Colors of the World: Fast Separation of Dyes with Ascentis Express

By: Rudolf Köhling, Reporter US Volume 27.3

Rudolf Köhling

Dyes surround us everywhere every day. They can be found in common places like the printing ink in magazines or books and in plastics, textiles, and leather, but also in unusual places like diesel fuel and tattoo color. Most of these synthetic colors are based on aromatic ring structures containing heteroatoms and tend to have a high potential for causing cancer; as a result, they are not intended for use in food coloring. But since 2003, there have been several incidents of Sudan I contamination in chili powder. This situation necessitates the analysis of spice mixtures to determine if they have been adulterated (1, 2). Further, a sensitive HPLC method is needed for quality control testing of dyes and the identifi cation of byproducts. Supelco’s Ascentis® Express HPLC columns provide outstanding sensitivity and resolution for such applications.

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Method Development for Dyes

Table 1 contains a list of dyes added to one sample and dissolved in a mixture of methanol and acetonitrile. The sample was injected on an Ascentis Express C8 HPLC column under varying mobile phase conditions to determine the best separation parameters. Temperature, injection volume, detector settings, and flow rate were kept constant.

Table 1. Structure and Mass of the Dyes in the Sample Mixture. Most of the Compounds are Detected as [M+H]+ Ions except (5), which gives [M]+ Ions

The chemical and physical properties of the dyes differ strongly, so the fi rst step in developing a suitable HPLC method was the use of a gradient run ranging from 25% acetonitrile to 100% acetonitrile (B) and 0.1% formic acid in water as an aqueous counterpart (A). The UV chromatogram of the combined wavelengths 360, 550, and 620 nm showed good chromatography of all compounds except for the poor peak shape of Sudan 410 at 17.35 minutes (Figure 1A).

Figure 1. UV Chromatograms of Sudan III and Sudan 410. (A) Initial Conditions, (B) Addition of Methanol to Mobile Phase, (C) Final Conditions after Adjusting Gradient

To optimize that peak shape, methanol was added to the organic mobile phase (acetonitrile:methanol, 90:10); the gradient run was repeated, resulting in better peak shape for Sudan 410 (Figure 1B). In a fi nal experiment, the gradient profi le was changed and optimum conditions were attained (see Table 2). Figure 1C shows the step-bystep improvements in the chromatography.

Table 2. Initial and Final HPLC Method Settings for Separation of the Seven Dyes Listed in Table 1, After Optimization

Only three runs were needed to get the fi nal method, showing how easy and fast it is to develop methods with Ascentis Express columns. Further, Ascentis Express columns contain Fused-Core™ particles that allow for faster run times; even separations performed on standard HPLC systems can be sped up by up to 30% with Ascentis Express.

Figure 1 shows UV chromatograms of Sudan III (Peak 6) and Sudan 410 (Peak 7) after three optimization steps. An organic phase mixture of methanol:acetonitrile (10:90) and a fi nal gradient composition of 98% organic mobile phase resulted in the best overall peak shapes with a minimum of tailing of compounds (Peak 7). Figure 3 shows the fi nal chromatogram with very good separation of all analytes. Figure 2 is a UV chromatogram of the final HPLC method. Resolution, sensitivity, and peak symmetry were optimal for all analytes. The total run time on a standard HPLC instrument (Agilent 1100) was 25 minutes, but the separation could easily be performed faster on ultra-performance instruments.

Figure 2. UV Chromatogram of the Final HPLC Method

Figure 3. Expanded View of UV Chromatogram Showing Unknown Impurity at 7.7 min. and Malachite Green (5)

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Ascentis Express HPLC Columns: High Efficiency and LC-MS Compatibility

Using Ascentis Express columns on standard HPLC, fast LC, and ultra-performance instruments can yield heightened sensitivity (Figure 2). Mass detectors in LC-MS systems are very sensitive to contaminants in solvents and column bleed, both of which are very low with Ascentis Express columns combined with the right set of LC-MS solvents and additives. Both aspects, high effi ciency and low column bleed, are basic requirements in trace analysis of small target analyte concentrations or in identifi cation of byproducts which may infl uence dramatically the quality and application of dyes. Figure 3 shows an example of the identifi cation of low concentrations of byproducts even in very complex mixtures. The unknown substance at the retention time of 7.68 minutes shows a nearly identical UV spectrum to Malachite Green (Peak 5 at 8.10 minutes), but the mass is 14 Da lower. This may correspond to the exchange of a methyl residue with a proton at a position in the Malachite Green molecule that has no infl uence on the chromophore. Only the displayed molecular structures fi t the UV and mass spectroscopic results.

To get optimal results from your LC-MS system and accurate UV and mass spectra of impurities with a high signal-to-noise level, it is best to use high purity LC-MS solvents and high performance HPLC columns such as Ascentis Express.

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  1. Commission Decision. Offi cial Journal of the European Union. L154/114. June 10, 2003.
  2. Rapid Alert System for Food and Feed (RASFF). 2004. Annual Report. European Commission of Health & Consumer Protection Directorate General.

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