Four simple, rapid, and accurate spectrophotometric methods were developed for the simultaneous determination of two food colorants, Carmoisine (E122) and Ponceau 4R (E124), in their binary mixtures and soft drinks. The first method is based on recording the first derivative curves and determining each component using the zerocrossing technique. The second method uses the first derivative of ratio spectra. The ratio spectra are obtained by dividing the absorption spectra of the binary mixture by that of one of the components. The third method, derivative differential procedure, is based on the measurement of difference absorptivities derivatized in first order of solution of drink samples in 0,1 N NaOH relative to that of an equimolar solution in 0,1 N HCl at wavelengths of 366 and 451 nm for Carmoisine and Ponceau 4R, respectively. The last method, based on the compensation method is presented for derivative spectrophotometric determination of E122 and E124 mixtures with overlapping spectra. By using ratios of the derivative maxima, the exact compensation of either component in the mixture can be achieved, followed by its determination. These proposed methods have been successfully applied to the binary mixtures and soft drinks and the results were statistically compared with the reference HPLC method (NMKL 130).
Color is a vital constituent of food and probably the first characteristic perceived by the human senses. Probably, for economic reasons, brightly colored and stable synthetic colorants have been widely used as food additives. These colorants are used to supplement and enhance natural colors destroyed during processing or storage and substantially increase the appeal and acceptability of food stuff. The term color additive can be applied to any dye, pigment, or other substances artificially made or obtained from a vegetable, animal, mineral, or another natural source [
Carmoisine (E122) and Ponceau 4R (E124) are synthetic dyes that contain azo and aromatic ring structures and are often used in dyeing food, drink, medicine, and cosmetics (Scheme
Chemical structures, common names, European community numbers (E), and color index (CI) numbers of synthetic food colorants studied.
The development of chemical and instrumental methods for the separation, identification, and quantitative analysis of synthetic food colorants has become extremely important for the food and beverages industry and academic and governmental institutions to assess the quality and safety of food products [
Various techniques have been introduced for the determination of mixtures of colorants with three or more component combination in food samples.
These include capillary electrophoresis [
In this present work, selective, sensitive, simple, and lowcost procedures were developed for the simultaneous determination of Carmoisine (C) and Ponceau 4R (P) in synthetic mixture and drink samples by using derivative spectrophotometry (DSM), ratio derivative spectrophotometry (RDM), derivative differential spectrophotometry (DDM), and compensation method (CM). These methods have been successfully applied to synthetic mixtures and drink samples and the results obtained were compared with those obtained by HPLC method according to NMKL 130 [
No extraction or evaporation step, no complexation agent, and no harmful chemicals are involved in the suggested methods, in the suggested methods, so can be used for routine analysis of both colorants in quality control and routine laboratories.
All spectral measurements and treatment of data were carried out using a Shimadzu UV2450; double beam UVVis spectrophotometer with two matched 1cm quartz cells, connected to a Hewlett Packard compatible personal computer and a HP 1102 Laser Jet Printer. Bundled UVPC personal spectroscopy software of version 2.21 was used to process the absorption and the derivative spectra. The spectral band width was 1 nm with wavelength scanning speed of medium. HPLC measurements were performed with an Agilent 1100 series HPLCDAD system with a vacuum degasser, quaternary pump, auto sampler, and injector with a 100
Separation was achieved using a Waters Spherisorb C_{18} column, (4.6 × 250 mm, 5
Distilled water was used to prepare the solutions and for mobile phase. All chemicals used were of analytical reagent grade and were purchased from Merck (Darmstadt, Germany). Ponceau 4R (E124, Cochineal Red A, molecular weight 604.47, CI 16255 CAS number 2611827) and Carmoisine (E122 Azorubine, molecular weight 502.44, CI 14720,CAS number 53026699) were from SigmaAldrich (Steinheim, Germany). Soft drinks were purchased from commercial markets.
Stock solutions of Carmoisine and Ponceau 4R (100
For DDM method, accurate volume aliquots of the stock solution were transferred into two sets of 10 mL volumetric flasks and the volume was made up with 0.1 N HCI and 0.1 N NaOH to give a series of equimolar solutions containing 2.0–10.0
For HPLC method, stock solutions of (25 mg/100 mL) Carmoisine and Ponceau 4R were prepared in a 1 : 1% v/v mixture of methanol—TBA buffer solution (0.005 M TBA + 0.005 M phosphate buffer, pH 6.5). The standard solutions were prepared containing 5–20
Commercial drink samples due to turbid are treatment with the Carrez I and II solutions.
Carrez solution I: it contains potassium hexacyanoferrate (II) trihydrate (K_{4}[Fe (CN)_{6}] × 3H_{2}O) 100 mL concentration: 15 g × 100 mL^{−1}
Carrez solution II: it contains zinc sulfate heptahydrate (ZnSO_{4} × 7H_{2}O) 100 mL concentration: 30 g × 100 mL^{−1}.
Final solutions for measurements were prepared over the concentration ranges given in Table
The derivative spectra were recorded over the wavelength range of 300–700 nm. The amplitude of the derivative curve was measured at the selected wavelengths for each method (Table
For the compensation method the absorbance difference spectra (sample versus reference) were recorded over the wavelength range of 300–700 nm, using different concentrations of the reference solution, prepared from the stock solution.
For the DD method, the Carmoisine and Ponceau 4R solutions in 0.1 N HCI difference derivative spectra were recorded against corresponding 0.1 N NaOH solutions of the colorants as a blank. The amplitude of the first derivative spectra (Table
Experimental parameters calculated for the simultaneous determination of Carmoisine and Ponceau 4R in binary mixture by compensation method.
Colorants  Concentration range (µg mL^{−1})  Ratio  Mean^{a}  RSD (%) 

Carmoisine  2–10  ^{ 1}D_{472}/^{1}D_{569}  0.600 ± 0.002  0.333 
Ponceau 4R  2–10  ^{ 1}D_{470}/^{1}D_{554}  0.690 ± 0.009  1.304 
Statistical parameters of the simultaneous determination of Carmoisine and Ponceau 4R by DSM, RDM, and DDM.
Methods  Analyte  Selected wavelength (nm)  Concentration range (µg mL^{−1})  Regression equations^{a}  Correlation coefficient 
Detection limit 
Quantification limit 

DSM  C  ^{ 1}D_{331}  2–10 

0.9995  0.086  0.286 
P  ^{ 1}D_{517}  2–10 

0.9996  0.091  0.304  


RDM  C  ^{ 1}D_{323}  2–10 

0.9994  0.079  0.263 
P  ^{ 1}D_{344}  2–10 

0.9996  0.082  0.273  


DDM  C  ^{ 1}D_{366}  2–10 

0.9996  0.071  0.236 
P  ^{ 1}D_{451}  2–10 

0.9998  0.081  0.270 
Aliquots of Carmoisine working solutions equivalent to 2.0–10.0
Appropriate aliquots of aqueous working solutions of Ponceau 4R were placed into 10 mL calibration flasks and the volume was made up with distilled water. The spectra of standard solutions in the concentration range 2.0–10
Accurately aliquots of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of colorants stock solution were transferred into 10 mL volumetric flasks and the volume was made up with distilled water. The spectra of the prepared standard solutions were scanned from 300 to 700 nm and stored in the computer. For the determination of Carmoisine, the stored spectra of Carmoisine were divided separately by the spectrum of 6
In order to determine Ponceau 4R, stored spectra of Ponceau 4R standard solutions were divided by a standard spectrum of Carmoisine of 6
The difference spectra between the acidic (0.1 N HCI) solution and equimolar basic (0.1 N NaOH) solution of pure colorants and drink samples were recorded from 300.0 to 700.0 nm by placing the acidic solution in the sample compartment and the basic solution in the reference compartment. A first derivative spectrum of each of the differential curves was subsequently recorded. The derivative absorbance values were measured at 366 and 451 nm for Carmoisine and Ponceau 4R, respectively.
By analogy, the same steps were followed by using solutions of pure Ponceau 4R in the reference cell to determine its concentration in the mixture at the balance point.
The same principle was also applied for the analysis of drink samples.
The same steps as for the compensation method were followed and the derivative ratio of the mixture (in the sample cell) was calculated at each time and plotted against the concentration of pure compound Carmoisine (in the reference cell). A line with slight curvature is usually obtained. The concentration of Carmosine can easily be interpolated from the graph by substituting the ratio of pure Ponceau 4R. At the point at which the ratio of the mixture is equal to that of pure Ponceau 4R, the concentration of Carmoisine in the reference cell is equal to that in the mixture in the sample cell. By analogy, the concentration of compound Ponceau 4R can be obtained.
This reference method is described in detail in Nordic Committee on Food Analysis [
Commercial samples were mixed with Carrez (I) and Carrez (II) solutions. Once precipitated, the samples were centrifuged. 50 mL of the sample that is clarified by Carrez precipitation was transferred to 100 mL volumetric flask and the volume was made up with distilled water. A series of dilution was prepared quantitatively with water from this solution to obtain standard solutions to reach the concentration ranges of calibration curves graphed for each of the proposed methods (DSM, RDM, and CM). For the DDM method, the same procedure was used, but in this case the final volume was made up with 0.1 N HCI and 0.1 N NaOH, separately for the determination of Carmoisine and Ponceau 4R. HPLC method, the same sample preparation procedure described above, was followed using a mixture 1 : 1, v/v of methanol—TBA buffer solution. After dissolution process, prepared solutions were filtered using 0.45
The validity and suitability of the proposed methods were assessed by accuracy (reported as percentage recovered), precision (reported as RSD%), linearity (evaluated by regression equation), limit of detection (LOD), and limit of quantification (LOQ) [
LOD and LOQ were based on the standard deviation of response and the slope of the corresponding curve using the following equations:
The linearity of the methods was established in the concentration ranges of 2–10
In order to demonstrate the validity and suitability of the proposed methods, intra and interday variability studies were performed at three different concentrations (2, 6, and 10
The precision was ascertained by carrying out five replicate determinations of synthetic mixtures at different concentration ratios of Carmoisine and Ponceau 4R. Recovery test also confirmed the accuracy and applicability of the proposed methods by analyzing several synthetic mixtures of Carmoisine and Ponceau 4R which reproduced different composition ratios within the linearity range. These ratios are also the ratios in drinks. The mean percentage recoveries and RSD values were calculated.
Recovery of the colorants of interest from a given matrix can be used as a measure of the accuracy or the bias of the method. The same ranges of concentrations as employed in the linearity studies were used.
In our study, the applicability of the derivative spectrophotometric methods was also validated by comparison with the HPLC technique.
Figure
Zeroorder spectra (a) of 10
Under computercontrolled instrumentation, derivative spectrophotometry played a very important role in the resolution of band overlapping in quantitative analysis [
Under the experimental conditions described, the calibration graphs obtained by plotting the derivative values versus concentrations for Carmoisine and Ponceau 4R, in the concentration range stated in Table
Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by the proposed methods.
Method  Accuracy (mean* ± RSD %)  Precision repeatability^{a} 
Intermediate precision^{b}  

DSM  C (^{1}D_{331}) 



P (^{1}D_{517}) 






RDM  C (^{1}D_{323}) 



P (^{1}D_{344}) 






DDM  C (^{1}D_{366}) 



P (^{1}D_{451}) 






CM  C (^{1}D_{472}/^{1}D_{569}) 



P (^{1}D_{470}/^{1}D_{554}) 



^{
b}The interday (
*The values of % recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2–10 µg mL^{−1}).
RSD %: relative standard deviation.
While the main disadvantages of the zerocrossing method in derivative spectrophotometry for resolving a mixture of components with overlapped spectra are the risk of small drifts of the working wavelengths and the circumstance that the working wavelengths generally do not fall in correspondence of peaks of the derivative spectrum, this may be particularly dangerous when the slope of the spectrum is very high with consequent loss of accuracy and precision, and the working wavelength is in proximity of the base of the spectrum which causes poor sensitivity. The main advantage of the RDM is the chance of doing easy measurements in correspondence of peaks so it permits the use of the wavelength of highest value of analytical signals (a maximum or a minimum), and moreover, the presence of a lot of maxima and minima is another advantage by the fact that these wavelengths give an opportunity for the determination of active compounds in the presence of other compounds and ingredients which possibly interfere in the assay [
This method is based on the use of the first derivative of the ratio spectra. The ratio spectra were obtained by dividing the absorption spectrum of the mixture by that of one of the components. As we depicted above, direct absorption measurements are not possible due to spectral overlap. This spectral overlapping was sufficiently enough to demonstrate the resolving power of the proposed method. Figure
First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6
Under the described experimental conditions, the calibration graphs obtained by plotting the derivative values of each colorant versus concentration in the concentration range stated in Table
Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the reference HPLC method (NMKL 130).
Methods  Analyte  Selected wavelengths (nm)  Assay results mean ± SD* ( 


Soft drink I (µg 100 mL^{−1}) 
Soft drink II (µg 100 mL^{−1}) 

DSM  C  ^{ 1}D_{331} 


P  ^{ 1}D_{517} 





RDM  C  ^{ 1}D_{323} 


P  ^{ 1}D_{344} 





DDM  C  ^{ 1}D_{366} 


P  ^{ 1}D_{451} 





CM  C  ^{ 1}D_{472}/^{1}D_{569} 


P  ^{ 1}D_{470}/^{1}D_{554} 





HPLC  C  520 


P  512 


**The corresponding theoretical value for
Differential spectrophotometry (ΔD_{1}) based on pH changes has also been reported to be useful in the determination of binary mixtures. This method that depends on utilization of difference absorption spectra corresponding to the same compound obtained at two different pH’s has been investigated for the analysis of binary mixtures. The procedure is comprised of the measurement of ΔD_{1} food colorants’ in acidic solutions against their alkaline solutions as blanks. The ΔD_{1} has been successfully used to eliminate interferences from drink samples. There are few reports on utilization of the above two combined techniques (difference and derivative) for the estimation of individual drug substances and for combined preparations [
The difference absorption spectra of Carmoisine, Ponceau 4R, and their binary mixture in 0.1 N NaOH and in 0.1 N HCI are shown in Figure
Difference absorption spectra of 10
Under the experimental conditions described, the calibration graphs obtained by plotting the derivative values of each colorant in the mixture versus concentration, in the range concentration stated in Table
The compensation method is a nonmathematical method for the detection and elimination of unwanted absorption during spectrophotometric analysis [
(a) First derivative spectra between the mixture solution (6
The developed and validated methods were successfully applied to the determination of Carmoisine and Ponceau 4R content in the commercial soft drinks. The obtained results are presented in Table
The linearity of the proposed methods was evaluated under different concentrations of Carmoisine and Ponceau 4R within the concentration range stated in Table
LOD is expressed as the analyte concentration corresponding to the sample blank value plus three standard deviations and LOQ is the analyte concentration corresponding to the sample blank value ten standard deviations [
The precision of the developed methods was expressed as a percentage of relative standard deviation (RSD%) for repeatability (intraday precision) and intermediate precision (interday precision). The data obtained were less than 1.06 (Table
Four derivative spectrophotometric methods for the simultaneous analysis of two common food colorants have been successfully researched, developed, applied, and compared by reference HPLC method. Proposed methods provide simple, accurate, and reproducible quantitative determination of Carmosine and Ponceau 4R in synthetic mixtures and soft drinks without any interference from ingredients present. The developed methods are simple (as there is no need for pretreatment), rapid (as they require measurements of ∆D1 and D1 values at a single wavelength) and direct (as they estimate each colorant independently of the other). UV methods offer a cost effective and time saving alternative to other methods, for example, colorimetric, complexometric and chromatographic analyses. The methods used in this study are more versatile and easy to apply than the HPLC, polarographic, and voltammetric methods. Also the methods did not require any sophisticated instrumentation, such as HPLC, which requires organic solvents and time or advanced methodologies (like chemometric methods).
We conclude that the sensitivities of the proposed methods are almost comparable and can be used for the determination of the two colorants in commercial drinks in the absence of official method.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research has been supported by Scientific Research Project Coordination Center of Yildiz Technical University (Project no. 20120102KAP07).