Food dyes, HPLC analysis

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

Several hundred dyes are known, and this diversity has always posed a problem of identity for the analysis, for many dyes have similar characteristics and some occur as a mixture (158). Among the different techniques available for the analysis of food colors, HPLC can replace many of the traditional techniques, providing rapid results that are much more specific for the determination of colorants (131,142). The technique has been shown to have great potential for synthetic food color analysis in terms of simultaneous separation, qualitative identification, and quantitation (159). 

Protein-rich foods can also be specially treated. According to Saag (135), in order to extract colorants from fish, samples are boiled, filtered, washed, with an ammonia solution to displace proteins, and then washed through Sephadex LH-20 with water. The colored zones are collected for HPLC analysis. Dairy products (ice cream, cheese, yogurt) are first mixed with acetone or ethanol to precipitate the protein, which is ground up with sea sand and Celite, and the slurry is placed in a column from which dyes are eluted with a solution of ammoniacal methanol (135,162). 

K Aitzemiiller, E Arzberger. Analysis of food dyes El 10, El 11 and E124 in fish samples by ion-pair partition HPLC. Z Lebensm Unters Forsch 169 335-338, 1979. 

The most recent paper on this topic has been published by Lu and Huang (213). The method consists of an online enrichment of the aromatic amines on a carboxymethyl-bonded silica precolumn and an HPLC-UV (at 254 nm) analysis. The mobile phase, ACN-acetate buffer (pH 4.66) (40 60, v/v), was used to desorb the analytes and for the subsequent separation. The method was applied to the determination of several compounds (4-aminoazobenzene (4-AAB), benzidine (Bz), 3,3 -methylbenzidine (DMBz), 4-aminobiphenyl (4-ABP), 3,3 -dichlorobenzi-dine (DCBz), and 2-naphthylamine (2-NA) together with some substituted naphthalens and phenols) in aqueous solution of four food dyes Direct Blue 6, Amaranth, Sunset Yellow FCF, and D C Orange No. 4. Detection limits ranged between 0.6 and 1.6 fig/g. Most part of the methods developed for this kind of determination are reported in Table 3. 

Owing to the varied structures of various food dyes, they can often be differentiated from one another by their characteristic ultraviolet/visible absorbance spectra. Using HPLC coupled with a diode array detector (HPLC-DAD) it is possible to collect a compound s absorbance spectrum as it elutes from the HPLC column, which greatly assists in identification. At Reading Scientific Services Ltd (RSSL) this type of detector is routinely used in a range of analyses of such substances as patulin, a mycotoxin found in apple juice, and in the analysis of colours and vitamins, which allows a more certain assignment of a particular peak to a specific compound to be made. 

Figure 7.6. HPLC analysis of food dyes in a beverage powder sample (Kool-Aid ) using gradient reversed-phase chromatography and UV detection at 290nm, which can detect many different colored dyes. Chromatogram courtesy of PerkinElmer, Inc.

All these methods give similar results but their sensitivities and resolutions are different. For example, UV-Vis spectrophotometry gives good results if a single colorant or mixture of colorants (with different absorption spectra) were previously separated by SPE, ion pair formation, and a good previous extraction. Due to their added-value capability, HPLC and CE became the ideal techniques for the analysis of multicomponent mixtures of natural and synthetic colorants found in drinks. To make correct evaluations in complex dye mixtures, a chemometric multicomponent analysis (PLS, nonlinear regression) is necessary to discriminate colorant contributions from other food constituents (sugars, phenolics, etc.). 

Similarly to the liquid chromatographic analysis of natural pigments, HPLC, especially RP-HPLC, plays a decisive role in the determination of synthetic dyes in a wide variety of accompanying matrices, such as human and animal tissues, food and food products, ground- and waste-waters, sludge, soil, etc. [100],... 

HPLC has been considered a powerful technique for the analysis of synthetic food color, for the detection of impurities in single dyes and also for the separation of a mixture of dyes (153). Re-versed-phase ion-pair chromatography has been found particularly useful for the separation and detection of a large number of food colors (159). 

The application of ion-pair HPLC in the analysis of food colors is summarized on Table 8. As indicated, TBA has been the most widely used ion pair. It can be observed that using gradient mobile phase elution, a larger number of synthetic dyes can be separated. However, the mobile phase programming should include a return to the initial condition as well as reequilibration of the column by maintaining the initial composition for a period of time. This procedure provides reproducible result (222). 

Methods for the analysis of aromatic amines in synthetic azo dyes and in food and beverages colored with these dyes have been developed. High-performance LC or GC methods are generally employed, often with the help of derivatization reaction and fluorimetric detection, with HPLC generally regarded as the best technique for the determination of aromatic amines. 

Applications of HPLC Of the bioanalytical separation technologies described in this book, arguably HPLC has the widest range of applications, being adopted for the purpose of clinical, environmental, forensic, industrial, pharmaceutical and research analyses. While there are literally thousands of different applications, a few indicators of how HPLC has been used are as follows (i) Clinical quantification of drugs in body fluids (ii) Environmental identification of chemicals in drinking water (iii) Forensic analysis of textile dyes (iv) Industrial stability of compounds in food products (v) Pharmaceutical quality control and shelf-life of a synthetic drug product (vi) Research separation and isolation of components from natural samples from animals and plants. 

If we are to examine any foodstuff using HPLC, we will need to remove the matrix prior to analysis. The Sudan red dyes will need to be extracted from the matrix. Food matter can be a complex mixture of animal and/or vegetable material containing a cocktail of many different compounds. Oil products contain complex mixtures of nonpolar hydrocarbon materials that are incompatible with most HPLC modes on their own. Thus, extraction of each matrix will be required before any analysis of the Sudan red dye is undertaken by HPLC. 

A method for the analysis of such dyes has been developed. The method is based on coupling of ionic liquid-based extraction with HPLC. In this way, Sudan dyes and Para Red in chib powder, chib oil, and food additive samples can be found. 

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