HPLC INVESTIGATION OF THE DEGRADATION OF SOME ARTIFICIAL AZO FOOD COLORANTS IN THE PRESENCE OF ASCORBIC ACID

A b s t r a c t: The influence of the concentration of ascorbic acid on some azo food colorant (Brilliant Black BN, Brilliant Blue FCF, Chinoline Yellow, Indigotine, Patent Blue V and Tartazine) degradation in the solution has been studied. Rate constant (k), time of colorant half-life (t1/2), as well as the type of the kinetic reaction have been determined. The colorants and the ascorbic acid have been determined by high performance liquid chromatography (HPLC) with diode array detector (DAD) and UV-VIS spectrometry. It was found that the higher concentration of ascorbic acid leads to the higher rate constant and lower colorants half-life. The values of the rate constants are 2.50–4.25 times higher when the colorant degradation was run in the presence of 500 mg L –1 of ascorbic acid than in the case of the presence of 100 mg L –1 ascorbic acid for Brilliant Blue FCF, Chinoline Yellow, Patent Blue V and Tartazine. From the other side, the degradation of Brilliant Black BN and Indigotine is very fast with a total degradation for 6–9 days. Therefore, the rate constants for those two food colorants are much higher than for the other investigated colorants. According to the linearity of the kinetics curves (ln(c/co) v.s. time) it appeared to be first order kinetics for the interval up to 4 days.


INTRODUCTION
The artificial food colorants are widely used in food industry, for coloring foodstuff matter, but in fact they have no any nutritional value.The use of particular food colorant in food industry depends primarily of its toxicity.However, at the same time, of major importance are also some other factors such as stability of the colorant used for coloring certain food product and whether there is a possible interaction between some component of the foodstuff matter and the colorant.There are several papers concern on the stability of some of the investigated colorants using different reducing agents or factors: by inorganic oxysulfur compounds [1], pH [2], microflora [3] or temperature [4].Some reducing agents present in the food may react especially with azo dyes forming degradation products, which may react further with other food components forming undesirable compounds [5][6][7][8][9][10][11][12][13].One of the agents, which can react with these dyes causing their degradation, is ascorbic acid [14 -17].The ascorbic acid is often used in the soft drinks as a nutritional supplement or as an antioxidant and provides a reducing environment in which azo dyes can easily be degraded.In real food, the sucrose, known as a stabilizer of ascorbic acid [14], is also present.These facts make the field of investigation of degradation of colorants used in food industry very actual.
In our previous paper [17] we presented data about degradation investigations of artificial food colorants with naphtalenic structure (Ponceau 4R, Amaranth, Allura Red, Brilliant Black BN, Carmoisine and Sunset Yellow) in the presence of ascorbic acid.In the present study, the influence of the concentration of ascorbic acid on the stability of some other azo dyes (Brilliant Blue BN, Brilliant Blue FCF, Chinoline Yellow, Indigotine, Patent Blue V, Tartazine), Fig. 1, has been presented.Rate constant (k), time of colorant half-life (t 1/2 ), as well as the type of the kinetic reaction have been determined.At the same time, degradation of ascorbic acid in the solutions has been also followed.Liquid chromatography column was used.Stainless steel 10 cm long and 4.6 mm inside diameter, packed with 5 μm Pecosphere C18, was used (Merck, Germany).

Reagents
Mobile phase was composed of solvent A (0.01 mol L -1 KH 2 PO 4 adjusted to pH 4.5 with o-phosphoric acid) and solvent B (methanol HPLC grade, Merck, Germany).
Acetate buffer (pH = 5.5) was prepared by dissolving 6.8 g of sodium acetate, adjusted with acetic acid in 1 L.
Reference food colorant standards were obtained from Etol (Slovenia) and Wurth (Austria).

Methods
The model solutions containing investigated dyes were made in acetate buffer (pH = 5.5).Concentration of added dyes in model solutions was 40 mg L -1 , and concentrations of the ascorbic acid were 100, 250 and 500 mg L -1 .The dissolved oxygen was removed from solutions by purging of helium.
The solutions were kept in dark at room temperature.The degree of degradation of ascorbic acid and food colorants was monitored within 4 weeks.The rate of ascorbic acid degradation was monitored by HPLC, and the rate of food colorant degradation was monitored simultaneously by HPLC and by visible spectrometry [18].
Gradient profile used for all colorants (except eritrozine) was as follows: 3 minutes 100 % A, 0 -100 % B in 4 min, held at 100 % B for 8 min, returned to 100 % A and held 10 min before next injection.Gradient profile for eritrozine was: 2 minutes 100 % A, 0 -100 % B in 1 min, 30-50 % B for 1 min, 70-100 % B for 1 min, held at 100 % B for 9 min, returned to 100 % A and held 10 min before next injection.HPLC determination of ascorbic acid was performed at 255 nm.Wavelengths for HPLC and spectrometric determination of artificial food colorants are given in Table 1.

RESULTS AND DISCUSSION
In our previous investigation [17] we found that the presence of ascorbic acid influences the degradation of the food colorants with naphtalenic structure (Ponceau 4R, Amaranth, Allura Red, Carmoisine and Sunset Yellow).In this paper, the influence of the ascorbic acid on the degradation of other azo food colorant (Brilliant Black BN, Brilliant Blue FCF, Chinoline Yellow, Indigotine, Patent Blue V, Tartazine) was studied.
The rate of degradation of the investigated food colorants was studied in the presence of ascorbic acid with different concentration (100, 250 and 500 mg L -1 ).The model solutions were previously preserved by adding of potassium sorbate (48 mg L -1 ).The concentration of all added dyes was 40 mg L -1 , because this concentration appeared to be most likely used in the preparation of soft drinks.The ascorbic acid is very rapidly oxidized by dissolved molecular oxygen in the model solutions.In order to decrease the degradation of ascorbic acid oxygen was removed from the solution by purged the samples by helium instead nitrogen proposed by other authors [19].The colorants and the ascorbic acid have been determined by high performance liquid chromatography (HPLC) with diode array detector (DAD) and UV-VIS spectrometry.
From the obtained results (Fig. 2, Table 2) it can be seen that the rate of degradation of the investigated colorants increases with the concentration of the ascorbic acid.Namely, when the degradation is followed in the presence of 100 mg L -1 ascorbic acid, after 21 days more than 85 % of colorants remains in solution (from 86.5 % for tartazine to 93.8 % for brilliant blue FCF).In the presence of 250 mg L -1 ascorbic acid, after 28 days nondegradated colorants are present from 70.5 % for tartazine to 79.2 % for chinoline yellow and brilliant blue.In the model solution with 500 mg L -1 ascorbic acid, the corresponding value ranges from 61.2 % for tartazine to 74.5 % for chinoline yellow.The data for the rate constants are similar to the azo dyes with one naphthalene structure [17].However, the presence of ascorbic acid increases significantly degradation rate of Brilliant Black BN and Indigotine (Fig. 2).For example, the rate constant for Brilliant Black BN and Indigotine in the presence of 500 mg L -1 of ascorbic acid is 86 or 44 times higher than that for chinoline yellow, respectively (Table 2).According to the results for the degradation of investigated food colorants in the presence of various concentrations of ascorbic acid vs. time, rate constant (k), time of the colorants half life (t 1/2 ) and correlation coefficient (r) have been determined (Table 2).The values of the rate constants were calculated from the slopes of the straight lines obtained by plotting lnγ vs. time.This dependence has the form of the linear equation (lnγ = -kt + lnγ ο ) which shows that the plot of lnγ versus t yields a straight line, so the reactions are first order [20].As it can be seen from these results, the rate constant (k) and half-life (t 1/2 ) are higher by increasing the initial concentration of ascorbic acid in the model solution.Correlation coefficients for degradation in the presence of 100 mg L -1 of ascorbic acid, were calculated for the 3 days (except for Indigotine were it is calculated for 24 h, and for Brilliant Black BN for 4 days) period.The other kinetics parameters (k, t 1/2 ) refer to period of 3 weeks.All parameters for the colorant degradation in the presence of 250 and 500 mg L -1 of ascorbic acid were obtained for the period of 4 weeks.

Kinetics of degradation of the synthetic food colorants in the presence of ascorbic acid
As it can be seen, the higher concentration of ascorbic acid leads to the higher rate constant and lower colorants half-life.The values of the rate constants are 2.50-4.25 times higher when the colorant degradation was run in the presence of 500 mg L -1 of ascorbic acid than in the case of the presence of 100 mg L -1 ascorbic acid for Brilliant Blue FCF, Chinoline Yellow, Patent Blue V and Tartazine.