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Fundamentals of Spectrophotometry
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
Non-Instrumental Methods
(spectrophotometry)
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]
Fundamentals of Spectrophotometry
Properties of Light
- 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
Fundamentals of Spectrophotometry
Properties of Light
- Energy (E): the energy of one particle of light (photon) is proportional to its
frequency
E = hn
where: E = photon energy (Joules)
n = frequency (sec-1)
h = Planck’s constant (6.626x10-34J-s)
ln = c n = c / l
where: c = speed of light (3.0x108 m/s in vacuum))
n = frequency (sec-1)
l = wavelength (m)
hc ~
E= = hcn
l
where: n~ = (1/l) = wavenumber
➢ The Color of Light Absorbed and Observed passing through the Compound are
Complimentary
Fundamentals of Spectrophotometry
Absorption of Light
➢ 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
Fundamentals of Spectrophotometry
Absorption of Light
Increasing [Fe2+]
P
T=
Po
P
Percent transmittance %T = 100
Po
P
A = - log = - log(T ) = - log(%T / 100 )
Po
A = ebc
where: A = absorbance (no units)
e = molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)
•
"- e - e " I e . - e e e e I .
unsaturated etones have high Emax values from lo,
000 2 0 , 000.
lA lB
Fundamentals of Spectrophotometry
Spectrophotometer
- Filter:
Fundamentals of Spectrophotometry
Spectrophotometer
2. Quartz – ultraviolet
Process:
Photomultiplier tube
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
Fundamentals of Spectrophotometry
Spectrophotometer
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
4.) Example:
‚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
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
Ground State
I = kPo c
where: k = constant
Po = light intensity
c = concentration of analyte (mol/L)
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?