# Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40

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1 Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40 Note: there is a second document that goes with this one! Absorbance Spectrophotometry - Calibration Curve Procedure. The second document has instructions for determining the slope and intercept of a linear data set. You will need to do this for the calculations in the second week of this experiment. Introduction The purpose of this experiment is to determine the concentration of red dye in an unknown sample, provided by your instructor. Students will familiarize themselves with use of volumetric pipettes, volumetric flasks and the absorption spectrophotometer. A review of molarity calculations will be helpful in performing this lab! Discussion Absorbance Spectrophotometry is a commonly used laboratory technique for determining the concentration of substances in solutions. In this experiment we will prepare several samples of FD&C Red #40, a commercial food dye, and use them to calibrate the absorbance spectrophotometer. Using the calibrated spectrophotometer, we will then analyze a sample of Kool-Aid brand drink mix to determine the quantity of Red #40 in the sample. Many compounds are colored due to their absorption of visible light. Our eyes are naturally capable of detecting many different wavelengths or energies of light, and we perceive these different wavelengths as different colors, as summarized in the table below: Color violet blue green yellow orange red Wavelength nm nm nm nm nm nm So, for instance, light with wavelengths between 620 and 750 nm are perceived by the human eye as red light. White light, such as that produced by the sun or many artificial light sources, contains all of the visible wavelengths of light combined together; our eye perceives this as white but it is really a blend of all of the colors of the spectrum (red, orange, yellow, green, blue, indigo and violet). When white light passes through a colored liquid, some of the wavelengths are absorbed and some are transmitted. The result is that that the liquid will appear colored to our eye. If a liquid absorbs all of the light in the violet, blue, green and yellow wavelength ranges, but allows orange and red light to pass through, then the liquid will appear orange-red 1

2 because its only transmitting orange and red light. All of the other colors are filtered out by the molecules in the liquid. On the other hand, a substance that absorbs in the wavelength range nm will appear blue or purple to our eyes, because all of the red, orange, yellow and green light have been absorbed. If a sample absorbs no light within the visible spectral range, then it will appear as a clear, colorless liquid. Water, for instance, absorbs such a small amount of visible light that our eyes generally cannot perceive the absorbance; unless you have a very large volume of water, it appears completely clear. (Please note that water is NOT truly clear - it DOES absorb visible light to a small degree. That s why it gets darker as you move to the bottom of a pool of water, even if the water is clean). If a sample absorbs ALL of the visible wavelengths then it is opaque; no visible light will pass through it at all. Many aqueous solutions of chemicals are transparent (meaning that they transmit light) but not colorless; they will absorb certain wavelengths and transmit others, resulting in a colored solution as described above. Aqueous solutions of copper ion, for instance, appear blue or green because they absorb light in the range from about nm. Aqueous solutions of certain iron ions are orange because they absorb in the range from about nm. To illustrate these ideas, consider the figure below. Light is shown on the left as incident on a sample and then emerging from the sample at the right. In general, P o, the radiant power of the incident light, will be larger than P, the power of the emerging light, because the sample will absorb some of the light. This is referred to as the absorbance of the solution. Not only is P o > P, but the colors (wavelengths) of the emerging light may be different than the incident light. Po sample P b A simple and logical relationship exists between the absorbance of the solution and the concentration of the solution; namely, that the more sample molecules we place in the path of the incident light, the greater will be the absorbance of the light. Low concentrations will transmit more light (and the color will appear lighter or less intense), and high concentrations will transmit less light (and the color will appear darker or more intense). This relationship is the basis of Beer s Law, usually expressed as follows: A = bc (equation 1) 2

3 where A is the absorbance, is a mathematical proportionality constant called the molar absorptivity, b is the pathlength of the light through the sample (as shown in the figure above), and c is the concentration of the absorbing species. Beer s Law can be used to determine concentration of a compound in solution if we measure A and know both and b, or their product b. The law s most common application in chemistry is to liquid-phase solutions; the concentration, c, is usually expressed in molarity (M) and the pathlength, b, is usually expressed in centimeters. The constant is specific to each wavelength and units of M -1 cm -1. If you look at the units of absorbance in Beer s Law, you can see that the value of A is unitless (or dimensionless). A spectrophotometer is a simple device that measures the amount of light which is absorbed at a particular wavelength; in terms of the figure above, it compares P (the light which passes through the sample) to P o (the light which is incident on the sample). In theory, absorbances can range from zero (a sample which is totally transparent at the wavelength tested) to infinity (a sample which is completely opaque at the wavelength tested). In practice, however, absorbances above about 2 are not measurable; above an absorbance of 2, the sample transmits so little light that it is difficult to measure. (You will notice as you perform the experiment that if the reading rises above 1.999, the display will flash, indicating an error readings above this value are not reliable). In very simple terms, the spectrophotometer measures how dark the color of a solution is and Beer s Law allows us to mathematically relate the darkness of the color to the concentration of the dissolved solute. Look again at Beer s law, above you will notice that it has the form of a linear equation. The form of a linear equation is shown below, compared to Beer s law: y = mx + intercept A = (b)c + 0 You will notice that a plot of absorbance on the y-axis vs. concentration on the x-axis is predicted to be a linear plot, with a slope equal to b, and an intercept of zero. In absorbance experiments, this is referred to as the calibration curve. The first step in performing an absorbance experiment is to determine the calibration curve, by preparing samples with known concentration (x-values) and measuring their absorbances (y-values). The calibration curve is a description or representation of the mathematical relationship between A and c. Thus, once the calibration curve is complete, you will be able to interconvert between A and c. 3

4 For example, the graph below is a calibration curve for iodide ion, I 1-. Several solutions of iodide ion were prepared, and their absorbances measured on the spectrophotometer: Notice that the intercept of the y-intercept of the line is close to zero this makes sense, because if the concentration the light-absorbing compound iodide is zero, then the absorbance should also be zero. In practice, the intercept will be a very small number, close to zero, but may not be exactly equal to zero. {Don t be surprised when you do your calculations if your intercept is not zero!}. Also, the slope of the line has been calculated and is shown in the bottom left corner of the figure, along with the calculated intercept. Now that the calibration curve is known, the concentration of any iodide solution is easily determined by measuring its absorbance. For instance, if an unknown solution containing iodide has a measured absorbance of 0.400, then you can see from the graph that the concentration of iodide must be around M; that s the x-value that corresponds to a y-value of Rather than approximating the concentration, however, we can precisely calculate it. Knowing that on this linear plot the slope is , and the intercept is , the relationship is: y = mx + b ABS = [I 1- ] If the absorbance is 0.400, the concentration of iodide is calculated to be M. Note that calculating the molarity gives a much more precise value than simply estimating it from the graph. This is why the slope and intercept of the line must be determined. The calculation process outlined in this example is very similar to the method you will use to determine the concentration of FD&C Red Dye #40 in an unknown sample. 4

9 Absorbance Spectrophotometry: Data Page Name Lab partner Spectrophotometer number Week 1: Absorbance of Standards The stock solution, which was provided, was prepared by dissolving grams of Red 40 in water to make L of solution. Standard Concentration of Red 40 (M) Absorbance Questions be sure to show ALL CALCULATIONS on your calculation page! 1. Calculate the molarity of the standard Red 40 solution that was provided. The molecular formula for Red 40 is C 18 H 14 N 2 S 2 O 8. M 2. Calculate the concentration of Red 40 in the four standards you prepared. Enter them in the table above. 3. The pathlength of the cuvettes we used in this experiment is 1.00 cm. Use the formula for Beer s Law (equation 1 in the discussion) to determine the molar absorptivity of Red 40. (For the purpose of this question, you may assume that the intercept of the calibration curve is zero). Report your answer for all four standards; they should be very similar, although they may not be exactly the same. 9

10 Data Page (continued) Week 2: Kool-Aid analysis Mass of powder mix used: grams Absorbance of Unknown Slope of calibration curve Intercept of calibration curve Questions be sure to show ALL CALCULATIONS on your calculation page! 4. Based on the absorbance of the Kool-Aid, determine the concentration of Red 40 in Solution 2. M 5. How many moles of Red 40 were in your Kool-Aid Solution 2? 6. How many grams of Red 40 were in your Kool-Aid Solution 2? moles grams 7. How many grams of Red 40 were in Solution 1? (This is a very simple question! What is the relationship between the amount of solute in solution 1 and solution 2?) 8. Calculate the % of Red 40 (by mass) in the Kool-Aid powder mix. grams % Remember to submit a copy of your calibration curve with this report. 10

11 Absorbance Spectrophotometry: Advance Study Assignment Name 1. A solution is prepared by dissolving grams of FD&C Red #40 in water to produce Liters of solution. Given that the molecular formula of Red 40 is C 18 H 14 N 2 S 2 O 8, determine the molarity of this stock solution. M ml of the dark red stock solution described question #1 is transferred to a volumetric flask and diluted with water to ml. a. how many moles of Red 40 are in the ml sample that was transferred? moles b. After the dilution, what is the molarity of the Red 40 in the ml of solution? M note: the preceding problem (#2) is a dilution calculation. A convenient formula for calculating concentrations after dilution is M 1 V 1 = M 2 V 2, where M 1 and V 1 are the molarity and volume of the concentrated solution, respectively, and M 2 and V 2 are the molarity and volume of the dilute solution. 3. a. The solution described in problem #2 is placed in the spectrophotometer. Based on the calibration curve shown on the next page, what would you predict the absorbance to be? Note: like the example given in the discussion, the absorbance should be calculated, not just estimated from the graph. Notice that the slope and intercept of the calibration curve are provided in the figure on the next page. Absorbance = 11

12 The following calibration curve was produced for Red 40. The slope was calculated to be x The intercept of the calibration curve is b. A sample of powered drink mix with a mass of grams is dissolved in water to make ml of solution. A sample of this solution is placed in the spectrophotometer and its absorbance is measured as Based on the calibration curve above, calculate the molarity of Red 40 in the unknown. c. How many grams of Red 40 are in the drink mix solution? M d. What is the % by mass of Red 40 in the drink mix powder? g % 4. A sample of powdered drink mix with a mass of g is dissolved in water to make ml of solution ml of the resulting solution is transferred to a new container and diluted to a total volume of ml. How many grams of the drink mix powder are in the second solution? (hint: there is a very simple relationship between the concentrations of these two solutions... this is a basic one-step calculation!) grams 12

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