🎨 Synthetic Food Dyes (HPLC)
In short
Identification and quantitative determination of synthetic food dyes (tartrazine, sunset yellow, azorubine, allura red etc. The test is performed according to PN-EN 12878:2014 (oraz metody wewnętrzne oparte na HPLC-DAD); Determination range: 0.5–500 mg/kg (depending on dye and matrix). The procedure comprises 10 steps (about 1 h 45 min in total); it is used for: Official food control by Sanitary Inspection (SANEPID), Quality control of carbonated drinks, juices, syrups, Testing of sweets, candies, jellies for azo dyes.
At a glance
- Standard: PN-EN 12878:2014 (oraz metody wewnętrzne oparte na HPLC-DAD)
- Category: Food Chemistry
- Procedure steps: 10
- Total time of stages: 1 h 45 min
- Determination range: 0.5–500 mg/kg (depending on dye and matrix)
- Limit of detection (LOD): 0.1–0.5 mg/kg
- Limit of quantification (LOQ): 0.5–1.0 mg/kg
Overview
Synthetic food dyes (E100–E199) are among the most frequently controlled food additives due to potential health risks — allergic reactions, hyperactivity in children (so-called "Southampton six") and possible carcinogenic effects of some azo dyes. Regulation (EC) 1333/2008 specifies permitted dyes and their maximum doses in individual food categories.
The HPLC method with UV-Vis (DAD — diode array detector) detection is the reference analytical technique for simultaneous identification and quantitative determination of many synthetic dyes in one analysis. Reversed-phase chromatography (RP-HPLC) on a C18 column with gradient elution allows separation and quantification of all permitted synthetic dyes in approximately 20–30 minutes.
Most commonly determined dyes are: tartrazine (E102), quinoline yellow (E104), sunset yellow (E110), azorubine (E122), ponceau 4R (E124), allura red (E129), brilliant blue FCF (E133), indigotine (E132) and green S (E142). The method requires preliminary extraction of dyes from the food matrix, purification on SPE cartridges and chromatographic analysis.
Accredited laboratories performing these tests participate in proficiency testing programs (PT) organized by FAPAS and LGC, ensuring reliability of results at the international level.
Method principle
Synthetic dyes are extracted from a food sample with a mixture of water/methanol/ammonia, purified on SPE cartridges (polyamide or C18) and separated by liquid chromatography on a C18 column with gradient elution (mobile phase: ammonium acetate buffer pH 7 / acetonitrile). Detection using a DAD (diode array detector) at multiple wavelengths (427 nm, 482 nm, 520 nm, 630 nm) allows dye identification based on absorption spectrum and retention time. Quantitative determination — from calibration curve of standards.
Applications
- Official food control by Sanitary Inspection (SANEPID)
- Quality control of carbonated drinks, juices, syrups
- Testing of sweets, candies, jellies for azo dyes
- Analysis of spices and sauces (curcumin vs tartrazine)
- Control of children's food (EU restrictions — additional labeling)
- Testing of imported food for unauthorized dyes (Sudan I–IV)
- Monitoring of dyes in dietary supplements
Key parameters
| Parameter | Value |
|---|---|
| Determination range | 0.5–500 mg/kg (depending on dye and matrix) |
| Limit of detection (LOD) | 0.1–0.5 mg/kg |
| Limit of quantification (LOQ) | 0.5–1.0 mg/kg |
| Column | C18 (250 × 4.6 mm, 5 µm) or UHPLC C18 (100 × 2.1 mm, 1.8 µm) |
| Detection wavelengths | 427, 482, 520, 630 nm (DAD: full spectrum 200–700 nm) |
| Analysis time | 20–30 min (HPLC), 8–12 min (UHPLC) |
Standard
- Standard number
- PN-EN 12878:2014 (oraz metody wewnętrzne oparte na HPLC-DAD)
- Title (PL)
- Oznaczanie syntetycznych barwników w żywności metodą HPLC
- Title (EN)
- Determination of synthetic food colorants by HPLC
Step-by-step procedure
-
Sample preparation
Homogenize food sample (blender). Weigh 2–5 g into volumetric flask.
-
Dye extraction
Add 20 mL of methanol/water/ammonia mixture (70:25:5). Place in ultrasonic bath for 15 min. Filter.
-
SPE cleanup
Condition SPE cartridge (5 mL methanol, 5 mL water). Load extract. Rinse 5 mL water. Elute dyes with 5 mL methanol with ammonia.
-
Concentration and dissolution
Evaporate eluent to dryness under nitrogen stream (40°C). Dissolve residue in 1 mL mobile phase A. Filter through 0.22 µm into vial.
-
Standard preparation
Prepare calibration curve (5 points) from certified dye standards in range 0.5–50 µg/mL.
-
HPLC analysis
Inject 10–20 µL of sample onto C18 column. Gradient elution: 0→50% ACN in ammonium acetate buffer pH 7 in 25 min. Flow rate 1.0 mL/min.
-
Detection and identification
Record chromatogram at 427, 482, 520 and 630 nm. Identify dyes by retention time and UV-Vis absorption spectrum (DAD library).
-
Quantitative determination
Calculate dye concentrations from calibration curve. Include dilution factor and recovery.
-
Quality control
Analyze blank sample, spiked sample and CRM. Recovery 90–110%, repeatability RSD ≤ 5%.
-
Report
Prepare report with results in mg/kg, uncertainty and comparison with limits according to Regulation 1333/2008.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with DAD detector | Agilent 1260 Infinity II + DAD G7117C, Shimadzu Nexera + SPD-M40, Waters Alliance + 2998 PDA | 150 000–350 000 PLN |
| Chromatographic column C18 | Agilent Zorbax Eclipse Plus C18, Waters Symmetry C18, Phenomenex Kinetex C18 | 1 500–3 500 PLN |
| SPE system (solid phase extraction) | Supelco Visiprep SPE manifold, Oasis HLB or polyamide cartridges | 2 000–5 000 PLN (manifold) |
| Ultrasonic bath | Bandelin Sonorex, Elma S, Branson 2800 | 3 000–8 000 PLN |
| Analytical balance | Mettler Toledo MS205DU, RADWAG AS 220.R2 | 8 000–25 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Dye standards (certified CRM) | — | Tartrazine E102, sunset yellow E110, azorubine E122, ponceau 4R E124, allura red E129, brilliant blue E133 — Sigma-Aldrich, Dr. Ehrenstorfer |
| Acetonitrile (HPLC grade) | 75-05-8 | Purity ≥ 99.9%, for mobile phase and extraction |
| Methanol (HPLC grade) | 67-56-1 | For extraction of dyes from food matrix |
| Ammonium acetate | 631-61-8 | For preparation of mobile phase buffer (pH 7.0, concentration 0.02 mol/L) |
| Ammonia 25% | 1336-21-6 | For alkalinization of extraction phase — facilitates dye dissolution |
| SPE cartridges (polyamide/C18) | — | For purification of extracts from fat and protein matrices, 500 mg/6 mL |
Health and safety (OHS)
- Acetonitrile — toxic (inhalation, skin contact), work under fume hood
- Methanol — toxic, flammable (flash point 11°C) — fume hood, no open flame
- Ammonia — corrosive, irritating to respiratory tract — fume hood, glasses, gloves
- Dye standards — some azo dyes may be sensitizing, use gloves
- HPLC waste (mobile phase) — collect in organic waste containers
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN 12878:2014 (oraz metody wewnętrzne oparte na HPLC-DAD) — “Determination of synthetic food colorants by HPLC”.
How does this method work?
Synthetic dyes are extracted from a food sample with a mixture of water/methanol/ammonia, purified on SPE cartridges (polyamide or C18) and separated by liquid chromatography on a C18 column with gradient elution (mobile phase: ammonium acetate buffer pH 7 / acetonitrile). Detection using a DAD (diode array detector) at multiple wavelengths (427 nm, 482 nm, 520 nm, 630 nm) allows dye identification based on absorption spectrum and retention time.
What is the measuring range and accuracy?
Determination range: 0.5–500 mg/kg (depending on dye and matrix); Limit of detection (LOD): 0.1–0.5 mg/kg; Limit of quantification (LOQ): 0.5–1.0 mg/kg; Column: C18 (250 × 4.6 mm, 5 µm) or UHPLC C18 (100 × 2.1 mm, 1.8 µm).
How long does the test take?
The times given for the individual stages add up to approximately 1 h 45 min. The procedure comprises 10 steps.
What equipment is required?
HPLC system with DAD detector, Chromatographic column C18, SPE system (solid phase extraction), Ultrasonic bath, Analytical balance. Indicative cost of the core instrument (HPLC system with DAD detector): 150 000–350 000 PLN.
Where is this test used?
Official food control by Sanitary Inspection (SANEPID); Quality control of carbonated drinks, juices, syrups; Testing of sweets, candies, jellies for azo dyes; Analysis of spices and sauces (curcumin vs tartrazine); Control of children's food (EU restrictions — additional labeling); Testing of imported food for unauthorized dyes (Sudan I–IV); Monitoring of dyes in dietary supplements.
What safety precautions apply?
Acetonitrile — toxic (inhalation, skin contact), work under fume hood; Methanol — toxic, flammable (flash point 11°C) — fume hood, no open flame; Ammonia — corrosive, irritating to respiratory tract — fume hood, glasses, gloves; Dye standards — some azo dyes may be sensitizing, use gloves; HPLC waste (mobile phase) — collect in organic waste containers.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN 12878.