Spectra of Conjugated Dyes & Beer's Law Overview Getting Started Techniques Procedure FAQ Full Lab Manual Introduction & Goals Chemistry & Background Key Questions Prelab Problems Safety Procedure In Your Write-up Experiments Index ChemLab Home		Measuring the spectrum of conjugated dyes Obtain a solution of one of the two conjugated dyes in a cuvet and note its color in your lab notebook. Estimate the wavelength of maximum absorbance assuming that the dye has one absorption peak. You can use the picture of a color wheel in your lab manual or the Spectral Colors Applet to help you estimate the wavelength of maximum absorption for a solution of the observed color. Use the estimate of the maximum absorbance as a starting wavelength to measure and plot the absorption and transmission spectra, using a Spec 20. You and your partner must decide at what wavelengths to measure the absorbance and transmission to map out the spectrum adequately in a reasonable period of time. The Spec 20 must be calibrated with a blank at each wavelength. For information about how to use the two types of Spec 20 spectrometers in the lab, see the following pages in the techniques section of this website. Spectrometer: Analog Spectrometer: Digital Plot both the transmission and absorbance spectra as you measure them. Your two spectra should include the entire visible range, but need not have the same wavelength resolution in all regions. You will use a scanning spectrometer to measure the absorption spectrum of the second conjugated dye. For information about this instrument, go to the following page in the techniques section of this website. Spectrometer: Scanning Measuring the absorption spectrum of Koolaid Obtain a packet of the drink mix supplied in your lab and record its flavor. Make up 25 mL of solution at the concentration given on the label using an analytical balance and volumetric flask. Record the mass of drink mix used and calculate the concentration of the solution in g/L. For detailed information about how to make up a solution with a volumetric flask, see this week's Techniques overview page. Place some of your drink solution in a cuvet and measure its absorption spectrum with the scanning spectrometer. Your TA will show you how to operate the instrument, but you should make the measurements. Label the spectrum with the wavelength(s) of maximum absorption. More information about how to use the scanning spectrometer can be found on the following page in the Techniques section of this website. Spectrometer: Scanning Paper chromotography Cut a piece of chromatography paper an inch or two longer than the height of the beaker you plan to use. Make the cut perpendicular to the strip of paper, so the bottom of the strip is straight. Place a small amount of powdered drink mix on a watch glass and add a drop or two of water. Mix the water and Koolaid together into a uniform, concentrated slurry. Use a rectangular strip of filter paper and apply a small spot of the concentrated drink solution with a wooden stick, about 1 inch from the end of the strip. Allow the spot to dry and apply another spot of solution to the same place until the color of the spot is clearly visible. Fold the strip of paper and suspend it, with the spot end down, in a beaker containing a thin layer of water. The bottom of the paper should just touch the water and the spot should be above the surface of the water. Allow the water to diffuse up the paper, until you are satisfied with the results and have a clear answer to your questions. After you have completed the chromatogram and the spectrum, identify the dye(s) in the mix, as observed by each technique. Record your results in your lab notebook. Beer's Law experiment for food dyes To start, you will produce a "standard curve" for each dye found in your drink mix. Your data will be absorbance values for several solutions of precisely known concentration of the individual dye(s) under study. Dilutions of the initial stock solution should be done with a volumetric flask, volumetric pipets, and your best analytical technique. To make a series of solutions of known concentration, you can pipet different amounts of the stock solution into a set of volumetric flasks. Click on the link for information about how to use a pipet You should pipet different volumes of stock solution into each flask so that you will have at least four different concentrations, plus the original stock solution. Next, you can dilute the solution in each volumetric flask to make a set of solutions with different concentrations. Dilute by adding deionized water until the meniscus reaches the mark on the neck of the flask. More details about how to use a volumetric flask are given on the page below. Volumetric flask When your dilutions are complete, you should have a set of solutions with precisely known concentrations and different absorbances. Don't forget to inlcude the original stock solution in your measurements. Use a Spec 20 to measure the absorbance at the wavelength of maximum absorbance for each of your solutions. After you have completed the measurements of absorbance vs. concentration for the set of known dye solutions, graph a Beer's Law plot(s) by hand or on a graphing calculator, to check for linearity. If more than one dye is present in your drink mix, plan to share data with another group of students. You will need a set of Beer's Law data at the appropriate wavelength for each dye present in your drink mix. Absorbance measurements of Koolaid solution Your final goal is to determine the concentration of the dye(s) in the Koolaid solution. To do this, use a Spec 20 to measure the absorbance of your drink solution at all relevant wavelengths. Don't forget to calibrate with a water blank at each wavelength. For your uncertainty analysis, you will need to record the uncertainty with which you can measure the absorbance of your solution.
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