Spectrophotometry: An Analytical Tool CHM 103 Sinex Spectrophotometry: An Analytical Tool

Io I The process of light being absorbed by a solution concentration 2 with sample I < Io concentration 1 blank where Io = I light source detector Io I As concentration increases, less light is transmitted (more light absorbed). b Cell with Pathlength, b, containing solution PGCC CHM 103 Sinex/Gage

Some terminology I – intensity where Io is initial intensity of light entering a solution and I is the intensity of light exiting a solution T – transmission (no units, ratio) T = I/ Io %T = 100 x T (absorption: Abs = 1 – T or %Abs = 100 - %T) A – absorbance (no units) A = - log T = -log I/ Io PGCC CHM 103 Sinex/Gage

Remembering the “More Lights, Color, Absorption” lab activity, what factors affect the amount of light that is absorbed by a solution in a spectrometer? PGCC CHM 103 Sinex/Gage

A = abc Beer’s Law a = molar absorptivity b = pathlength where a = molar absorptivity (actually the symbol ε is the correct symbol for this but a is easier to remember) b = pathlength c = molar concentration See the Beer’s Law Simulator PGCC CHM 103 Sinex/Gage

Molar absorptivity Depends on the electronic structure of the substance being analyzed (analyte) Varies with the wavelength of light because a compound absorbs different amounts at different wavelengths Units = L mol-1 cm-1 (a = A/bc = 1/(mol/L x cm)) PGCC CHM 103 Sinex/Gage

Analyze at what wavelength? Scan visible wavelengths from 400 – 650 nm (detector range) to produce an absorption spectrum (A vs. l) lmax phototube detector range lmax - wavelength where maximum absorbance occurs PGCC CHM 103 Sinex/Gage

The BLANK The blank contains all substances except the analyte. Is used to set the absorbance to zero: Ablank = 0 This removes any absorption of light due to these substances and the cell. All measured absorbance is due to analyte. PGCC CHM 103 Sinex/Gage

The components of a Spec-20D Light source - white light of constant intensity slits filter occluder Grating slits Separates white light into various colors Phototube detects light & measures intensity Rotating the grating changes the wavelength going through the sample Sample When blank is the sample Io is determined otherwise I is measured PGCC CHM 103 Sinex/Gage

What does the absorbed light (electromagnetic radiation) do to the molecule? IR UV 700 nm visible 400 nm Energy increasing high energy UV – ionizes electrons low energy UV and visible – promotes electrons to higher energy orbitals (absorption of visible light leads to a colored solution) IR – causes molecules to vibrate (more later) PGCC CHM 103 Sinex/Gage

UV/visible light absorption Valence electrons In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: p  p* In transition metals, hydrated ions such as Cu2+ have splitting of d orbital energies and electronic transitions – weak absorption In complexed transition metals, charge transfer of electrons from metal to ligand as Cu(NH3)42+ – strong absorption PGCC CHM 103 Sinex/Gage

Uses of visible spectrophotometry Analysis of unknowns using Beer’s Law calibration curve Absorbance vs. time graphs for kinetics Single-point calibration for an equilibrium constant determination Spectrophotometric titrations – a way to follow a reaction if at least one substance is colored – sudden or sharp change in absorbance at equivalence point, a piece-wise function (Been there, done that!) PGCC CHM 103 Sinex/Gage

Standard Curves Concentration (mol/L or M) 0.01 0.02 0.05 0.06 0.07 0.03 0.04 Absorbance regression equation If you know the absorbance of an unknown you can determine the concentration. PGCC CHM 103 Sinex/Gage

purple colorless colorless Kinetics of Crystal Violet Reaction CV+ + OH-  CV-OH purple colorless colorless Follow concentration of crystal violet over time as it reacts by measuring its absorbance. How will absorbance change with time? For a absorbance vs. time plot, how will you determine the rate of the reaction? Chime structures PGCC CHM 103 Sinex/Gage

purple colorless colorless CV+ + OH-  CV-OH purple colorless colorless This is tracking reaction progress over time. Since the absorbance is related to concentration, rate or DA/Dtime is the slope of a regression line. absorbance Short run times to get initial rates. time STELLA model PGCC CHM 103 Sinex/Gage

Single-point calibration Standard with measured absorbance Astd = abcstd Unknown with measured absorbance Aunk = abcunk Ratio the two equations Aunk/ Astd = abcunk /abcstd Aunk/ Astd = cunk /cstd Solve for cunk PGCC CHM 103 Sinex/Gage

Equilibrium Constant Determination Fe+3 + SCN- = Fe(SCN)++ colorless colorless orange K = (Fe(SCN)++)/(Fe+3)(SCN-) Using the reactants, shift reaction based on Le Chatelier’s principle. Fe(SCN)++ + SCN- = Fe(SCN)2+ We start with a high concentration of Fe+3 and lower its value by dilution. Interactive Excel spreadsheet PGCC CHM 103 Sinex/Gage

When calibration curves go bad! The linear Beer’s Law relationship starts to show curvature at high concentrations Single-point calibration assumes a linear calibration curve Non-linear PGCC CHM 103 Sinex/Gage

Spectrophotometric titration Let’s consider the analysis of hydrogen peroxide with potassium permanganate in an acidic solution. The potassium permanganate or MnO4- is the only colored substance in the reaction. (It can serve as its own indicator.) How would the absorbance change as titrant was added? PGCC CHM 103 Sinex/Gage

5H2O2 + 2MnO4- + 6H+  5O2 (g) + 2Mn+2 + 8H2O purple Notice you do not need to have a data point at the equivalence point. Equivalence point located by extrapolation of the two lines. absorbance Equivalence point MnO4- reacting, color disappears xs MnO4- accumulates Volume of titrant (mL KMnO4) PGCC CHM 103 Sinex/Gage