additive (E160a), it is widely used to impart yellow to orange shades in margarine, cheese, beverages, and baked goods. It is also commercially significant as a pro-vitamin A source. Testing β-Carotene for purity involves dissolving a precisely weighed sample in an organic solvent – typically hexane, petroleum ether, or cyclohexane – under carefully controlled, low-light conditions to prevent oxidation and isomerisation, both of which alter absorbance readings.

The absorbance of the prepared solution is measured using a UV-Vis spectrophotometer. Purity is confirmed when the specific absorbance (E1%, 1cm) at the target wavelength exceeds 2375, which corresponds to a spectrophotometric purity greater than 95%. A spectrophotometric method using an isobestic wavelength – at approximately 421 nm – allows quantification of total β-carotene content independently of the ratio of geometric isomers, making it particularly useful for commercial preparations that may contain mixtures of all-trans and cis-isomers. Other studies confirm maximum UV-Vis absorption for β-carotene at around 450-455 nm in acetone or hexane-based solvents, with the exact peak shifting slightly depending on the solvent system used. Any deviation from the expected absorption profile signals the presence of degradation products or impurities, triggering further investigation.

Annatto (bixin and norbixin) 🔗

Annatto (E160b) is extracted from the seeds of the achiote tree (Bixa orellana) and provides red-to-orange hues commonly used in cheddar cheese, butter, and snack foods. Its two primary colouring compounds are bixin (oil-soluble) and norbixin (water-soluble), which behave differently in solution and must be assessed separately.

Testing annatto involves dissolving the sample in an appropriate solvent – alkaline solutions for norbixin, and organic solvents such as chloroform or DMSO for bixin – and measuring absorbance at around 480 nm, where these carotenoid compounds show maximum absorption. The FAO/WHO Joint Expert Committee on Food Additives (JECFA) has issued specific tentative methods for determining total colouring matter content in annatto extracts, as well as separate protocols for residual solvents, reflecting the complexity of assaying this colour accurately. The test determines both the total colour strength and the relative proportion of bixin to norbixin – a ratio that affects how the colour behaves in oil-based versus water-based food systems.

Saffron 🔗

Saffron (E164) is the most expensive spice in the world by weight, derived from the dried red stigmas of Crocus sativus L. flowers. Because of its extraordinary value and the labour-intensive process of harvesting – requiring approximately 150,000 flowers per kilogram – saffron is also the most adulterated spice in history. Common adulterants include dyed safflower petals, calendula, turmeric powder, and synthetic dyes added to boost apparent colour strength.

Quality testing of saffron targets three key chemical markers: crocin (colour), picrocrocin (bitter taste), and safranal (characteristic aroma). The internationally recognised quality standard for saffron is ISO 3632, which classifies saffron into three commercial categories based on UV-Vis spectrophotometric measurements at 440 nm (crocin/colouring power), 270 nm (picrocrocin/bittering power), and 330 nm (safranal/odorous power). The test procedure involves dissolving approximately 125 mg of dried saffron in water under low-light, low-temperature conditions, filtering the solution, and then measuring absorbance across the 200-700 nm spectral range. The absorbance at 440 nm – the “colouring power” – is the principal indicator of crocin concentration and directly reflects saffron’s colour quality grade.

The spectrophotometric method under ISO 3632 identifies the unique spectral fingerprints of crocin, picrocrocin, and safranal, allowing laboratories to verify saffron’s authenticity and detect the presence of foreign materials or synthetic dyes. When saffron samples fall outside expected absorbance values at these three wavelengths, it is a clear signal of adulteration or substitution. For more precise compound-level quantification, high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are used alongside spectrophotometry.

Testing synthetic colouring matters 🔗

Synthetic food dyes offer consistency, cost-effectiveness, and high colour intensity, which makes them widely used across the processed food industry. However, certified colour manufacturers are required to submit every batch of synthetic colour to regulatory authorities for testing to ensure it meets purity standards before use in food. The purity tests for synthetic colours are technically similar to those for natural colours – both use spectrophotometric optical density measurements – but each dye has its own target wavelength, reference solvent, and maximum permitted levels for subsidiary dyes and chemical impurities.

Tartrazine (E102 / FD&C Yellow No. 5) 🔗

Tartrazine is a synthetic lemon-yellow azo dye widely used in processed foods, beverages, confectionery, and pharmaceutical products. It is water-soluble and has a maximum absorbance in aqueous solution at 425 nm. To test tartrazine for purity, a sample of known weight is dissolved in distilled water, and absorbance is measured at this 425 nm wavelength using a UV-Vis spectrophotometer. The measured optical density is then compared against certified reference standards to calculate the total colour content as a percentage.

Beyond the primary colour content measurement, analysts also assess tartrazine for subsidiary dyes – related azo compounds produced as by-products during manufacturing – as well as insoluble matter, residual salts, and heavy metals. The FDA’s batch certification process for synthetic dyes includes at least 10 separate analyses covering purity, moisture, unreacted intermediates, subsidiary colours, and the heavy metals lead, arsenic, and mercury. A batch is only certified once all parameters fall within the specifications set out in the listing regulation.

Sunset Yellow FCF (E110 / FD&C Yellow No. 6) 🔗

Sunset Yellow FCF is a petroleum-derived orange azo dye with a pH-dependent maximum absorption at approximately 480 nm at pH 1, shifting to around 443 nm at pH 13. This pH sensitivity is a key consideration during testing, which means all measurements must be conducted under tightly controlled, standardised pH and temperature conditions to ensure reproducibility. The dye is widely used in orange-coloured soft drinks, confectionery, and baked goods.

Testing Sunset Yellow FCF follows the same general approach as tartrazine: the sample is dissolved in an appropriate aqueous buffer solution, and optical density is measured at the dye’s characteristic absorption wavelength – typically 480 nm. The calculated absorbance value is used alongside the Beer-Lambert equation to determine the concentration and percentage purity of the colouring matter. Because food dyes are prepared from chemical compounds, spectrophotometry is essential to detect the concentration of each component and ensure no compound in the dye is present at a dangerous level. Subsidiary dyes formed during the manufacturing process of Sunset Yellow are also quantified, as their presence above permitted thresholds can affect both the shade of the final colour and regulatory compliance.

How optical density measurement translates to purity 🔗

The concept of optical density (OD) is central to all these tests. Optical density is another term for absorbance – both express how much light at a specific wavelength is absorbed by a solution. A higher OD value at the target wavelength indicates more of the active colouring compound is present. By applying Beer-Lambert’s Law, analysts calculate the precise concentration of the colouring matter from the OD reading, then express this as a percentage of the theoretical maximum – giving the purity figure.

For both natural and synthetic colours, purity calculations involve comparing the observed absorbance against the specific absorptivity coefficient for that compound – a standardised reference value unique to each colouring matter. Spectrophotometry can detect minute quantities of substances with high sensitivity and specificity, and its speed and minimal sample preparation requirements make it suitable for high-throughput quality testing environments. The method does have limitations – complex food matrices can interfere with measurements – but for testing isolated colour ingredients rather than finished food products, it remains the most cost-effective and widely recognised analytical approach.

Regulatory context and quality standards 🔗

Quality testing of colouring matters is not voluntary – it is mandated by food law in most jurisdictions. Colour additives must comply with individual listing regulations. Using a colour additive that does not conform to identity and purity specifications may cause a product to be deemed adulterated, exposing manufacturers to enforcement action. In the European Union, colour additives are regulated under Regulation (EC) No 1333/2008 on food additives, with purity criteria set in separate Commission directives. Internationally, the FAO/WHO Joint Expert Committee on Food Additives (JECFA) publishes Combined Compendium specifications for food colour additives, covering identification by chromatography and spectrophotometry, colouring matter content by spectrophotometry, and limits for water-soluble and organic solvent-soluble colour fractions. These internationally harmonised methods form the backbone of quality assurance testing protocols used by food manufacturers and testing laboratories worldwide.

For synthetic dyes specifically, regulatory frameworks differ across regions in terms of naming conventions and approved uses, but the requirement for batch-level purity testing prior to food use is a near-universal standard. This means the same Tartrazine approved as FD&C Yellow No. 5 in the United States is verified under equivalent analytical criteria to the E102 used in European food products – the chemistry of the purity test is consistent even where labelling rules differ.

What do you think? Given that both natural and synthetic food colours undergo similar spectrophotometric purity tests, does the source of a colour additive – natural versus synthetic – meaningfully change the level of trust you place in its safety? And as adulteration techniques become more sophisticated, particularly for high-value ingredients like saffron, should routine food colour testing go beyond spectrophotometry to include more advanced methods like HPLC or mass spectrometry as the standard baseline?

References

1. https://www.fao.org/4/a0691e/a0691e00.htm
2. https://www.fda.gov/industry/color-additives/color-additives-history
3. https://www.bfias.eu/scientific-methods-to-measure-colors
4. https://sensing.konicaminolta.us/us/blog/identifying-food-dyes-with-spectrophotometers/
5. https://pmc.ncbi.nlm.nih.gov/articles/PMC7022967/
6. https://pmc.ncbi.nlm.nih.gov/articles/PMC2586117/
7. https://link.springer.com/article/10.1007/s00217-002-0547-5
8. https://pmc.ncbi.nlm.nih.gov/articles/PMC8857520/
9. https://contractlaboratory.com/saffron-testing-ensuring-the-quality-of-flavor-scent-and-color/
10. https://www.mdpi.com/2304-8158/11/20/3245
11. https://www.hunterlab.com/blog/spectrophotometric-determination-of-saffron-adulteration-helps-prevent-food-fraud/
12. https://iacmcolor.org/safety-of-synthetic-certified-colors/
13. https://en.wikipedia.org/wiki/Tartrazine
14. https://en.wikipedia.org/wiki/Sunset_yellow_FCF
15. https://www.testronixinstruments.com/blog/spectrophotometers-for-food-dye-analysis/