Experiment 5: Crystal Violet Virtual Lab

Experiment 5: Crystal Violet Virtual Lab

Name: _____________________________________________________ Date: ________________ The Kinetics of Fading Crystal Violet by Reaction with Sodium Hydroxide Question What is the order of the reaction between crystal violet and sodium hydroxide with respect to crystal violet? Introduction Crystal violet (CV+ ) belongs to a class of intensely colored organic compounds called triphenylmethane dyes. In aqueous solution, it has a brilliant violet color. Its resonance structures are shown in figure 1a and 1b. Crystal violet molecules will react with hydroxide ions to form a neutral compound, which is colorless in solution (“c” in figure 1). Figure 1. Resonance structures of crystal violet and structure after reaction with a hydroxide ion. The rate law for this reaction has the general form: 𝑅𝑎𝑡𝑒 = 𝑘[𝐶𝑉 +] 𝑛 [𝑂𝐻−] 𝑚 Equation 1. where 𝑘 is the rate constant and 𝑛 and 𝑚 represent the orders of the reaction with respect to the concentrations of corresponding species and must be experimentally determined. The hydroxide ion concentration will essentially remain constant if the reaction is carried out under conditions of high molar ratio of hydroxide ion to crystal violet. The rate law can then be simplified to a pseudo rate law of: 𝑅𝑎𝑡𝑒 = 𝑘 ′ [𝐶𝑉 +] 𝑛 Equation 2. where 𝑘 ′ = 𝑘[𝑂𝐻−] 𝑚. Under these conditions, if concentration of crystal violet is monitored over time, the value of 𝑛 can be determined by application of integrated rate laws. If the reaction is zero order with respect to crystal violet the integrated rate law is: [𝐶𝑉 +]𝑡 = −𝑘𝑡 + [𝐶𝑉 +]0 Equation 3. and a plot of [𝐶𝑉 +]𝑡 versus time will yield a linear plot with a slope equal to −𝑘. If the reaction is first order with respect to crystal violet the integrated rate law is: 𝑙𝑛[𝐶𝑉 +]𝑡 = −𝑘𝑡 + 𝑙𝑛[𝐶𝑉 +]0 Equation 4. and a plot of 𝑙𝑛[𝐶𝑉 +]𝑡 versus time will yield a linear plot with a slope equal to −𝑘. If the reaction is second order with respect to crystal violet the integrated rate law is: 1 [𝐶𝑉 +] ⁄ 𝑡 = 𝑘𝑡 + 1 [𝐶𝑉 + ⁄ ]0 Equation 5. and a plot of 1 [𝐶𝑉 +] ⁄ 𝑡 versus time will yield a linear plot with a slope equal to 𝑘. By obtaining concentration versus time data, plotting [𝐶𝑉 +]𝑡 versus time, 𝑙𝑛[𝐶𝑉 +]𝑡 versus time, and 1 [𝐶𝑉 +] ⁄ 𝑡 versus time, and assessing the linearity of the plots, the order of the reaction with respect to crystal violet can be determined. One way to monitor concentration of a colored solution is by colorimetry. Colorimetry is the measurement of color intensity. Colorimetry can be accomplished by various instruments. In this lab, a smartphone or other camera capable device will be used to acquire images of the reaction over time. The pixels in the image will be analyzed with a color picker application that can determine a red, green, or blue (RGB) color intensity. The green channel will be used as green is the most complementary of the three channels as compared to the purple color of the crystal violet solution; thus, it is absorbed the most by the solution and will yield the most quantitative data. The color intensity data will be converted into absorbance data by the following equation: 𝐴 = −log(𝐼⁄𝐼0) Equation 6. where 𝐴 is the absorbance, 𝐼 is the intensity from the green channel of a sample and 𝐼0 is the intensity from the green channel of a blank. Ultimately, concentration will be calculated by using a standard calibration curve prepared from standard solutions and the Beer-Lambert law. The BeerLambert law (Beer’s law) relates absorbance to the concentration of a solution by the following equation: 𝐴 = 𝜀𝑏𝑐 Equation 7. where 𝐴 is the absorbance, 𝜀 is the molar absorptivity coefficient, 𝑏 is the path length of light through the solution, and 𝑐 is the molar concentration of the solution. If 𝜀 and 𝑏 are known, concentration can be found from absorbance data. When the absorbances of standard solutions are plotted versus the concentration of the crystal violet solutions a linear plot results with a slope equal to 𝜀𝑏. This is the standard calibration curve, and the equation for the curve can be used to determine the concentration of an unknown by measuring its absorbance. Hazards and Safety Crystal violet is a strong dye and may stain clothes and skin. Crystal violet is moderately toxic by ingestion and is a body tissue irritant. Sodium hydroxide is caustic and may irritate the skin and eyes. Safety glasses and gloves should be worn at all times. An apron is suggested. Clean up spills immediately and consult MSDS for complete safety information. Preparation of Standard Solutions for Calibration Curve Background: A calibration curve can be used to determine the unknown concentration of a substance. The standard calibration curve is determined by obtaining data from a set of standard solutions with known concentrations. A 25.0 µM crystal violet stock solution will be diluted in order to prepare the standard solutions. Dilution involves reducing the concentration of a solution by adding a known volume of distilled water to a known volume of stock solution. To calculate how much stock solution and distilled water are required to make the concentrated solution, the dilution equation can be used: 𝑀1𝑉1 = 𝑀2𝑉2 Equation 8. where 𝑀1 is the molarity of the stock solution, 𝑉1 is the volume of the stock solution required to make the desired diluted solution, 𝑀2 is the molarity of the desired diluted solution, and 𝑉2 is the volume of the desired diluted solution. Example: If 10.0 mL of a 12.5 µM solution is desired the amount of 25.0 µM stock solution and distilled water can be calculated as follows: 𝑀1𝑉1 = 𝑀2𝑉2 𝑀1 = 25.0 µM 𝑉1 = ? 𝑀2 = 12.5 µM 𝑉2 = 10.0 mL 25.0 µM (𝑉1) = 12.5 µM (10.0 mL) 𝑉1 = 5.0 mL 5.0 mL of the 25.0 µM crystal violet stock solution is needed to prepare 10.0 mL of the 12.5 µM solution. To reach the total volume of 10.0 mL, 5.0 mL of distilled water must be added. Fill in the table below with the volumes of 25.0 µM stock solution and distilled water needed to make 10.0 mL of each of the standard solutions listed. Table 1. Volumes of stock solution and distilled water for standard solutions. 12.5 µM solution 10.0 µM solution 7.5 µM solution 5.0 µM solution 2.5 µM solution Volume of 25.0 µM stock solution 5.0 mL Volume of distilled water 5.0 mL Total volume 10.0 mL 10.0 mL 10.0 mL 10.0 mL 10.0 mL Procedure: 1. Using a 10 mL disposable graduated pipet and pipet pump measure the above amounts of 25.0 µM stock solution and distilled water and combine them in small test tubes to make each standard solution. Label each solution as it is made. 2. Using a 10 mL disposable graduated pipet and pipet pump transfer 4.0 mL of each standard solution to a square plastic cuvette. Place 4.0 mL of distilled water in a cuvette for a blank. Reaction between Crystal Violet and Sodium Hydroxide and Collection of Images Background: In order to study the order of the reaction with respect to crystal violet a large concentration of hydroxide ions must be used relative to the concentration of crystal violet. A 1:2000 mole ratio of crystal violet to hydroxide ion will be used for this experiment. To achieve this ratio, 4.0 mL of 25.0 µM crystal violet solution and 5.0 mL of 0.040 M sodium hydroxide solution will be mixed. Procedure: 1. Line up cuvettes containing the 25.0 µM solution, 12.5 µM solution, 10.0 µM solution, 7.5 µM solution, 5.0 µM solution, 2.5 µM solution, blank, empty cuvette, and a timer with a white background and foreground, as in figure 3. 2. Position a smartphone or other camera capable device approximately 12 inches from the solutions. If possible construct a holder for the smartphone or other device to minimize movement during image collection as in figure 3. 3. Adjust the positions of the solutions and smartphone (or other device) and the lighting to minimize shadows or glare in the images. 4. In a small test tube, mix 4.0 mL of the 25 µM crystal violet stock solution with 5.0 mL of the 0.040 M sodium hydroxide solution. Quickly transfer 4.0 mL the reaction mixture to the empty cuvette. 5. Immediately upon mixing, start the timer and then start the image acquisition. If possible, collect images using a free application, such as Camera FV-5 Lite, that automatically collects images at timed intervals and can lock the focus and white balance. Collect images every 10 seconds for 10 minutes. Analysis of Images Procedure: Analysis of the images with a color picker application can be completed on the smartphone or other camera capable device used for image collection or images can be transferred to a computer for analysis. To assist with data analysis an example data table and calculations are provide below. 1. Start image analysis once hands or other objects do not interfere with consistent lighting. Figure 3. Reaction set up. Figure 4. Example image. 2. Starting with the 25.0 µM solution, use a color picker application to measure the green color intensity at three locations in the sample. 3. Repeat this for the 12.5 µM solution, 10.0 µM solution, 7.5 µM solution, 5.0 µM solution, 2.5 µM solution and blank. 4. Calculate the average green color intensity for each solution and blank. 5. Convert each of the average green color intensities to an absorbance using equation 6. 6. Prepare the calibration curve by plotting the absorbance versus concentration of each solution and blank. 7. Determine the slope and y-intercept for the line of best fit and put them into y = mx + b format. In this equation y represents the absorbance and x represents the concentration. This equation will allow for determining the concentration of the reaction mixture given an absorbance of the mixture at each time point. 8. Starting with the image used for the calibration curve measure the green color intensity of the reaction mixture at three locations. 9. Repeat this for each image at 30 second intervals from the starting time. 10.Calculate the average green color intensity of the reaction mixture at each time point. 11.Convert the average green color intensity from each time point to an absorbance using equation 6. 12.Convert the absorbance values to concentration values using the equation determined by the calibration curve in step 7. 13.Calculate ln(concentration) for each time point 14.Calculate inverse concentration for each time point. 15.Construct three plots using Excel, Google spreadsheets, or a similar program: a. concentration versus time b. ln(concentration) versus time c. inverse concentration versus time 16.Assess the linearity of each plot to determine the integrated rate law and the order of the reaction with respect to crystal violet. A correlation coefficient (R2 ) may be useful in assessing the linearity of the plots. 17.If the plots are inconclusive, additional time points in between the 30 second interval images can be assessed. 18.Construct a table similar to table 2. Example calculations can be found below. Table 2. Example data table. Time (seconds) Measured Green Intensity #1 Measured Green Intensity #2 Measured Green Intensity #3 Calculated Average Intensity Calculated Absorbance Calculated Concentration Calculated ln(Conc.) Calculated Inverse Conc. 30 64 67 69 66.667 0.383 8.517 2.142 0.117 60 90 …