Fundamentals of Spectrophotometry

Fundamentals of Spectrophotometry 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

Fundamentals of Spectrophotometry Introduction

3.) Illustration Measurement of Ozone (O3) Above South Pole

- O3 provides protection from ultraviolet radiation- Seasonal depletion due to chlorofluorocarbons

Spectra analysis

of [O3]

Chain Reaction Depletion of O3

O3 cycle

Fundamentals of Spectrophotometry 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 (): distance (nm, cm, m) from peak to peak

- Frequency (): number of complete oscillations that the waves makes each second Hertz (Hz): unit of frequency, second-1 (s-1) 1 megahertz (MHz) = 106s-1 = 106Hz


1.) Particles and Waves Parameters used to describe light

- Energy (E): the energy of one particle of light (photon) is proportional to its frequency

hE where: E = photon energy (Joules)

= frequency (sec-1)h = Planck’s constant (6.626x10-34J-s)

As frequency () increases, energy (E) of light increases


1.) Particles and Waves Relationship between Frequency and Wavelength

Relationship between Energy and Wavelength

/cc where: c = speed of light (3.0x108 m/s in vacuum))

= frequency (sec-1) = wavelength (m)

~hchc

E

where: = (1/) = wavenumber~

As wavelength () decreases, energy (E) of light increases


2.) Types of Light – The Electromagnetic Spectrum Note again, energy (E) of light increase as frequency () increases or

wavelength () decreases


2.) Types of Light – The Electromagnetic Spectrum

Fundamentals of Spectrophotometry 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 Complimentary


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 electron 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:


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 - )

Increasing [Fe2+]

Absorbance is directly proportional to concentration of Fe+2


3.) Beer’s Law The relative amount of light making it through the sample (P/Po) is known as

the transmittance (T)

oPP

T

oPP

T% 100Percent transmittance

T has a range of 0 to 1, %T has a range of 0 to 100%


3.) Beer’s Law 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)

)()( 100/T%logTlogPP

logAo

A has a range of 0 to infinity


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

bcA where: A = absorbance (no units)

= 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)


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 absorbance (or ) vs. wavelength for a compound

The greater the absorbance of a compound at a given wavelength (high ), the easier it will be to detect at low concentrations


4.) Absorption Spectrum By choosing different wavelengths of light (A vs. B) different compounds

can be measured

AB

Fundamentals of Spectrophotometry 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


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- Affect on equilibrium, (HA and A- have

difference 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 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

Bad choice for either compound (a) or (b)Best choice compound (a)Best choice compound (b)


4.) Example:

A 3.96x10-4 M solution of compound A exhibited an absorbance of 0.624 at 238 nm in a 1.000 cm cuvet. A blank had an absorbance of 0.029. The absorbance of an unknown solution of compound A was 0.375.

Find the concentration of A in the unknown.

Fundamentals of Spectrophotometry 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

* transition in vacuum UV

n * saturated compounds with non-bonding electrons

n *, * requires unsaturated functional groups (eq. double bonds) most commonly used, energy good range for UV/Vis

Fundamentals of Spectrophotometry What Happens When a Molecule Absorbs Light?

1.) Molecule Promoted to a More Energetic Excited State Two Possible Transitions in Excited State

- Single 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

10-5 to 10-8 s 10-4 to 10 sLife-times:


2.) Infrared and Microwave Radiation Not energetic enough to induce electronic transition

Change vibrational, translational 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 rocking

In-plane scissoring

Out-of-plane waggingOut-of-plane twisting


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 stateS1 So

Phosphorescence – emitting a photon by relaxing from an excited triplet electronic states to a ground singlet state

T1 So


5.) 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


5.) Fluorescence and Phosphorescence Fluorescence and phosphorescence come at lower energy than absorbance Emission spectrum is roughly mirror image of absorption spectrum

Color Change Due to Fluorescence at Higher Wavelength


5.) Fluorescence and Phosphorescence Emission spectrum are of lower energy or

higher wavelength because of the efficiency of vibrational relaxation- Absorption to an excited vibrational

state will relax quickly to a ground vibrational state before the electronic relaxation


1.) Excitation and Emission Spectra

Excitation Spectra – measure fluorescence or phosphorescence at a fixed wavelength whilevarying the excitation wavelength.

Emission Spectra – measure fluorescence or phosphorescence over a range of wavelengths using a fixed varying excitation wavelength.


2.) 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

ckPI owhere: k = constant

Po = light intensityc = concentration of analyte (mol/L)


3.) Example

In formaldehyde, the transition n *(T1) occurs at 397 nm, and the n*(S1) transition comes at 355 nm. What is the difference in energy (kJ/mol) between the S1 and T1 states?

Fundamentals of Spectrophotometry

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