Quantitative Spectroscopy Beer’s Law A 1 = e 1 bc e is molar absorptivity (unique for a given compound at 1 ) b is path length c concentration
Standard Practice Prepare standards of known concentration Measure absorbance at lmax Plot A vs. concentration Obtain slope Use slope (and intercept) to determine the concentration of the analyte in the unknown
Absorption measurement Must make measurements at two wavelengths A 1 = e 1HIn bc HIn + e 1In bc In- A 2 = e 2HIn bc HIn + e 2In bc In- Must have the slopes of four Beer’s law plots. Then you have two equations with two unknowns.
The four slopes Measure standard solutions of Indicator adjusted to high pH (three pH units above pKa) [In-]/[HIn] = 1000 Measure standard solutions of Indicator adjusted to low pH (three pH units below pKa) [HIn]/[In-] = 1000 These are provided for you.
In the lab Prepare Indicator solution adjusted to the proper pH Measure its pH and Absorbance at 430 nm and 565 nm Adjust pH with dilute HCl or NaOH by.2 pH units and remeasure Continue until you have five measurements above and below the starting pH
Spreadsheet Type in pH and corresponding absorbance measurements and the spreadsheet will calculate Ka and pKa values automatically –Calculate [H + ] from the pH –Solves simultaneous equation for [HIn] and [In-] –Solves for Ka, Ka = [H + ][In - ]/[HIn] –Solves for pKa, pKa = -logKa –Calculates a standard deviation