Separating and Identifying Food Dyes by Paper Chromatography - Lab Report Example

SEPARATING AND IDENTIFYING FOOD DYES BY PAPER CHROMATOGRAPHY The resolution of blue green 3, yellow 5, yellow 6, red 40, blue 2, and red 3, determined through the differences in Rf values, was best achieved by paper chromatography using 0.10% NaCl solution, instead of water or 70% isopropyl alcohol as mobile phase. Difference between the polarity of the solutions relative to the stationary phase caused the observed differences among the chromatograms obtained using the different solvents. On the other hand, the relative polarity of the dyes resulted to differences in their interactions with the phases, and subsequently in their distances traveled along the chromatography paper and in their Rf values. Unknown mixtures of dyes were also resolved into their respective components using paper chromatography. INTRODUCTION Chromatography is a separation technique that determines the components of a mixture. Drug tests of blood and urine samples as well as determining the presence of harmful substances in drinking and groundwater are the most common uses of chromatography. The solid or liquid stationary phase holds the mixture to be separated. In addition, it is the medium to which the mobile phase passes through. On the other hand, the liquid or gaseous mobile phase passes along the stationary phase and in the process carries the components of the mixture with it. In the end of the experiment, separate bands are observed, representing the different components that move along the stationary phase at different rates. The chromatogram is the pattern of separated bands produced after chromatography. Each band is characterized by its retention factor, which is the ratio of the distance between the origin line and the band of the component and the distance between the origin line and the solvent front. In paper chromatography, a specialized paper made of cellulose is used as the stationary phase. The best mobile phase for a particular separation experiment depends on which solvent system the sample components have different Rf values. Thus, measurement of Rf values of the individual sample components in a variety of solvents is necessary in deciding which solvent to use as the mobile phase. This experiment was conducted to determine, through paper chromatography, the retention factors (Rf) of the seven pure food dyes, red 3 (R3), red 40 (R40), blue 1 (B1), blue 2 (B2), yellow 5 (Y5), yellow 6 (Y6), and green 3 (G3), approved by the Food and Drug Administration (FDA) and dissolved in three different solvent systems, distilled water, 70% isopropyl alcohol, and 0.10% sodium chloride (NaCl) solution. The solvent solution that resulted to the best separation was then be used to identify the dyes in unknown mixtures. This experiment would then demonstrate the use of paper chromatography in analysis of purity and determination of components of mixtures. EXPERIMENTAL METHOD Identification of best solvent The procedure was adapted from the manual prepared by Markow (1997). Briefly, 7 ml of each solvent system is placed in its corresponding 250 ml beaker. They were covered with Petri dish covers. Three 7.5 x 13.5 cm chromatography papers were each marked to delineate the origin line, which is 1 cm from the bottom edge of the paper along the long axis, and along this line eleven points that are 1 cm away from one another were marked as well. These points corresponded to the seven individual dyes and three two-dye combinations (B1 and R40, B1 and Y5, R3 and Y6, and B2 and Y5). Using a toothpick, the corresponding individual dyes or two-dye mixtures were applied five times on their corresponding spots on the three chromatography papers, which were then rolled on its horizontal axis. The edges were then stapled to become adjacent to each other. The papers were then placed inside their corresponding beakers, with their bottom edge touching the solvent. The beakers were covered. The starting times were recorded. When the solvent front was 1.5 cm from the top, the paper was removed, and the ending time for that experiment was recorded as well. Rf values were then calculated for each band. Identification of composing dyes in unknown mixtures There were four unknown dye mixtures (UN1-UN4) provided. UN1 was green, UN2 was red, UN3 was black, and UN4 was yellow. A 7.5 x 13.5 cm chromatography paper (chromatogram 4) was then spotted with B1, B2, unknown 1 (UN1), Y5, Y6, UN2, R3, R40, UN3, G3, and UN4. The chromatography was performed using 0.10% NaCl solution as mobile phase. The Rf values of the components of the unknown mixtures were compared with the Rf values of the pure dyes. The unknown component was designated to be a particular pure dye if their Rf values match. RESULTS Identification of best solvent In the identification of the best solvent system for resolving dyes, the chromatography experiments using water, 70% isopropyl alcohol and 10% NaCl solution as mobile phases were run for six minutes. The characteristics of the chromatograms are summarized in table 1. Table 1. Distances, in centimeter (cm), of dyes travelling along the chromatography paper from the origin line. Dyes separated Water 70% isopropyl alcohol 0.10% NaCl solution Distance Rf Distance Rf distance Rf Solvent front 5.2 -- 1.4 -- 5.6 -- B1 5.1 0.98 1.2 0.86 5.4 0.96 B2 4.6 0.88 0.6 0.43 2.3 0.41 Y5 5 0.96 0.5 0.36 4.5 0.80 Y6 5.2 1 1 0.71 3.5 0.62 R3 4.1 0.79 1.4 1 1 0.18 R40 5 0.96 1.2 0.86 3.4 0.61 G3 5.2 1 1.4 1 5.2 0.93 The best solvent in resolving these dyes is the one that has resulted to the most differentiation of the bands. In calculating the standard deviation of the Rf values of each chromatogram, the one run with water had 0.07, that with 70% isopropyl alcohol had 0.26, and the chromatogram obtained using 0.10% NaCl solution had 0.28. Comparing the three, the most difference was obtained using the 0.10% NaCl solution. It was thus used as the mobile phase for the next part of the experiment. Identification of composing dyes in unknown mixtures In the chromatogram, it was observed that UN1, UN2 and UN4 had two components, while UN3 had three. In further characterizing the chromatogram mixtures, the values in table 2 were obtained. Also included in the table are the appropriate Rf values obtained in the first part of the experiment (refer to table 1). They were placed to calculate the average Rf values for the seven pure dyes resolved using 0.10% NaCl solution. The averages will be used to compare with the Rf values of the unknown components. Table 2. Characteristics of the chromatogram of four unknown dye mixtures Dyes separated Distance (cm) Rf Rf (Part I) Average Rf Solvent front 6.4 -- -- -- Pure dyes B1 6.4 1 0.96 0.98 B2 3 0.47 0.41 0.44 Y5 5 0.78 0.80 0.79 Y6 4 0.62 0.62 0.62 R3 1 0.16 0.18 0.17 R40 3 0.47 0.61 0.54 G3 6.4 1 0.93 0.96 UN1 co1 5 0.78 -- -- co2 1.5 0.23 -- -- UN2 co1 1.5 0.23 -- -- co2 3.5 0.55 -- -- UN3 co1 3.9 0.61 -- -- co2 2 0.31 -- -- co3 0.5 0.08 -- -- UN4 co1 2.5 0.39 -- -- co2 3.4 0.53 -- -- If the average Rf of the pure dyes were matched to the Rf values of the unknown components then the corresponding pure dye for each unknown component are as follows: Table 3. The identities of the unknown components. ‘??’ denotes unmatched Rf value to those of the pure dyes. UN1 (green) co1 Y5 co2 R3 UN2 (red) co1 R3 co2 R40 UN3 (black) co1 R40 co2 ?? co3 ?? UN4 (yellow) co1 B2 co2 R40 DISCUSSION It is the capillary action among water molecules from the liquid solvent (mobile phase) and those trapped among the cellulose molecules of the chromatography paper (stationary phase) makes possible the ascension from the origin line to the top of the paper. Thus, in the experiment, the solvent that contained more water, pure water and 0.10% NaCl went farther along the paper compared to 70% isopropyl alcohol. Thus, rubbing alcohol was not chosen as the best mobile phase because it would provide a relatively small room for resolution of dyes. Comparing the two in terms of the resolution of dyes, it was observed that the salt solution resulted to better resolution. This is because the stationary phase and mobile phase is the same when water is used as solvent. The separated substances would not have preferential interaction to stationary phase or mobile phase, and the dyes, as observed in the experiment, would only travel as far as the solvent front. The separation mechanism of this type of liquid chromatography is absorption. As the solvent passes through the spotted sample, it dissolves its components. A component’s interaction between the stationary phase and the liquid phase determines its Rf value, such that the substance that is more alike, in terms of polarity, to that of the stationary phase, tends to travel less farther than others. The Rf thus turns out lower as well. Differences in distances traveled were caused by the differences in the chemical structures of the dyes. If the Rf values of the dyes are ranked from highest to lowest, then the ranking is B1, G3, Y5, Y6, R40, B2, R3. Looking at their chemical structures, B1 and 63 are the most nonpolar in the group, each containing 5 benzene rings. G3 is more polar than B1 because its benzene rings are farther away from one another. The rest of the dyes have 4-3 benzene rings, thus they are relatively more polar. Furthermore, the more nonpolar dyes are relatively immiscible with the polar stationary phase. They are thus dragged along by the mobile phase to the solvent front. The more polar dyes, on the other hand, interact with the stationary phase more easily, and thus their distances traveled with the mobile phase were less than the other two. This lead to lower Rf values. Such differences between the dyes make the separation of unknown mixtures into components with Rf values that can be compared with the reference data obtained in the first part of the experiment. However, there were bands that did not have similar Rf values to any of the pure dyes. It is thus possible that the bands were still impure, and as such can be broken down into components. References Fox, M. A. & Whiteshell, J. K. (2003). Organic Chemistry (3rd ed.). Canada: Jones and Barlett Publishers. Markow, P. G. (1997). Separating and Identifying Food Dyes by Paper Chromatography. In C. L. Stanitski. Modular Laboratory Program in Chemistry Series. Stanford: Cengage Learning. Read More