Analytica Chimica Acta 464 (2002) 237–247

Automatic screening method for the rapid andsimple discrimination between synthetic

and natural colorants in foods

Mónica González, Mercedes Gallego, Miguel Valcárcel∗Analytical Chemistry Division, Campus de Rabanales, University of Córdoba, E-14071 Córdoba, Spain

Received 28 February 2002; received in revised form 13 May 2002; accepted 30 May 2002

Abstract

A simple screening method was developed to discriminate between synthetic and natural colorants present in foods in orderto reduce the use of expensive instruments such as a liquid chromatograph with diode array detection. A rapid flow systemwas proposed in which samples containing natural and synthetic colorants in an acetic acid medium were passed through awool/cotton column, where only synthetic colorants were retained and were then eluted with dilute ammonia. Yellow, red andgreen–blue–brown additives can be monitored at 400, 530 and 610 nm, respectively, with a conventional spectrophotometer.Complete discrimination (no false positives) between natural and synthetic colorants can be obtained for molar concentrationsof natural colorants (in the absence of synthetic ones) up to 2000 (yellow), 2000 (red) and 10 000 (brown) times that of thedetection limit (DL) of synthetic additives. The reliability of the method was established at five concentrations (between 0.5and 3 DL) of the synthetic colorants tartrazine (yellow), erythrosin B (red) and brilliant black BN (brown). For a cut-offconcentration of 2 DL, the percentage of false negatives ranges from 8 to 12%. Finally, the method was applied to screeningseveral fruit drinks and candies for the determination of synthetic colorants with a sampling frequency of 10 h−1.© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Colorants; Screening method; Photometry; Foods

1. Introduction

Since the way food appears has always been asimportant as how it tastes, modern food manufactur-ing has become very concerned with preserving theaspect of foods that have lost their natural colors dur-ing processing. By adding colorants, the appearancecan be preserved while also reducing batch-to-batchcolor variations, enhancing the natural color and pro-ducing consumer appeal for products that have no

∗ Corresponding author. Tel.:+34-957-218616;fax: +34-957-218606.E-mail address:qa1meobj@uco.es (M. Valcarcel).

natural color[1]. Food colorants are usually classifiedas either natural (or nature-identical) or synthetic.Natural colorants generally have a lower tinctorialstrength than synthetic colorants and are generallymore sensitive to light, temperature, pH and redoxagents[2]. Because over the last 20 years syntheticcolorants have increasingly been perceived as unde-sirable or harmful by consumers[3] and the EuropeanUnion and the United States have restricted the useof synthetic colorants as additives in foods, banningthe harmful ones, the application of natural pigmentshas become extremely important to the food industry[4,5]. Simple and rapid methods for the separationand determination of natural and synthetic colorants

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0003-2670(02)00494-4

238 M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247

are required in order to (i) establish whether thereare synthetic dyes present in foods and whether theyare permitted, (ii) determine their concentrations, (iii)confirm the absence of added dyes in foods wherethey are not declared and (iv) check on the stabilityof colorants during processing and storage.

Different methods for separating colorants fromfoods have been developed using organic solvent ex-traction procedures[6,8] and solid-phase extractionwith RP-C18 [9–12] or amino materials[12] (for nat-ural and synthetic colorants), and Amberlite XAD-2(for synthetic colorants)[13], although for qualita-tive studies the easiest option is dying polyamide[14] or wool [15,16]. The adsorption of syntheticcolorants (tartrazine, sunset yellow, ponceau 4R,amaranth and brilliant blue FCF) from drinks andcandies, on a polyamide sorbent[14] was carried outat pH 4 (adjusted with citric acid) and elution withan alkaline–ammoniacal solution. In another method[15], thirteen synthetic food colorants were isolatedfrom foods by adsorption on wool. After desorptionwith 10% ammonia solution and gentle warming,a spectrum of the resulting colorant solution wasrecorded and compared to reference spectra of purecolorants. Linear regression analysis was then usedto resolve the mixture spectrum into its constituentcomponents. The Spanish Ministry of Food, Fishand Agriculture’s (MAPA) official method for deter-mining synthetic colorants in wine involves manualadsorption on wool after laborious manipulation ofthe sample including boiling, cooling, neutralizationand extraction with ether prior to separation by paperchromatography[16].

The individual determination of colorants in foods isusually carried out using a spectrophotometric detec-tor [6,14,15,17]but the resolution of complex mixturesrequires separation in different extracts[6], the useof dual-wavelength and derivative methods[6,14,17],or chemometric approaches[14,15]. However, for theresolution of mixtures, the usual alternatives are chro-matographic techniques such as liquid chromatogra-phy (LC) with UV–VIS [8,10,11,18–20]detection,paper chromatography[16] or thin-layer chro-matography[12,13,21]and capillary electrophoresis[7,9,22].

Government bodies, inspection services and rou-tine laboratories are increasingly interested in ob-taining analytical systems that provide rapid and

reliable yes/no responses rather than detailed chemicalinformation. Screening systems are an interesting al-ternative because they are designed to filter samplesand select only those where the target analytes arepresent above or below a pre-set concentration thresh-old [23]. The advantages of sample screening systemsinclude reduction of costs, rapidity and simplicity[24,25]. In this work, the combined use of a continu-ous flow system and a spectrophotometer for samplescreening to discriminate between synthetic and natu-ral colorants is described for the first time. The methodis based on the classical characteristics of syntheticcolorants and natural colorants: synthetic colorantsdye natural materials such as cotton or wool whilenatural colorants do not. Therefore, a very simpleflow system that includes a column packed with thosenatural materials discriminates between both types ofcolorants; the natural ones (not retained) can be deter-mined in the first step and synthetic ones (retained) inthe second step, after their elution. Natural and syn-thetic colorants for yellow, red and green–blue–brownwere chosen as models, selecting a maximum wave-length for each color (400, 530 and 610 nm, respec-tively). The use of a diode array detector that measuresthe three wavelengths simultaneously does not sim-plify the method because green–blue–brown colorantsabsorb light at the same wavelengths as yellow and redcolorants.

2. Experimental

2.1. Apparatus

All spectrophotometric measurements were madeon a Shimadzu UV–VIS 160A recording spectropho-tometer (Kyoto, Japan) equipped with a Hellma(Jamaica, NY) flow cell (path length 10 mm, in-ner volume 18�l). The absorbance was individuallyrecorded at 400, 530 or 610 nm for yellow, red orgreen–blue–brown colorants, respectively.

The flow system consisted of a Gilson (Villiers-le-Bel, France), Minipuls-3 peristaltic pump furnishedwith poly(vinyl chloride) pumping tubes, a Rheodyne(Cotati, CA) 5041 injection valve, PTFE tubing of0.5 mm i.d., and laboratory-made adsorption columnspacked with sorbent. The columns were constructedfrom PTFE capillaries of 3 mm i.d., packed with 80 mg

M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247 239

of cotton or wool. A water bath (Selecta, Barcelona,Spain) was also employed.

2.2. Reagents and standard solutions

All experiments were performed with analytical-reagent grade chemicals or better. Synthetic colorants(tartrazine, erythrosin B, lissamine green B, brilliantblue FCF, indigo carmine and brilliant black BN), nat-ural colorants (curcumin, riboflavin,trans-�-caroteneand carminic acid) and glucose were all supplied bySigma–Aldrich (Madrid-Spain). All other reagents(ammonia, acetic acid, sodium acetate, aluminumsulfate, potassium sodium tartrate, petroleum etherand ethanol) were purchased from Merck (Darmstadt,Germany).

As caramel color (Class I; E-150a) was not com-mercially available, it was obtained by treatment ofglucose as follows: glucose (100 g) in Milli-Q wa-ter (10 ml) was heated in a stainless steel vessel upto 170◦C for 170 min with continuous stirring[26];color formation was monitored at 610 nm until the ab-sorbance (color intensity) ranged from 0.010 to 0.140.The color intensity was defined as the absorbance ofa 0.1%(w/v) solution of caramel color solid in waterin a 1 cm cell at 610 nm. When the color target wasreached, additional water (50 ml) was added to cooldown the caramel.

All wool and cotton packings were washed withpetroleum ether to eliminate the natural grease of thefibers[15,16]. In order to increase the adsorption ca-pacity of the wool and the reproducibility of the re-sults, the wool was treated[16] as follows: 10 g ofwool was stirred for 1 h with 500 ml of a solutionof 1 g l−1 aluminum sulfate and 1.2 g l−1 potassiumsodium tartrate in Milli-Q water; the mixture was leftstanding for 2–3 h and finally washed with water anddried at room temperature.

Stock standard solutions containing 1 mg ml−1 in-dividual colorants (tartrazine, erythrosin B, lissaminegreen B, brilliant blue FCF, indigo carmine, brilliantblack BN or caramel) and 0.2 mg ml−1 riboflavin wereprepared in Milli-Q water and 1 mg ml−1 of curcumin,trans-�-carotene and carminic acid in ethanol. Allstock solutions were stored in glass-stoppered bottlesat 4◦C in the dark. Solutions of various concentrationswere prepared daily by diluting the stock standard so-lutions in 1 mol l−1 acetic acid/sodium acetate buffer.

2.3. Sample preparation

Fruit drinks and candies were purchased at localmarkets in Spain. The samples were prepared bythe following methods. Fruit drinks were degassedin an ultrasonic bath for 15 min and then 1.5–4.0 ml(n = 5) was diluted in 25 ml of 1 mol l−1 aceticacid/sodium acetate buffer. Candies were ground in aceramic mortar and an amount accurately weighed at(ca. 0.25–0.5 g,n = 5) was dissolved in 10 ml of thebuffer solution by heating at 60◦C for 3 min; whenthe solution was cool, it was diluted to 25 ml with1 mol l−1 buffer. For both types of samples, aliquots of3 ml were completely aspirated into the flow systemshown inFig. 1.

2.4. Screening procedure

A continuous flow system coupled on-line to aphotometer was used (Fig. 1). A volume of 3 ml ofeither the natural/synthetic colorant standards mix-ture or of the treated samples, both in 1 mol l−1

acetic acid/sodium acetate at pH 4.7, containing0.2–6.0�g ml−1 colorant, was gently warmed in a wa-ter bath at 40◦C and continuously introduced into thesystem at 1.5 ml min−1 and propelled through the sor-bent column of wool or cotton, also heated at 40◦C. Inthis step, synthetic colorants were retained in the sor-bent column, but natural colorants were not retainedand, therefore, were able to be determined directly inthe effluent. The column was then rinsed with dis-tilled water for 1 min to remove potentially adsorbedinterferents and the remainder of the natural colorsthat could have impregnated the column. By switch-ing the injection valve, a 1 mol l−1 NH3 stream wasdriven toward the column, in the opposite direction tothe sample at 3.5 ml min−1 to elute the synthetic col-orants. The colorants were monitored at 400 (yellow),530 (red) or 610 nm (green–blue–brown). The eluentstream (1 mol l−1 NH3) was used as a blank, givingnegligible absorbance. Peak height was used as theanalytical signal. The column was conditioned with3 ml of 1 mol l−1 buffer, pH, 4.7, and occasionallywashed with 3 ml of ethanol (viz. after every 10 realsamples). Under these conditions, the sorbent columnremained active for 1 week (ca. 400 extractions). Tocheck whether the “yes” response corresponded toone or several compounds of similar colors, a liquid

240 M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247

Fig. 1. Manifold for screening synthetic (S)/natural (N) colorants in food samples: IV, injection valve; W, waste; LC–DAD, liquidchromatograph–diode array detector for confirmatory analyses.

chromatographic (LC) technique is necessary to sep-arate the target compounds, which can be monitoredsimultaneously at the three mentioned wavelengths.

3. Results and discussion

The proposed method is based on the fact that syn-thetic colorants dye pure wool or natural cotton whilenatural colorants do not; although natural colorantsmight initially adhere to these materials, they areeasily washed away with water[16]. However, foodsoften contain several colorants that together con-tribute to the final color: colorants of different colorsor colorants that are categorized under the same color.In order to resolve these mixtures of colorants (bothnatural and synthetic), they are grouped in accor-dance with the spectral zone where they demonstratewavelengths of maximum absorption. Preliminaryexperiments were focused on obtaining the absorp-tion spectra of all synthetic colorants studied in the1 mol l−1 NH3 medium. For the yellow colorant (tar-trazine) the absorption maximum was at 400 nm,for the red colorant (erythrosin B) at 530 nm andfor the green–blue–brown colorants (lissamine greenB, brilliant blue FCF, indigo carmine and brilliantblack BN), which show similar visible spectra, the

absorption maximum was located between 570 and630 nm. Wavelengths of 400, 530 and 610 nm for thethree large groups were selected for this study. Thespectra of the natural colorants (curcumin, riboflavin,trans-�-carotene, carminic acid and caramel) wereobtained in an acetic acid/sodium acetate medium andthe wavelengths cited above could be also employed.In addition, the 10 colorants studied are permittedand in fact are widely used in a great number offoodstuffs such as dairy products, sauces, mustards,bouillon cubes, soft drinks, liquors and candies.

3.1. Optimization of the flow system

Optimization studies were carried out for each col-orant and from the results compromise values of theexperimental variables were selected. The variablesinfluencing the system can be divided intro threegroups: those related to chemical variables, retentionunit and flow. The variables were studied by prepar-ing aqueous standard solutions of individual synthetic(tartrazine, erythrosin B, lissamine green B, brilliantblue FCF, indigo carmine and brilliant black BN)and natural (curcumin, riboflavin,trans-�-carotene,carminic acid and caramel) colorants, using the flowsystem ofFig. 1. For each standard, parallel testswere done with a column (100 mg) of treated wool or

M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247 241

Fig. 2. Effect of sample pH on the adsorption of synthetic colorants on a cotton sorbent column. Sample: a standard solution containing1�g ml−1 of each synthetic colorant.

cotton. To discriminate between synthetic and naturalcolorants the first variable studied was the samplepH, with the aim of obtaining maximum retention ofsynthetic colorants and minimal retention of naturalcolorants. For this purpose, 3 ml of colorant standardsolutions at concentrations of 1�g ml−1 were used;in all instances the eluent was a dilute ammonia so-lution. The effect of pH on the natural and syntheticcolorant’s sorption was studied over the range 1–10by adjusting each standard solution with dilute HNO3or NaOH as required; in all instances the absorbancewas measured in the eluate. No natural colorants wereretained over the entire range of pH for both columns.On the other hand, as can be seen inFig. 2, all syn-thetic colorants studied were completely retained be-tween pH 2 and 5 (adsorption efficiency ca. 100%), theoptimum range being shorter for tartrazine and indigocarmine. An acetic acid/sodium acetate buffer at pH4.7 was selected to ensure a more precise preparationof the sample with the detriment of a slightly decreasein the tartrazine signal. Because natural colorantswere not retained by both sorbent materials, they canbe identified qualitatively in the effluent of the column(acetic acid/sodium acetate buffer), and therefore, thefollowing variables were optimized only for individ-ual synthetic colorants by group of colors (yellow,

400 nm; red, 530 nm and green–blue–brown, 610 nm)at concentrations of 1�g ml−1. The concentration ofthe buffer only affected the retention of tartrazine anderythrosin B, which need at least a concentration of0.7 mol l−1 for optimal retention. Ammoniacal so-lutions at variable concentrations were employed aseluent (pH 7–12); erythrosin B is the colorant thatrequired the most restricted range (pH 10–12), andtherefore, this range was selected for all the colorants.So, 1.0 mol l−1 NH3 (pH 11.5) was chosen as elu-ent to ensure complete elution of the colorants, ormixtures of colorants, at high concentrations. The op-timum range of values as well as the selected valuesof all chemical variables are listed inTable 1.

In previous manual methods the colorants wereadsorbed/desorbed by wool by warming or boilingthem in water[15,16]; for this reason the influence oftemperature on the adsorption of synthetic colorantson cotton or wool was studied. The temperature ofadsorption was studied in the range of 15–60◦C byimmersing the sample and the wool/cotton columnin a water bath at the required temperature. The in-fluence of this variable on adsorption proved to benegligible but it was observed that the precision ofthe results was better at certain temperatures and forthis reason both the sample and the column were

242 M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247

Table 1Chemical and flow variables of the colorant screening method

Variable Optimumrange

Selected value

Sample pH 2.0–5.0 Acetic acid/sodium acetatebuffer pH 4.7

Acetic acid/sodium acetateconcentration (mol l−1)

0.5–2.0 1.0

Eluent (NH3) pH 10.0–12.0 11.5Adsorption temperature (◦C) 25.0–45.0 40.0Amount of cotton/wool (mg) 60.0–150.0 80.0Sample breakthrough (ml) 0.5–3.5 3.0Sample flow rate (ml min−1) 0.2–2.0 1.5Eluent flow rate (ml min−1) 2.8–4.5 3.5

maintained at 40◦C. Various columns containing dif-ferent amounts of sorbent (20–150 mg) were tested.Neither sorbent (wool or cotton) nor its amount af-fected the system greatly. No significant changes inabsorbance were noted above 60 mg of material butit was observed that changes in the compactness ofthe columns containing more than 100 mg of sorbentaltered the flow of the solutions through the columns.A serious limitation for systems with mini-columnsis the small amount of sorbent that they contain, af-fecting the breakthrough volume, which is defined asthe maximum sample volume which can be perco-lated through the sorbent column with a theoreticalrecovery of at least 99%. The sample’s breakthroughvolume was studied by fixing the amount of analytesat three different quantities (2, 5 and 8�g) and vary-ing the sample volume (0.5–10 ml). For both types ofcolumns and for all synthetic colorants good recov-eries were obtained up to 3.5 ml. The values selectedfor retention unit variables are listed inTable 1.

The influence of the sample flow rate on the sorp-tion efficiency resulted in very small variations for allsynthetic colorants up to 2 ml min−1. The flow rate ofthe ammoniacal solution (eluent) was varied between0.5 and 4.5 ml min−1. At a low flow rate the colorantwas more dispersed than at a high flow rate; on theother hand, when the flow rate was very high the res-idence time of the eluent in the column was not longenough for efficient elution, therefore, a medium rangeflow rate was employed. This variable had the great-est effect on tartrazine, erythrosin B, lissamine greenB and brilliant blue FCF. The selected flow rates arelisted inTable 1.

Finally, the adsorption efficiency for all syntheticcolorants was calculated by using the optimized vari-ables listed inTable 1and the manifold shown inFig. 1. First, 3 ml of the individual standard solutionscontaining 1�g ml−1 of each colorant was passedthrough the sorbent column. After each standard so-lution was processed, the column (wool or cotton)was cleaned with 3 ml of 1 mol l−1 NH3 to eliminatesorbed analytes. The adsorption efficiency was as-sessed (using absorbance measurements) by compar-ing the amount of each additive present in the standardsolutions before and after passing them through thesorbent column. The adsorption efficiency was 100%for all colorants (excepting tartrazine, 94%).

All the variables were optimized for the two natu-ral sorbents (wool and cotton). The results were sim-ilar for both sorbents but reproducibility was slightlyworse for wool than for cotton. It is also easier to packthe column with cotton and, once packed, the cottonstays more homogeneous within the column. More-over, cotton does not require treatment to be used. Forthese reasons cotton was chosen to calculate the ana-lytical characteristics, selectivity and reliability of themethod.

3.2. Sensitivity and selectivity of the method

Under the selected chemical and flow conditions(Table 1), the manifold depicted inFig. 1 was usedto obtain calibration results for the colorants studied.The graphs were constructed from results obtainedby introducing 3 ml of aqueous standard solutionscontaining various concentrations of each colorant(0.2–6�g ml−1 in the 1 mol l−1 acetic acid/sodiumacetate buffer (pH 4.7)); seven standards, each withthree replicates, were used for each calibration graph.The results obtained are listed inTable 2. Detectionlimits (DLs) (calculated as three times the standarddeviation of the background noise, determined usinga stream of 1 mol l−1 NH3, divided by the slope ofeach calibration graph) ranged from 6 to 15 ng ml−1.The repeatability of the screening method, expressedas relative standard deviation (R.S.D.), was checkedon 11 individual samples containing 1 or 2�g ml−1

of the colorants and was found to be ca. 4.5%.Natural colorants can be determined by color

group (yellow, red, green–blue–brown) in the cottoncolumn’s effluent because they are not retained in the

M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247 243

Table 2Analytical characteristics of synthetic colorant screening

Analytes 400 nm 530 nm 610 nm Linear rangea

(�g ml−1)Detection limita

(ng ml−1)R.S.D. (%)b

Slope Intercept Slope Intercept Slope Intercept

Tartrazine 0.376 0.009 – – – – 0.3–3.0 11 2.8Erythrosin B – – 0.392 0.008 – – 0.3–3.0 8 5.4Lissamine green B 0.080 0.003 0.069 0.002 0.331 0.006 0.3–3.5 9 4.7Brilliant blue FCF 0.128 0.002 0.093 −0.004 0.472 0.006 0.2–2.5 6 2.4Indigo carmine 0.039 −0.003 0.058 0.007 0.194 0.007 0.5–6.0 15 5.4Brilliant black BN 0.143 −0.003 0.205 0.004 0.271 0.004 0.4–4.0 11 6.1

a The linear range and detection limit (DL) correspond to the most sensitive wavelength; for detection of DL see text.b At mid-linear rangen = 11.

column itself; however, when present in high con-centrations they could be partially retained and notcompletely eliminated in the washing step. There-fore, the influence of natural colorants in the deter-mination of synthetic colorants of the same color,was investigated. For this purpose three syntheticcolorants—tartrazine, erythrosin B and brilliant blackBN—were selected as the model. Interferents in-creased the synthetic colorants’ absorbance in allinstances; the tolerance limit of each natural colorantwas taken as the largest amount yielding a responseless than the analytical signal plus three times itsstandard deviation for 1�g ml−1 of each syntheticcolorant. The natural yellow colorants curcumin,riboflavin and�-carotene were tolerated up to con-centrations of 150, 100 and 80�g ml−1, respectively,in the determination of 1�g ml−1 of tartrazine; thenatural red colorant carminic acid can be tolerated upto 100�g ml−1 in the determination of 1�g ml−1 oferythrosin B; caramel did not interfere in the deter-mination of 1�g ml−1 of brilliant black BN at highconcentration such as 600�g ml−1. The high selec-tivity of the method for synthetic colorants versusnatural colorants of the same color can be ascribed tothe characteristics of the cotton.

In summary, the proposed method can discriminatebetween natural and synthetic colorants present in afood, however, if there are mixtures of synthetic col-orants of different colors in the food (yellow withgreen–blue–brown or red with green–blue–brown) thequantification of the colors must be done mathemati-cally in agreement with the molar absorptivities (slopeof the calibration graphs) of those colorants as indi-cated inTable 2.

3.3. Discrimination between natural and syntheticcolorants

A substantial portion of the analytical informationrequested is qualitative responses. Because the evalua-tion norms (technological and toxicological) that con-trol natural colorants are generally more permissivethan those that control synthetic colorants, qualitativeresponses to yes/no questions such as “does the sam-ple contain synthetic colorants?”, or “does this foodcontain a banned synthetic colorant?” have becomeextremely relevant. The most basic qualitative answerwithin this screening system is the binary response tothe question, “is it synthetic colorant in the food?”. Themain error occurs when there is no synthetic colorantin the food but there are natural colorants that givefalse positives. To avoid false positives, it was neces-sary to establish at what threshold the concentrationof natural colorants might cause an erroneous “yes”response to the presence of synthetic colorants in thefood sample. First, it was indispensable to set up animaginary concentration of synthetic colorants to bethe cut-off concentration[27]. The cut-off is the con-centration corresponding to twice the detection limit(DL) of each synthetic colorant at each wavelength(22 ng ml−1 for yellow (tartrazine, 400 nm) and brown(brilliant black BN, 610 nm) colorants and 16 ng ml−1

for red colorants (erythrosin B, 530 nm)). A false pos-itive arises when the signal yields a “yes” response fora standard solution containing only natural colorantsat a concentration above the cut-off value (of syntheticcolorants). A systematic study was carried out using 50standard solutions for each concentration of the threenatural colorants chosen (curcumin, carminic acid and

244 M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247

Fig. 3. Discrimination between natural and synthetic colorants. Per-centage of false positives (n = 50) obtained for standard solutionscontaining only individual natural colorants (curcumin, carminicacid and caramel with yellow, red and brown colors, respectively)with regard to the detection limits (DL) of synthetic colorants.

caramel as yellow, red and brown, respectively). Asper the tolerated ratio obtained in the selectivity study,three concentration levels of the natural colorants cor-responding to 2000 DL, 5000 DL and 10 000 DL forcurcumin and carminic acid and 10 000 DL, 25 000DL and 50 000 DL for caramel were chosen. The re-sults obtained are shown inFig. 3 where at each con-centration level the reliability is given at a confidencelevel of 95%. As can be observed, the proportion offalse positives was only 12% at 5000 DL (which corre-sponds to ca. 50�g ml−1) for curcumin and carminicacid and 8% at 25 000 DL (which corresponds to ca.270�g ml−1) for caramel. No false positives for syn-thetics were obtained up to concentrations of 15 and20�g ml−1 for natural red and yellow colorants, re-spectively, or 110�g ml−1 for natural brown colorant.These concentrations are high enough to prove thatthe screening method is able to accurately discrimi-nate between natural and synthetic colorants.

3.4. Reliability of the screening method forsynthetic colorants

The confidence level of the proposed sample screen-ing system was established on the basis of the level offalse positives and negatives through a simple chemo-metric study[27], in which only synthetic colorants

were present in the sample. As in the aforementionedexperiments, the cut-off concentration was set at twicethe detection limit. A false positive arises when thesignal yields a “yes” response for a standard solu-tion containing the synthetic colorant at a level be-low the cut-off value and a false negative is producedwhen a standard solution containing the synthetic col-orant at a level above the cut-off level gives an ab-sorbance measurement lower than the cut-off signal.Three synthetic colorants (tartrazine, erythrosin B andbrilliant black BN with detection limits of 11.0, 8.0and 11.0 ng ml−1, respectively) were selected for thisstudy, with the objective of determining the reliabil-ity of the screening method for each color group atthe three wavelengths defined earlier (400, 530 or610 nm). A systematic study at five concentration lev-els (0.5 DL, 1 DL, 1.5 DL, 2 DL and 3 DL), wascarried out by using 50 samples at each concentrationof the three colorants.Fig. 4 shows the distributionof false positives and negatives obtained for each syn-thetic colorant. The percentage of false positives roseas the concentrations of each food colorant increased,being zero for the three synthetic colorants at 0.5 DL,4% (for tartrazine and erythrosin B) and 8% (for bril-liant black BN) at 1 DL and reaching 16% for thethree colorants at 1.5 DL. The confirmation of falsepositives is not problematic in screening methods be-cause they can be detected by the later confirmationmethod but the existence of false negatives affects thereliability of the method more directly because oncequalified as negative a sample is not re-tested. As canbe observed inFig. 4, the percentage of false nega-tives decreases with increase in the concentration ofthe three synthetic colorants, being zero at 3 DL inall instances. At the cut-off concentration of 2 DL theproportion of false negatives was higher for erythrosinB (12%) than for the other colorants (8%), probablydue to the fact that erythrosin B had the lowest DLvalue.

3.5. Application to food samples

The proposed method was applied to the screeningof synthetic and natural colorants in fruit drinks andcandies. These samples gave positive responses forsynthetic colorants quantified at 400, 530 or 610 nmaccording to their color. The labels on the fruit drinksand candies showed what natural or synthetic colorants

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246 M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247

Table 3Determination of synthetic colorants after food sample screening

Sample Label Screeningresponse

Concentrationfounda

Colorant Analytes

Fruit drinkApple Synthetic yellow Tartrazine Positive 14.0± 0.8

Synthetic blue Brilliant blue FCF Positive 6.2± 0.4

Pineapple Synthetic yellow Tartrazine Positive 33± 2Fruits Synthetic green Lissamine green B Positive 11± 1Orange Natural yellow �-Carotene Negative –Cola Natural brown Caramel Negative –Guava Natural red Carminic acid Negative –

CandyLemon Synthetic yellow Tartrazine Positive 99± 5

Natural yellow Curcumin Negative –

Orange Synthetic yellow Tartrazine Positive 75± 3Natural red Carminic acid Negative –

Blackberry Synthetic brown Brilliant black BN Positive 65± 5

a Average values± S.D. (n = 15) in mg l−1 for fruit drinks and mg kg−1 for candies.

were apparently contained but not the amounts or con-centrations; the positive list of colorant additives (nat-ural and synthetic) in soft drinks generally limits theconcentrations to 100 mg l−1 while the limit for can-dies is 300 mg kg−1 [4]. Samples that contained eithersynthetic or natural colorants were selected but only afew were found with a mixture of both (candies). Theresults of the screening method are listed inTable 3.As can be seen, positive responses were obtained forall samples that contained synthetic colorants andnegative responses were obtained for all samples thatcontained only natural colorants. Individual syntheticcolorants were easily quantified using their calibrationgraphs. When there were mixtures of colorants fromdifferent color groups present in the samples, as wasthe case in the apple drink (yellow and blue colorants),absorbance measurements at the wavelengths of eachcolorant were taken. Next, the concentration of theblue colorant was determined at 610 nm, which wasused to calculate its absorbance at the wavelength ofthe color group of the second colorant in the mixture(yellow at 400 nm) and then subtract the first colorant’scontribution to the total absorbance of the mixture(both yellow and blue colorants); finally the concen-tration of the second colorant was calculated using itscalibration graph. Average concentrations, calculatedfrom five individual amounts of each food sample and

analyzed in triplicate (n = 15), are listed inTable 3.Natural colorants were not quantified because the cot-ton column’s effluent was not monitored in the photo-meter.

As the data were consistent, it can be concludedthat the proposed screening method is accurate andcan, thus, be used to determine synthetic colorants ac-cording to their family color (viz. yellow, red, green–blue–brown) even when natural colorants are presentalthough it cannot directly identify the synthetic col-orant. As with all screening methods, positive resultsmust be confirmed by other, more sophisticated,techniques—for example, liquid chromatographycoupled with either a diode array detector or, ideally,with a mass spectrometer—that are able to separateand identify the colorants.

Acknowledgements

The authors gratefully acknowledge financial sup-port from Spain’s Dirección General de InvestigaciónCientıfica y Técnica (Grant BQU2001-1815). M.González also wishes to thank the Department ofChemical Engineering and Pharmaceutical Technol-ogy, University of La Laguna, Spain, for their supportin this research.

M. Gonzalez et al. / Analytica Chimica Acta 464 (2002) 237–247 247

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