Beruflich Dokumente
Kultur Dokumente
Received 1 August 2006; received in revised form 29 September 2006; accepted 2 October 2006
Available online 6 October 2006
Abstract
A reversed-phase high performance liquid chromatographic method for the successful separation and determination of 13 synthetic food colorants
(Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Amaranth E 123, Ponceau 4R E 124, Erythrosine E 127, Red
2G E 128, Allura Red AC E 129, Patent Blue V E 131, Indigo Carmine E 132, Brilliant Blue FCF E 133 and Green S E 142) was developed. A C18
stationary phase was used and the mobile phase contained an acetonitrile–methanol (20:80 v/v) mixture and a 1% (m/v) ammonium acetate buffer
solution at pH 7.5. Successful separation was obtained for all the compounds using an optimized gradient elution within 29 min. The diode-array
detector was used to monitor the colorants between 350 and 800 nm. The method was thoroughly validated. Detection limits for all substances
varied between 1.59 (E 142) and 22.1 (E 124) g L−1 . The intra-day precision (as R.S.D.r ) ranged from 0.37% (E 122 in fruit flavored drink
at a concentration of 100 mg L−1 ) to 4.8% (E 142 in icing sugar at a level of 0.9 mg kg−1 ). The inter-day precision (as R.S.D.R ) was between
0.86% for E 122 in fruit flavored drink at 100 mg L−1 and 10% for E142 in jam at a concentration of 9 mg kg−1 . Satisfactory recoveries, ranging
from 94% (E 142 in jam) to 102% (E 131 in sweets), were obtained. The method was applied to the determination of colorants in various water-
soluble foods, such as fruit flavoured drinks, alcoholic drinks, jams, sugar confectionery and sweets, with simple pre-treatment (dilution or water
extraction).
© 2006 Elsevier B.V. All rights reserved.
Keywords: Liquid chromatography; Diode-array detector; Synthetic dyes; Food analysis; Confectionary; Soft drinks
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2006.10.002
104 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110
Fig. 1. Chemical structures, common names, E (European Community) and CI (Colour Index) numbers of synthetic food colorants studied.
and 10 mg L−1 were also prepared by mixing and dilution of 2.6. Validation of the method
appropriate aliquots from standard stock solution of each sub-
stance. All solutions were stored at 4 ◦ C in the dark and were The instrument calibration was carried out using different
stable at least for 2 months. concentrations between 0.10 and 60 mg L−1 of each compound,
All samples were obtained from market control and included with two replicates per concentration, in order to determine the
fruit flavored drinks, alcoholic drinks, jams, sweets and sugar linear region of each colorant. Calibration curves were also pre-
confectionary. A 10-mL sample of drink was diluted with water pared with the mixed standards solutions at concentration levels
in a volumetric flask of 50 mL. The samples were degassed, if 0.10, 0.20, 0.50, 0.80 and 1.0 mg L−1 to check the selectivity of
necessary, by strong stirring. The solid samples were homoge- the method. The calibration curve of each colorant was used for
nized. A portion of 10 g of jam, sweet or a confectionary product the validation experiments and quantification.
was accurately weighted and dissolved in 50 mL of water. The The limit of detection (LOD) of each compound was calcu-
sample solution was placed in ultrasonic bath for 15 min for the lated as three times the standard deviation of the response of
complete extraction of the colorants. These solutions were fil- 10 independent replicate analyses of a 0.060 mg L−1 standard
tered through a folded paper filter and the filtrate was collected solution.
in volumetric flask of 50 mL. Intra-day and inter-day precision was assessed by analyzing
All solutions were injected after filtering through 0.45-m six replicates of a sample during one day (n = 6, intra-day pre-
disposable syringe filters. cision) or two replicates by three analysts in two different days
(n = 2, k = 3, j = 2, inter-day precision), correspondingly. Differ-
2.5. Determination of colorant purities ent colorants (E 102, E 110, E 122, E 124, E 129, E 131 and
E 142) and food matrices (flavoured drinks, liqueurs, cherries,
Purities of colorants were determined according to the iden- jams, sweets and icing sugar) were chosen for these experiments.
tification method reported in Regulation 95/45/EC [34]. This In order to evaluate the trueness of the method, recovery
method is based on spectrophotometric absorbance measure- experiments were performed. Three different food samples (fruit
ment of a diluted standard solution (i.e. 10 mg L−1 ), for which flavoured drink, jam and flavoured sweets) were spiked with E
Beer–Lambert law is valid. The percentage amount of a colorant 122, E 142 and E 131, respectively, at fortification levels of 10,
purity is given by the equation: 50 and 100 mg kg−1 and analyzed accordingly (each sample six
times at each fortification level).
DF × A
%purity = × 105 (1)
A1%
1 cm × C 3. Results and discussion
where DF is the dilution factor of measured solution from stock
3.1. Determination of colorant purities
standard solution, A the absorbance of measured solution (A < 1)
with reference to water, the expression A1%
1 cm represents the spe- All colorant standards were analyzed to determine their col-
cific absorbance of an 1% (10 g L−1 ) aqueous solution of the
orant content. The procedure using a spectrophotometer enabled
colorant at the prescribed wavelength using a path length of
easy calculation of colorant purities, presented in Table 2. The
1 cm at 20 ± 1 ◦ C, and C is the concentration of stock solution
purity of the individual colorant standards ranged from 52.5% (E
expressed as the mg of the unpurified colorant in 100 mL dis-
102) to 97.0% (E 123). The low colorant content is often a conse-
tilled deionized water. The A1%1 cm value for each compound is quence of the specific manufacturing process. The by-products
presented in Table 2 [34].
are usually inorganic salts, such as NaCl [22].
Table 2
Dissociation constants, specific absorbance, A1% 3.2. Optimization of the separation
1 cm , and calculated purities of
synthetic colorants studied
A simple method for the determination of all permitted syn-
Colorant pKa ± S.D.a A1%
1 cm Purity (%)
thetic food colorants is advantageous for practical application.
E 102 9.40 ± 0.01 530 52.5 Reversed-phase liquid chromatography (RP-LC) is suitable for
E 104 n.a.b 865 69.9 the analysis of these compounds, if conditions are chosen in
E 110 10.36 ± 0.01 555 89.2
E 122 n.a. 510 83.6
which the ionized analytes are able to form neutral molecules,
E 123 10.36 ± 0.02 440 97.0 taking into account their available dissociation constants, shown
E 124 11.24 ± 0.01 430 56.9 in Table 2 [35]. Hence, the addition of an ion-pairing reagent,
E 127 n.a. 1100 84.2 which makes the resolution more complicate and causes a vari-
E 128 n.a. 620 67.5 ety of problems [36], is not always necessary.
E 129 11.35 ± 0.01 540 84.6
E 131 7.67 ± 0.02 2000 88.1
The main characteristics of the colorants that should be taken
E 132 n.a. 480 62.3 into account for an efficient separation are their hydrophobic-
E 133 n.a. 1630 60.3 ity and the presence of acidic and alkaline groups (Fig. 1). The
E 142 n.a. 1720 78.5 hydrophobicity of non-azo compounds is weaker than those of
a Dissociation constants ± standard deviations. azo colorants. In addition, the hydrophobicity of colorants with
b n.a.: not available. naphthalene ring is stronger than those with benzene ring [37].
K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110 107
Under a RP-LC separation, the more polar compounds elute the width (w) and the peak purity factor (PP) of each peak were
first. However, since the chromatographic determination of the calculated. The peak purity factor is produced from the compar-
colorants is performed, normally, at pH around 7, the acidic ison of the absorbance spectrum of a compound in mixture with
and alkaline groups present in the molecules can change the the corresponding spectrum in its individual standard solution.
elution sequence [37]. Thus, under the conditions of the cur- In addition, the resolution coefficient (Rs ) for two successive
rent experiments, colorants are carried along with the organic peaks was determined.
solvent (mixture of methanol:acetonitrile). The addition of ace- Two main resolution problems were observed when the first
tonitrile was found to improve significantly the asymmetry of gradient elution program was applied. The colorants E 128 and
the peaks, which has also been noticed when used in methanol E 142 were co-eluted and, although they were determined in
[38]. However, the addition of an inorganic electrolyte to the different wavelengths, a mutual interference appeared. The res-
mobile phase, as a modifier, is necessary in order to improve olution of colorants E 122 and E 133 was also unsatisfactory.
the separation of ionizable species and to obtain the separation However, in this case, only E 133 interfered with the determi-
in reasonably short analysis time. Ammonium acetate buffer nation of E 122. These conclusions were also verified from the
has been used as a modifier for the purification and separa- low peak purities values of E 128, E 142 and E 122, the high
tion of seven azo colorants by RP-HPLC and preparative liquid peak purity value of E 133 and the unsatisfactory Rs values for
chromatography [39]. It was also used (at a pH 5) for the sep- these two couples of colorants (0.30 and 0.49, respectively).
aration and determination of two (E 102 and E 110) [38] or The resolution problems were solved by decreasing grad-
five azo food dyes (E 102, E 104, E 110, E 122 and E 124) ually the amount of the organic solvent (mobile phase B) in
in soft drinks by RP-HPLC [23]. A gradient elution prepared gradient programs. Consequently, the time of analysis increased.
by a mixture of sodium acetate buffer (0.1 M and pH 7) with Seven experiments were performed for which the rate of increase
acetonitrile was used for the successful separation of 14 syn- of solvent B gradually decreased from 4.3% B per min in the
thetic food colorants in standard solutions [22]. It was also used first experiment to 2.6% B per min in the last experiment (data
for the successful determination of eight azo-colorants in food- not shown). Finally, a combination of two experiments resulted
stuff by liquid chromatography–electrospray-mass spectrometry in the final optimized gradient program, which is presented in
(LC–ESI-MS), where it was found that excellent separation, Table 2. Use of this program resulted in an efficient separa-
with symmetrical peak shapes, was achieved only in the pres- tion with no interferences for all colorants. The chromatogram
ence of ammonium acetate and acetic acid [40]. The optimum and the chromatographic characteristics obtained from the opti-
concentration of ammonium acetate in terms of plate number mized gradient program are presented in Fig. 2 and Table 3,
and peak symmetry was reported to be around 0.1 M [39]. At respectively. The peak purity (PP) factor is greater than 0.999
higher concentrations of ammonium acetate, retention times are for all compounds. The simultaneous determination of the col-
increased, likely due to an enhanced “salting-out” effect and so orants E 128 and E 142 have been achieved and E 133 no longer
enhance the interaction of the analyte with the stationary phase. interferes with the determination of E 122. The elution order of
This effect was also observed in the separation of Tartrazine (E E 133 and E 104III has been changed. These compounds seem
102), Brilliant Blue (E 133) and Sunset Yellow (E 110) by a C18 to be co-eluted (small Rs value), but from their high PP values
column with the use of phosphate as buffer in the mobile phase and the fact that they have different absorption spectra and they
[37]. The acetate buffer solution is also stable; it does not inter- are determined in different wavelengths, obviously, they do not
act with the HPLC system and is adequate for UV absorbance interfere each other. Along with the PP factor (the purity of the
measurements. All colorants are present as neutral compounds absorption spectra), retention times could also be used for qual-
at pH 7.5, in order a reversed phase mechanism to take place. itative analysis for all the compounds since their repeatability is
Decreasing the pH of the aqueous portion of the mobile phase very good. The R.S.D. of tR (n = 10) was always less than 0.32%
resulted in poor resolution of the LC separation of the azo dyes (E 102) (Table 3).
and the data could not be used for quantitative analysis [37,39].
Therefore, no further pH optimization of the mobile phase A
was attempted.
A 10 mg L−1 mixed standard solution containing the 13 col-
orants was used to study the optimum conditions of separation.
The initial gradient elution program used was as follows. For
the initial 2 min, the mobile phase A (ammonium acetate buffer)
goes through the column for elution of inorganic compounds.
Between 2 and 25 min, a gradient is carried out and for the last
2 min (25–27 min) the mobile phase B passes through the col-
umn for the elution of possible organic impurities. Finally, a
2-min equilibrium phase of the column is taking place for the
next run.
The elution of the colorants was observed in a chromatogram Fig. 2. Chromatogram of a mixed standard solution using the optimized gra-
that was obtained by scanning the wavelength range from 350 dient program of Table 2. The concentrations of all colorants were 10 mg L−1 .
to 800 nm, continuously. From software, the retention time (tR ), Identification of the peaks and their corresponding tR are given in Table 3.
108 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110
Table 3
Chromatographic data of the colorants with the optimized gradient program of Table 1
Peak Colorant λmax (nm) tR ± S.D. (min) (n = 10) w (min) Rs Peak purity
Table 4
Limits of detection, linear range, calibration equations and coefficients of determination (R2 ) of all colorants
Colorant LOD (g L−1 ) Linear range (mg L−1 ) Calibration equation R2
Many of colorants have isomers that appear in the chro- E 127, E 131, E 133 and E 142, have been characterized from
matogram as small peaks. The colorant Quinoline Yellow (E shorter linear ranges. Limits of detection for all substances var-
104) exhibits three peaks corresponding to isomers, probably ied between 1.59 g L−1 for E 142 and 22.1 g L−1 for E 124.
disulfo- and monosulfo-derivatives (E 104I, peak 4, E 104II, In case of Quinoline Yellow (E 104) only the E 104I isomer was
peak 7 and E 104III, peak 13 in Fig. 2) [22]. The unspec-
ified peaks shown in chromatogram were identified by their
absorbance spectra and were found to correspond to isomers Table 5
of E 132 (15.8 min), E 142 (19.4 min), E 133 (25.8 min), E 127 Precision data (intra-day, as R.S.D.r and inter-day, as R.S.D.R ) and concentration
levels (C) of seven colorants in various food matrices
(26.1 min) and E 131 (26.3 min) (Fig. 2).
Colorant Matrix Concentration R.S.D.r R.S.D.R
level (C) (%) (%)
3.3. Validation of the method
13.6 1.2 3.9
Sour cherry flavoured
Calibration equations of mixed standard solutions, coeffi- E 122 47.5 0.53 1.1
drink (mg L−1 )
97.6 0.37 0.86
cients of determination (R2 ), linear range and the limits of
detection of all colorants are presented in Table 4. Calibration E 102 Icing sugar (mg kg−1 ) 12.4 1.4 2.2
equations were calculated using the peak area of the substances. E 110 Icing sugar (mg kg−1 ) 10.1 1.4 3.9
E 142 Icing sugar (mg kg−1 ) 0.94 4.8 9.3
In case of E 104, the sum of the peak areas of the three iso- E 124 Liqueur (mg kg−1 ) 14.4 2.4 3.5
mers was used. The slopes of calibration equations of mixed E 129 Cherries (mg kg−1 ) 73.6 1.9 6.5
solutions were compared with t-test with those calculated from
4.7 2.7 6.2
the measurement of individual standard solutions (concentra- E 131 Sweets (mg kg−1 )
72.2 1.9 3.6
tion levels 0.10–60 mg L−1 ) and it was found that they do not
E Jam 9 1.9 10
differ significantly. The linear range is different between the ana- (mg kg−1 ) 84.5 0.90 3.4
142
lytes. In general, colorants that have high A1% 1 cm value, such as
K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110 109
Table 6
Mean recoveries and R.S.D.s (n = 6) of three colorants from spiked food matrices at various fortification levels
Colorant Food matrices Concentration level
100 50 10
Recovery (%) R.S.D. (%) Recovery (%) R.S.D. (%) Recovery (%) R.S.D. (%)
E 122 (mg L−1 ) Fruit flavoured drink 97.9 0.44 99.6 0.73 97.7 1.19
E 142 (mg kg−1 ) Jam 94.4 0.90 95.1 1.87
E 131 (mg kg−1 ) Mint flavoured sweets 101.8 1.98
used for the determination of the LOD. The LODs of the col- Table 7
orants obtained in this study are in the same range as or slightly Determination of colorants in water-soluble foodstuffs collected from the Greek
market
better than those reported in other studies using ion-pair HPLC
methods [27–29]. Sample Colorant found Mean content of colorant R.S.D. (%)
The precision experiments resulted in good R.S.D.s for both A (mg L−1 ) E 110 0.35 3.3
intra-day and inter-day precision, for the colorants and food E 122 0.63 7.3
matrices chosen. Precision was determined for colorants with E 124 0.89 3.9
different polarity, which eluted at the beginning (E 102), in the B (mg L−1 ) E 110 2.7 2.7
middle (E 110, E 124 and E 129) and near the end of the chro- E 122 125 1.9
matogram (E 142, E 122 and E 131). Six different food matrices, C (mg kg−1 ) E 110 12.2 1.5
in which the addition of these colorants is frequent, were cho- E 122 23.2 1.3
sen (Table 5). The intra-day precision (as R.S.D.r ) ranged from E 124 15.3 1.5
0.37% for E 122 in fruit flavored drink at a concentration of E 132 44.9 1.2
100 mg L−1 , to 4.8% for E 142 in icing sugar at a level of D (mg kg−1 ) E 131 4.7 2.7
0.9 mg kg−1 . The inter-day precision (as R.S.D.R ) was between E (mg kg−1 ) E 104 15.8 1.6
0.86% for E 122 in fruit flavored drink at 100 mg L−1 and 10% E 110 5.9 2.1
for E142 in jam at a concentration of 9 mg kg−1 (Table 5). The E 122 0.97 2.5
results obtained from this work are in general agreement with E 129 18.1 1.8
E 133 0.5 3.1
those obtained from other studies, where maximum R.S.D.s
ranged between 2.1% [23] and 6.4% [40]. F (mg kg−1 ) E 102 0.5 3.2
E 104 10.8 1.6
The results from recovery experiments in three different
E 110 10.8 1.2
matrices at levels ranged from 10 to 100 mg kg−1 are shown in E 122 2.3 2.0
Table 6. The high level (100 mg kg−1 ) was chosen in accordance E 129 27.8 1.9
to the legislation limits [2]. E 122 and E 142, which had shown E 133 4.8 1.7
poor resolution, and E 131, a compound with low polarity (thus, G (mg kg−1 ) E 124 52 1.6
with a possibly low affinity to water extraction), were chosen for
recovery experiments. The developed method resulted in satis-
factory recoveries for all the tested compounds, ranging from
94% for E 142 in jam to 102% for E 131 in sweets. The recov- at low concentrations. In general, colorants were determined in
eries are more than adequate for the developed application and foods collected from the Greek market in a more wide concen-
slightly better than those reported in literature [26–28]. tration range than those reported in other studies [23,26–29].
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