5 Quantitative Spectroscopy Beer’s LawAl1 = el1bce is molar absorptivity (unique for a given compound at l1)b is path lengthc concentration
6 Standard Practice Prepare standards of known concentration Measure absorbance at lmaxPlot A vs. concentrationObtain slopeUse slope (and intercept) to determine the concentration of the analyte in the unknown
11 Absorption measurement Must make measurements at two wavelengthsAl1 = el1HInbcHIn + el1InbcIn-Al2 = el2HInbcHIn + el2InbcIn-Must have the slopes of four Beer’s law plots. Then you have two equations with two unknowns.
12 The four slopesMeasure standard solutions of Indicator adjusted to high pH (three pH units above pKa)[In-]/[HIn] = 1000Measure standard solutions of Indicator adjusted to low pH (three pH units below pKa)[HIn]/[In-] = 1000These solution will be prepared and measured to produce the four Beer’s Law Plots (each set at both wavelengths)
13 In the lab Prepare Indicator solution adjusted to the proper pH Measure its pH and Absorbance at 430 nm and 565 nmAdjust pH with dilute HCl or NaOH by .2 pH units and remeasureContinue until you have five measurements within 1.0 pH units above and below the starting pH
14 Spreadsheet Use data to produce four Beer’s Law Plots Determine slopes of Beer’s Law PlotsCalculate [H+] from the pHUse slopes to solve the system of two equations for [HIn] and [In-]Solve for Ka, Ka = [H+][In-]/[HIn]Solve for pKa, pKa = -logKaCalculate averages and 95 % CL