Analysis of synthetic dyes in food samples by capillary zone electrophoresis Application Note Foods and Flavors Authors Rainer Schuster, Angelika Gratzfeld-Hüsgen Agilent Technologies, Waldbronn, Germany Abstract Synthetic food dyes were separated by capillary zone electrophoresis using an alkaline phosphate buffer as background electrolyte. The precision had a relative standard deviation of less than 0.5 % for the run-to-run migration times and 2 % for the peak areas with buffer replenishment after each run. The detection limit for the individual dyes was about 1 ng using a 50-µm internal diameter Agilent extended path length capillary. Compounds were detected at different wavelengths— 215, 410, 520 and 598 nm—and the identities of the individual dyes were confirmed using peak-purity routines and a UV-visible spectral library. Introduction Azo Dyes Triphenylmethane Dyes Color is a vital constituent of foods OH NaO3S SO Na and probably the first characteristic 3 HO perceived by the human senses. NaO3S NN - Almost all foods— from raw agricultu- SO3 ral commodities to finished produc- ts—have an associated color. Further, NaO3S many tests have shown that color can Amaranth E123 (FD&C Red No. 2) + organoleptically dominate the flavor of (C2 H5 )N N (C 2 H5 ) a food. Colors have been added to Patent blue E131 foods since ancient times and include HO chlorophylls, carotinoids, flavonoids O O NaO3 S NN and anthocyans extracted from diffe- C C SO2 Na rent plants. Today synthetic dyes have C=C NaO3 S HN HN widely replaced natural colors. These SO Na dyes are used to supplement and 3 Sulfonated Indigo enhance the natural colors destroyed Sunset yellow FCF E110 (FD & C Yellow No. 6) Indigo carmine E132 (FD & C Blue No. 2) during processing or storage, and Figure 1 substantially increase the appeal and Chemical structures of some common synthetic dyes. acceptability of foodstuffs where no natural colors exist, for example, soft fonic acid moiety that often contains Table 1 shows some of the most widely drinks or ice cream. But, synthetic unwanted byproducts from correspon- used synthetic dyes with their EC dyes are also used to redye food to ding impurities. Triarylmethane colors name (E-number), the US name mask decay, and aging effects or to are distinguished by their brilliance of (FD&C) and the color index number disguise poor products. color and high tinc- torial strength, but (CI). have poor light stability. The United Colors permitted for food use can be States (US) and the European To ensure compliance with regulatory divided in three categories: Community (EC) are the two major requirements, analytical methods are 1. synthetic dyes—described in this geographical areas where color regu- required to determine nature and study, lations are enforced. The lists of concentration of a colorant in a food 2. natural colors (for example, caramel admitted colors are updated conti- product. Paper chromatography, or beetroot) and naturally identical nuously because of suspected carci- thin-layer chromatography, and high- colors (canthaxanthine), and nogenicity. performance liquid chromatography 3. inorganic pigments. (HPLC) have helped the analyst in the For example: examination of synthetic colors.1,2,3 Synthetic colors in food products are • amaranth (E123, FD&C Red No. 2)— predominantly azo and triarylmethane one of the most widelyused red Conventional HPLC separations use dyes. These are mostly acidic or anio- color—was delisted in the US in reversed-phase ion-pair chromatogra- nic dyes containing carboxylic acid, 1970, phy with silica ODS or RP-2 material sulfonic acid or hydroxy groups, which • indigo carmine (E132, FD&C Blue and a quaternary ammonium salt as 4,5 form negatively-charged colored ions No. 2) was delisted in 1980, the counterion. at basic pH ranges. Capillary zone • tartrazine (E102, FD&C Yellow No. 5) Several workers have adopted this electrophoresis (CZE) is an ideal tool was subjected to rigorous tests, and approach for different applications because it can separate all the diffe- • in 1990 the use of erythrosine (E127, using different sample-preparation rent functional groups in one analysis FD&C Red No. 3) was discontinued. techniques.6,7,8,9 For example, in the within a short run time. CZE separates Figure 1 shows the chemical structu- wool-fiber method the sample is compounds based on charge and size. res of some common synthetic dyes. extracted in a tartaric acid solution The range of color shades covered by In the EC colors are regulated by and a wool fiber is used to absorb the the azo group includes red, orange, council directives on coloring matters. colors of interest.8 After washing with yellow, brown and blue-black. These Of relevant interest are the proposals water and methanol the colors are dyes are prepared by coupling diazoti- in foodstuff directives for international washed off with a diluted ammonia zed sulfanilic acid to a phenolic sul- harmonization of colorant regulations. 2 solution, evaporated to dryness, and EC Name Food, Drug & Cosmetics CI dissolved in water or ethanol. E102 Tartrazine FD&C Yellow No. 5 19140 The polyamide method involves E103 Chryosine 14270 extracting the sample with acetic acid E104 Quinoline yellow FD&C Yellow No. 10 47005 and polyamide powder, whereby a E105 Yellow 13015 chromatographic column is filled with E110 Sunset yellow FCF FD&C Yellow No. 6 15985 polyamide and washed with water and E111 Sunset yellow FD&C Orange No. 2 15980 methanol.9 The colors are extracted E122 Azorubine Carmoisine 14720 with an ammonia solution, evaporated E123 Amaranth FD&C Red No. 2 16185 to dryness, and dissolved in water or E124 Ponceau 4R Ponceau 4R 16255 ethanol. E125 Scarlet red Scarlet red 14815 E126 Ponceau 6R 16290 Both wool-fiber and polyamide extrac- E127 Erythrosine FD&C Red No. 3 45430 tion are official methods in Germany E131 Patent blue V FD&C Violet No. 1 42051 and described in detail in §35 LMBG.10 E132 Indigo carmine FD&C Blue No. 2 73015 In the US individual methods for colors E142 Acid brilliant green FD&C Green No. 3 in food are described in the handbook E151 Black PN 28440 Ponceau SX FD&C Red No. 4 14700 of official methods of analysis of 11 Table 1 AOAC. Ion pair extraction with tetra- List of commonly-used colorings in EC and US classifications with color index numbers (CI). butyl-ammonium phosphate and back extraction using perchlorate has been 1 E 131 12 mAU reported by Puttemans et al. 40 1 2 2 E 142 3 E 127 4 E 132 5 E 105 Experimental 30 14 16 18 6 Carminic acid 6 3 12 7 E 103 Separations were performed using an 15 598 nm 20 8 E 110 Agilent CE system with a builtin diode- 13 9 E 111 array detector and Agilent 7 17 520 nm 10 E 125 ChemStation software. Separations 10 11 E 104 5 11 12 E 122 were achieved with fused-silica 50-µm 410 nm 13 E 124 id capillaries (64.5 cm total length, 56 cm 0 14 Naphthol yellow effective length) with an extended 910 15 E 123 8 16 E 151 path length or bubble cell at the 4 215 nm detector end. All separations were -10 17 E 102 18 E 126 performed at 30°C using a 10 mM 246 81012 sodium phosphate with 5 mM sodium Time [min] hydrogen carbonate buffer at pH 10.5. Figure 2 Capillaries were preconditioned by Electropherogram of a standard sample. flushing with 1 M sodium hydroxide for 3 minutes followed by running buffer Chromatographic conditions for 10 minutes. Samples were intro- Buffer: 10-mM sodium borate with 5-mM sodium hydrogen carbonate at pH 10.5 duced hydrodynamically with 100 and Electric field: 465 V/cm Capillary: I = 56 cm, L = 64.5 cm, id = 50 µm 200 mbars followed by a 200 mbars Injection: 100 mbar x s buffer plug. The samples were ana- Temperature: 30 ºC lyzed with an applied voltage of 30 kV Detection Signal: 215/50 nm, 520/60 nm red, 410/60 nm yellow, 598/4 nm blue and detected at 215/50 nm, 520/60 nm, Reference: off 410/60 nm and 598/4 nm. After each run the inlet and outlet vials were within 0.5 minutes was performed to figure 2 which shows the separation replenished and the column was avoid possible thermal expansion and of common synthetic dyes: patent rinsed with the separation buffer for 1 loss of sample. Details of the separa- blue E131, acid brilliant green E142, minute. A voltage ramp from 0 to 30 kV tion conditions are listed alongside erythrosine E127, indigo carmine E132, 3 chryosine E103, sunset yellow FCF a pilot wavelength for spectral acqui- ed at different pH values and concen- E110, sunset yellow E111, scarlet red sition and as a universal monitoring trations. The optimum buffer was E125, quinoline yellow E104, azorubine wavelength (for example, sweeteners found to be 10 mM phospate at pH E122, ponceau 4R E124, amaranth and preservatives). 10.5. E123, black PN E151, tartrazine E102 and ponceau 6R E126. The E numbers The spectral information of the com- However, the reliability data showed a correspond to the EC regulations for pound of interest was compared with clear trend to shorter migration times additives in food. the spectra in a library—compounds depending on the pH changes of the were analyzed not only by migration buffer even when replenishment of the time but also by spectral compari- two vials was used. This was mainly Results and discussion son—and a peak-purity check was due to the instable pH of this buffer performed by overlaying spectra taken system.