Module 4 - Thin-Layer Chromatography_ Analysis of Dyes

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September 26 2023 Thin-Layer Chromatography of a Set of Unknowns with Known Standard Dyes in 1-Propanol:Concentrated Ammonium Hydroxide and 1M HCl at the Microscale Level Objective: The goal of this experiment is to determine the two standard dyes used to create the unknown mixture by analyzing a known set of standard dyes with thin-layer chromatography as well as their respective pH’s and polarities. The techniques used in this experiment include the implementation of silica gel plates for dye spotting, elution with a slightly basic solvent, acid wash after elution, calculation of an R F values, and other technical skills including the use of forceps to hold the silica gel plates, blotting of liquid with paper towels, and the use of a 5μL micropipet. Experimental Procedure: The procedure for this experiment was provided by the canvas document “Thin_Layer_Chromatography_Modifications”. No additional modifications to the procedure needed to be made during the experimental process. Reaction Scheme: There is not a reaction scheme for this experiment because the primary objective of the lab isn’t concerned with a chemical reaction. Data & Results: Hydrochloric Acid Propanol Ammonium Hydroxide Molecular Formula HCl C 8 H 8 O NH 4 OH Amount (grams) N/A N/A N/A mmoles N/A N/A N/A Molecular Mass (g/mol) 36.48 60.1 35.05 Density (g/mL) 1.2 2.1 0.9 Melting Point (ºC) -26 -89.5 -3 through -69 Boiling Point (ºC) 110 81 through 83 25 SDS Can cause severe skin burns, do not inhale. Ignites easily, can irritate eyes with contact Can cause damage if ingested or inhaled, toxic to skin. Plate Spot Dye Used Solvent Spot R f Color Post Color Post

# Locatio n Dist. (cm) Dist. (cm) Elution HCl Immersion 1 Left Bromocresol Green 4 2.2 0.550 Bright Blue Yellow 1 Middle Methyl Red 4 2.4 0.600 Yellow-Oran ge Dark Red 1 Right Bromocresol Purple 4 2.3 0.575 Brown Light Red 2 Left Bromophenol Blue 2.6 1.4 0.538 Dark Blue Yellow 2 Middle Bromothymol Blue 2.6 1.7 0.654 Green Yellow 2 Right Unknown D 2.6 1.2 & 1.8 0.462 & 0.642 Yellow & Dark Blue Hot Pink & Yellow 3 Left Methyl Red 4.6 2.7 0.587 Yellow Dark Red 3 Middle Methyl Red & Unknown D 4.6 2.8 & 2.7 0.609 & 0.587 Blue & yellow Dark Red 3 Right Unknown D 4.6 2.8 & 2.7 0.609 & 0.587 Blue & Yellow Red & Yellow 4 Left Bromophenol Blue 3.4 2.2 0.647 Blue Yellow 4 Middle Bromophenol Blue & Unknown D 3.4 2.5 & 2.2 0.735 & 0.647 Yellow & Dark Blue Hot Pink & Yellow 4 Right Unknown D 3.4 2.5 & 2.1 0.735 & 0.617 Yellow & Blue Hot Pink & Yellow Observations: No picture was taken of plate 2.

Additional observations include: ● No heat change whatsoever for the entire experimental procedure ● Ammonium hydroxide is clear and has a pungent odor ● Hydrochloric acid is clear and has a slight odor ● The darker colors produced by the TLC were much more easily observable than the lighter colors. Calculations: Sample R f Calculation for Bromocresol Green (Plate 1) (distance traveled by the compound / distance traveled by the solvent front) (2.2 cm / 4 cm) = 0.550 Questions: 1a.The standard dyes present in my binary unknown, “Unknown D”, were Bromophenol Blue and Methyl Red. 1b. I concluded that the compounds listed in question 1a were present in my unknown by an analysis and comparison of the spot colors both post elution and post HCl immersion as well as the calculated R f values. I determined that the R f values for the suspected standard dyes and the unknown were identical (except for the co-spot of Bromophenol Blue, which was off by 0.100), and the colors were nearly identical which proved that these two standard dyes must have been the two that made up the binary unknown compound. The Bromophenol Blue was dark blue post elution and yellow post HCl immersion, and the methyl red was yellow-orange post elution and dark red post HCl immersion. The unknown was nearly identical to these dyes for the first and second plates as well as the co-spotting. The R f values obtained for the methyl red were 0.600 and 0.587 for the co-spot, the bromophenol blue was 0.538 and 0.647 for the co-spot, and the unknown D had R f values of 0.462 and 0.642, and 0.609, 0.587, 0.735, 0.617 for the co-spot.

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Due to the fact that the colors spotted were nearly identical between both dyes and the unknown D and the R f values were similar as well as closer than the other standard dyes, I was able to determine that the unknown D must be a mixture of Bromophenol Blue and Methyl Red. 2. 3. 4. The R f value for methyl red was calculated to 0.600 for the first elution, 0.609 for the co-spot and the R f value for bromophenol blue was calculated to 0.538 for the first elution, and 0.647 for the co-spot. Therefore, methyl red has a higher R f overall. Bromophenol Blue is more polar than methyl red because it has less mobility on the TLC plate, only traveling 1.4 cm compared to methyl red’s 2.4 cm.

Works Cited: “Thin_Layer_Chromatography_Modifications”, Canvas Document The acid dissociation constant of methyl red. A spectrophotometric measurement" S.W. Tobey. J. Chem. Educ. 1958, 35, 10, 514. The Equilibrium Constant for Bromothymol Blue: A General Chemistry Laboratory Experiment Using Spectroscopy" E. Klotz, R. Doyle, E. Gross and B.Mattson. J. Chem. Educ. 2011, 88, 5, 637.