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5. Spectroscopic Analysis of Two Analytes in a Single Sample Background and Principle: The concentrations of more than one analyte in a single solution can be determined spectrophotometrically if: (1) All of the analytes have different absorbance spectra which provide sufficient partial spectral selectivity (not full selectivity), that can be leveraged mathematically using more than one wavelength to distinguish one analyte from another. (2) Beer's Law applies to each analyte so their absorbance at each wavelength measured respond linearly to their concentration in solution. (3) The analyte components do not interfere with one another in solution causing the spectrum of the combination of components to be different from the sum of the spectra of the individual components. That is, Beer's Law must apply independently to each spectrum so a set of independent linear equations (one equation for each wavelength) can be written for all of the analytes present. For this laboratory, two coloring agents - Tartrazine and Sunset Yellow serve as the two analytes to be determined in a single sample. Tartrazine has the chemical name: Trisodium 1-(4-sulfonatophenyl)-4-(4- sulfonatophenylazo)-5-pyrazolone-3-carboxylate) and is also known as Tartrazine (MW = 534.36 g/mol). Its chemical structure is: Naooc -SO,Na OH Nao,s Sunset Yellow has the chemical name: Disodium 6-hydroxy-5-[(4-sulfophenyl)azo)-2- naphthalenesulfonate and is also known as Sunset Yellow (MW = 452.37 g/mol). Its chemical structure is: HO Naso, SO,Na You will use Beer's law to determine their concentrations in an unknown mixture. To do that, you will make solutions of known concentrations of Tartrazine and Sunset Yellow as calibration standards from which to determine the concentration of each compound at each of the two wavelengths. You will then analyze a reference solution with a known mixture of the compounds to verify your calibration and the absence of inter-analyte interference. Finally, you will determine the concentration of each analyte Tartrazine and Sunset Yellow in your "unknown” sample mixture. Pre-lab Questions: 1. How would you verify that the two compounds satisfy the three criteria for quantitative analysis? Describe the steps required for each criterion. (1pt) Materials: Tartrazine (Trisodium 1-(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-5-pyrazolone-3-carboxylate) Sunset Yellow (Disodium 6-hydroxy-5-[(4-sulfophenyl)azo)-2-naphthalenesulfonate) Reference Solution 1 Unknown mixture of tartrazine ad sunset yellow (S) LabQuest 321 Cuvette Kit 2x 250 mL Beaker 2x 100 mL Volumetric Flask