1. Non-Instrumental Methods
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

2. Fundamentals of Spectrophotometry
Introduction
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]

3. 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 (l): distance (nm, cm, m) from peak to peak
Frequency (n): 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

4. Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves
Parameters used to describe light
Energy (E): the energy of one particle of light (photon) is proportional to its frequency
where: E = photon energy (Joules)
n = frequency (sec-1)
h = Planck’s constant (6.626x10-34J-s)
As frequency (n) increases, energy (E) of light increases

5. Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves
Relationship between Frequency and Wavelength
Relationship between Energy and Wavelength
where: c = speed of light (3.0x108 m/s in vacuum))
n = frequency (sec-1)
l = wavelength (m)
where: = (1/l) = wavenumber
As frequency (l) decreases, energy (E) of light increases

6. Fundamentals of Spectrophotometry
Properties of Light
2.) Types of Light – The Electromagnetic Spectrum
Note again, energy (E) of light increase as frequency (n) increases or wavelength (l) decreases

7. Fundamentals of Spectrophotometry
Properties of Light
2.) Types of Light – The Electromagnetic Spectrum

8. 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

9. Fundamentals of Spectrophotometry
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 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:
DE = E1 - Eo

10. Fundamentals of Spectrophotometry
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 - e)
Increasing [Fe2+]
Absorbance is directly proportional to concentration of Fe+2

11. Fundamentals of Spectrophotometry
Absorption of Light
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%

12. Fundamentals of Spectrophotometry
Absorption of Light
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)
A has a range of 0 to infinity

13. Fundamentals of Spectrophotometry
Absorption of Light
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)
e = 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)

14. Fundamentals of Spectrophotometry
Absorption of Light
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 e) vs. wavelength for a compound
The greater the absorbance of a compound at a given wavelength (high e), the easier it will be to detect at low concentrations

15. Fundamentals of Spectrophotometry
Absorption of Light
4.) Absorption Spectrum
By choosing different wavelengths of light (lA vs. lB) different compounds can be measured lA lB

16. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
An instrument used to make absorbance or transmittance measurements is known as a spectrophotometer

17. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
Light Source: provides the light to be passed through the sample
Tungsten Lamp: visible light ( nm)
Deuterium Lamp: ultraviolet Light ( nm)
- based on black body radiation:
heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament
Low pressure (vacuum)
Tungsten 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

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

19. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
Wavelength Selector (monochromator): used to select a given wavelength of light from the light source
Reflection or Diffraction Grating:

20. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
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

21. Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
Light Detector: measures the amount of light passing through the sample.
Usually works by converting light signal into electrical signal
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 then 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
Photomultiplier tube

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

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

24. 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

25. Fundamentals of Spectrophotometry
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
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)

26. Fundamentals of Spectrophotometry
Chemical Analysis
3.) Precautions in Quantitative Absorbance Measurements
Choice of Wavelength
Choose a wavelength at an absorption maximum
Minimizes deviations from Beer’s law, which assumes e 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 (b)
Best choice compound (a)

27. Fundamentals of Spectrophotometry
Chemical Analysis
4.) Example:
A 3.96x10-4 M solution of compound A exhibited an absorbance of at 238 nm in a cm cuvet. A blank had an absorbance of The absorbance of an unknown solution of compound A was
Find the concentration of A in the unknown.

28. 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
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

29. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
1.) Molecule Promoted to a More Energetic Excited State
Geometrical Structure of the Excited State will Differ from the Ground State
Ground State
Excitation of an electron to the pi antibonding orbital (p*) in formaldehyde produces repulsion instead of attraction between the carbon and oxygen atom
Excited State

30. 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

31. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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

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

33. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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

34. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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)

35. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs 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

36. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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

37. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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

38. Color Change Due to Fluorescence at Higher Wavelength
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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

39. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
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

40. Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
5.) Fluorescence and Phosphorescence
Also, differences in stability of excited and ground state structure contribute to energy difference

41. Fundamentals of Spectrophotometry
Chemical Analysis
1.) 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 varying excitation wavelength.

42. Fundamentals of Spectrophotometry
Chemical Analysis
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
where: k = constant
Po = light intensity
c = concentration of analyte (mol/L)

43. Fundamentals of Spectrophotometry
Chemical Analysis
3.) Example
In formaldehyde, the transition n p*(T1) occurs at 397 nm, and the np*(S1) transition comes at 355 nm. What is the difference in energy (kJ/mol) between the S1 and T1 states?