Non-Instrumental Methods Introduction 1) Colorimetry An analytical technique in which the concentration of an analyte is measured by its ability to produce or change the color of a solution Changes the solution’s ability to absorb light 2) Spectrophotometry Any technique that uses light to measure chemical concentrations A colorimetric method where an instrument is used to determine the amount of analyte in a sample by the sample’s ability or inability to absorb light at a certain wavelength. Colorimetry Instrumental Methods (spectrophotometry) Non-Instrumental Methods

3) Illustration Measurement of Ozone (O3) Above South Pole O3 provides protection from ultraviolet radiation Seasonal depletion due to chlorofluorocarbons O3 cycle Chain Reaction Depletion of O3 Spectra analysis of [O3]

Properties of Light 1) Particles and waves • Light waves consist of perpendicular, oscillating electric and magnetic fields • Parameters used to describe light amplitude (A): height of wave’s electric vector Wavelength (λ): distance (nm, cm, m) from peak to peak Frequency (ν): number of complete oscillations that the wave makes each second Hertz (Hz): unit of frequency, second-1 (s-1) 1 megahertz (MHz) = 106s-1 = 106Hz

As frequency (ν) increases, energy (E) of light increases 1) Particles and Waves Energy (E): the energy of one particle of light (photon) is proportional to its frequency where: E = photon energy (Joules) ν = frequency (sec-1) h = Planck’s constant (6.626x10-34J s) As frequency (ν) increases, energy (E) of light increases where: c = speed of light (3.0x108 m/s in vacuum) ν= frequency (sec-1) λ = wavelength (m) where: = (1/λ) = wavenumber As wavelength (λ) decreases, energy (E) of light increases

2) Types of Light – The Electromagnetic Spectrum energy (E), frequency (ν), wavelength (λ)

2) Types of Light – motion of particle (atom and molecule)

Absorption of Light 1) Colors of Visible Light Many Types of Chemicals Absorb Various Forms of Light The Color of Light Absorbed and Observed passing through the Compound are Complementary

Color circle & Color index

Absorption of Light 2) Ground and Excited State When a chemical absorbs light, it goes from a low energy state (ground state) to a higher energy state (excited state) Only photons with energies exactly equal to the energy difference between the two energy states will be absorbed Since different chemicals have different electron shells which are filled, they will each absorb their own particular type of light Different electron ground states and excited states Energy required of photon to give this transition: DE = E1 - Eo

Absorption of Light 3) Beer’s Law The relative amount of a certain wavelength of light absorbed (A) that passes through a sample is dependent on: distance the light must pass through the sample (cell path length - b) amount of absorbing chemicals in the sample (analyte concentration – c) ability of the sample to absorb light (molar absorptivity - ε) Increasing [Fe2+] Absorbance is directly proportional to concentration of Fe2+

Percent transmittance 3) Beer’s Law The relative amount of light making it through the sample (P/Po) is known as the transmittance (T) Percent transmittance T has a range of 0 to 1, %T has a range of 0 to 100% Absorbance (A) is the relative amount of light absorbed by the sample and is related to transmittance (T) Absorbance is sometimes called optical density (OD)

3) Beer’s Law Absorbance is useful since it is directly related to the analyte concentration, cell pathlength and molar absorptivity. This relationship is known as Beer’s Law where: A = absorbance (no units) ε = molar absorptivity (L/mole-cm) b = cell pathlength (cm) c = concentration of analyte (mol/L) Beer’s Law allows compounds to be quantified by their ability to absorb light, Relates directly to concentration (c)

Beer’s Law

4) Absorption Spectrum Different chemicals have different energy levels different ground vs. excited electron states will have different abilities to absorb light at any given wavelength Absorption Spectrum plot of molar absorbance (ε) vs. wavelength for a compound The greater the absorbance  the easier it will be to detect at low concentrations

4) Absorption Spectrum lA lB By choosing different wavelengths of light (λA vs. λB), different compounds can be measured lA lB

Spectrophotometer 1) Basic Design An instrument used to make absorbance or transmittance measurements is known as a spectrophotometer

1) Basic Design Light Source: provides the light to be passed through the sample Tungsten Lamp: visible light (320-2500 nm) Deuterium Lamp: ultraviolet Light (160-375 nm) Low pressure (vacuum) Tungsten Filament - based on black body radiation: heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament In presence of arc, some of the electrical energy is absorbed by D2 (or H2) which results in the disassociation of the gas and release of light D2 + Eelect  D*2  D’ + D’’ + hn (light produced) Excited state

1) Basic Design Wavelength Selector (monochromator): used to select a given wavelength of light from the light source Prism: Filter:

Order of Interference (n): nl = d sinq Reflection or Diffraction Grating: Order of Interference (n): nl = d sinq

Sample Cell: sample container of fixed length (b). Usually round or square cuvet Made of material that does not absorb light in the wavelength range of interest Glass – visible region Quartz – ultraviolet NaCl, KBr – Infrared region

Light Detector: measures the amount of light passing through the sample. Usually works by converting light signal into electrical signal Photomultiplier tube Process: a) light hits photoemissive cathode and e- is emitted. b) an emitted e- is attracted to electrode #1 (dynode 1), which is 90V more positive. Causes several more e- to be emitted. c) these e- are attracted to dynode 2, which is 90V more positive than dynode 1, emitting more e-. d) process continues until e- are collected at anode after amplification at 9 dynodes. e) overall voltage between anode and cathode is 900V. f) one photon produces 106 – 107 electrons. g) current is amplified and measured

2) Types of Spectrophotometers Single-Beam Instrument: sample and blank are alternatively measured in same sample chamber.

2) Types of Spectrophotometers Double-Beam Instrument Continuously compares sample and blank Automatically corrects for changes in electronic signal or light intensity of source

Chemical Analysis 1) Calibration To measure the absorbance of a sample, it is necessary to measure Po and P ratio Po – the amount of light passing through the system with no sample present P – the intensity of light when the sample is present Po is measured with a blank cuvet Cuvet contains all components in the sample solution except the analyte of interest P is measured by placing the sample in the cuvet. To accurately measure an unknown concentration, obtain a calibration curve using a range of known concentrations for the analyte

Chemical Analysis 2) Limitations in Beer’s Law Results in non-linear calibration curve At high concentrations, solute molecules influence one another because of their proximity Molar absorptivity changes Affected by equilibrium, (HA and A- have difference in absorption) Analyte properties change in different solvents Errors in reproducible positioning of cuvet Also problems with dirt & fingerprints Instrument electrical noise Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)

3) Precautions in Quantitative Absorbance Measurements Choice of Wavelength Choose a wavelength at an absorption maximum Minimizes deviations from Beer’s law, which assumes ε is constant Pick peak in absorption spectrum where analyte is only compound absorbing light Or choose a wavelength where the analyte has the largest difference in its absorbance relative to other sample components Best choice compound (b) Best choice compound (a) Bad choice for either compound (a) or (b)

What Happens When a Molecule Absorbs Light? 1) Molecule Promoted to a More Energetic Excited State Absorption of UV-vis light results in an electron promoted to a higher energy molecular orbital s  s* transition in vacuum UV n  s* saturated compounds with non-bonding electrons n  p*, p  p* requires unsaturated functional groups (eq. double bonds) most commonly used, energy good range for UV/Vis

1) Molecule Promoted to a More Energetic Excited State Geometrical Structure of the Excited State will Differ from the Ground State Ground State Excited State Excitation of an electron to the pi antibonding orbital (p*) in formaldehyde produces repulsion instead of attraction between the carbon and oxygen atom

1) Molecule Promoted to a More Energetic Excited State Two Possible Transitions in Excited State Singlet state – electron spins opposed Triplet state – electron spins are parallel In general, triplet state has lower energy than singlet state Singlet to Triplet transition has a very low probability Singlet to Singlet Transition are more probable

2) Infrared and Microwave Radiation Not energetic enough to induce electronic transition Change vibrational and rotational motion of the molecule The entire molecule and each atom can move along the x, y, z-axis When correct wavelength is absorbed, Oscillations of the atom vibration is increased in amplitude Molecule rotates or moves (translation) faster Vibrational States of Formaldehyde Energy: Electronic >> Vibrational > Rotational

symmetric asymmetric In-plane scissoring Out-of-plane wagging Out-of-plane twisting In-plane rocking

3) Combined Electronic, Vibrational and Rotational Transitions Absorption of photon with sufficient energy to excite an electron will also cause vibrational and rotational transitions There are multiple vibrational and rotational energy levels associated with each electronic state Excited vibrational and rotational states are lower energy than electronic state Therefore, transition between electronic states can occur between different vibrational and rotational states Vibrational and rotational states associated with an electronic state

4) Relaxation Processes from Excited State There are multiple possible relaxation pathways Vibrational, Rotational relaxation occurs through collision with solvent or other molecules energy is converted to heat (radiationless transition) Electronic relaxation occurs through the release of a photon (light)

4) Relaxation Processes from Excited State Internal conversion – transition between singlet electronic states through overlapping vibrational states Intersystem crossing – transition between a singlet electronic state to a triplet electronic state by overlapping vibrational states

4) Relaxation Processes from Excited State Fluorescence – emitting a photon by relaxing from an excited singlet electronic states to a ground singlet state S1  So Phosphorescence – emitting a photon by relaxing from an excited triplet electronic states to a ground singlet state T1  So

4) Fluorescence and Phosphorescence Relative rates of relaxation depends on the molecule, the solvent, temperature, pressure, etc. Energy of Phosphorescence is less than the energy of fluorescence Phosphorescence occurs at a longer wavelengths than fluorescence Lifetime of Fluorescence (10-8 to 10-4 s) is very short compared to phosphorescence (10-4 to 102 s) Fluorescence and phosphorescence are relatively rare

1) Relation between Absorption and Emission Spectra Luminescence 1) Relation between Absorption and Emission Spectra Emission come at lower energy than absorbance Emission spectrum is roughly mirror image of absorption spectrum Absorption to an excited vibrational state will relax quickly to a ground vibrational state before the electronic relaxation

1) Relation between Absorption and Emission Spectra Also, differences in stability of excited and ground state structure contribute to energy difference

2) Excitation and Emission Spectra Excitation Spectra – measure fluorescence or phosphorescence at a fixed wavelength while varying the excitation wavelength. Emission Spectra – measure fluorescence or phosphorescence over a range of wavelengths using a fixed excitation wavelength.

5.) Fluorescence and Phosphorescence Intensity At low concentration, emission intensity is proportional to analyte concentration Related to Beer’s law At high concentrations, deviation from linearity occurs Emission decreases because absorption increases more rapidly Emission is quenched  absorption of excitation or emission energy by analyte molecules in solution where: k = constant Po = light intensity c = concentration of analyte (mol/L)