Experiment 1 Chemical Equilibria and Le Ch telier s Principle A local theatre company is interested in preparing solutions that look like blood for their upcoming production of Lizzie Borden They have hired Chemical Solutions Incorporated CSI to help them investigate the aqueous reaction of potassium thiocyanate with iron III nitrate that they have heard other companies are using as fake blood You will investigate this equilibrium for CSI both qualitatively and quantitatively The following useful information for these experiments is excerpted from reliable Web sites and is reproduced with permission of the authors You should also prepare for this experiment by reading about chemical equilibria and Le Ch telier s Principle Chapter 15 in your textbook The Iron Thiocyanate Equilibrium When potassium thiocyanate KNCS is mixed with iron III nitrate Fe NO3 3 in solution an equilibrium mixture of Fe 3 NCS and the complex ion FeNCS 2 is formed equation 1 The solution also contains the spectator ions K and NO3 The relative amounts of the ions participating in the reaction can be judged from the solution color since in neutral to slightly acidic solutions Fe 3 is light yellow NCS is colorless and FeNCS 2 is red If the solution is initially reddish and the equilibrium shifts to the right more FeNCS 2 the solution becomes darker red while if the equilibrium shifts to the left less FeNCS 2 the solution becomes lighter red or straw yellow You will add various reagents to this reaction at equilibrium to see if how those reagents shift the equilibrium position of the reaction using the color of the resulting solution Fe 3 NCS yellow colorless FeNCS 2 blood red 1 Quantitatively the relative amounts of the two reactants and the product are related by the equilibrium constant for the reaction in this case the formation constant Kf which is shown below To precisely control the red color of the solution it is necessary to know the value for Kf Kf can be calculated through an experimental determination of the FeNCS 2 eq using a standard curve week 2 and deduction of the Fe 3 eq and NCS eq by subtracting the amount of FeNCS 2 produced from the known added initial amounts of Fe 3 and NCS as that is how much was consumed during the reaction FeNCS 2 eq Fe 3 eq NCS eq Use of a Standard Curve In this technique a series of solutions with known concentrations is prepared and then a parameter such as absorbance is measured This parameter is then plotted versus concentration to yield a standard curve in this case how absorbance varies with concentration which is often a straight line with some degree of experimental error Analysis of the data allows determination of the best fit line e g with Excel A subsequent measurement of the absorbance for an unknown sample allows determination of its concentration using the equation for the standard curve The unknown concentration should lie in the concentration range of the standard solutions used to construct the standard curve for an accurate concentration determination Beer s Law Beer s Law relates the experimental absorbance value for a chromophore a substance that absorbs light to the concentration of that chromophore in solution Beer s law has many forms the most common is A l C In this equation A is the measured absorbance of the chromophore at a given wavelength usually at a peak maximum or lmax determined from a spectrum spanning ultraviolet and or visible wavelengths of light The Greek letter epsilon stands for the molar extinction coefficient M 1 cm 1 an experimentally determined constant for the specific chromophore at a specified wavelength The molar extinction coefficient is a quantitative measure of the light absorbance by the chromophore at that wavelength for a one molar solution and a one centimeter path length The value l is the path CH 142 Experiment 1 Spring 2012 length or the distance the light travels through the solution in the cuvette container used for the absorbance measurement Lastly C is the molar concentration of the chromophore mol L used for the measurement Beer s law says that the relationship between the absorbance of the chromophore and its concentration is linear allowing construction of a standard curve by plotting absorbance versus concentration such as shown in Figure 1 This particular curve is most reliable for absorbance values between 0 0 1 0 The concentration for an unknown solution can be determined by measuring its absorbance first Then use the known absorbance value and the equation for the standard curve to solve for x the concentration Figure 1 Absorbance vs concentration M of a chromophore 2 CH 142 Experiment 1 Spring 2012 Pre Laboratory Assignment Week 1 Buy your lab notebook see Lab Syllabus for specific requirements This week and every week come to lab with a hand written outline of the experimental procedure in your lab notebook Week 2 In addition to the usual outline of the procedure in your lab notebook please answer the following questions Due before 9 AM Fri Feb 10th in white bookcase by Keyes 310 A Calculate the concentrations of NCS and Fe 3 in each of the following solutions Note that the final total volume is 10 0 mL in each case due to the addition of H2O As always all work must be shown to receive full credit Watch those units and sig figs volume 0 0020 M KNCS mL volume 0 0020 M Fe NO3 3 mL 1 0 5 0 2 5 5 0 total solution volume mL 10 0 10 0 B 1 Construct a standard curve in Excel using the following data and 0 0 micromolar concentration gives 0 000 absorbance based on the absorbance at 275 nm of protein sample Print your graph once it is titled Y vs X includes the equation of the line and the R2 value has a label for each axis and includes any appropriate units 2 Use this graph to determine the unknown concentrations of two samples of the protein showing your work 3 Would you expect these determinations to be accurate or not Explain your answer Concentration of Protein Sample 2 0 micromolar 1 0 micromolar 0 50 micromolar 0 20 micromolar unknown concentration 1 unknown concentration 2 3 Absorbance at 275 nm 0 460 0 251 0 117 0 067 0 178 0 603 CH 142 Experiment 1 Spring 2012 th th Experimental Procedure Week 1 Feb 6 10 For each of the external stresses described below necessary information is provided regarding the manner in which one or more of the chemical species is affected You will use a spot plate containing multiple wells and use a different well for each of the operations described recording your