1. SPECTROPHOTOMETRY
Applied Chemistry

2. 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

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
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

8. 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

9. 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

10. 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%

11. 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

12. 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)

13. 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

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

15. 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

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

17. 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

18. 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

19. Fundamentals of Spectrophotometry
Chemical Analysis
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

20. Single-beam Spectrophotometer
Double-beam Spectrophotometer