Chemical Measurement
Instrumental Analysis:
Absorption
Ab
ti S
Spectrometry
t
t
Chapter 18
Emission and Absorption
Light emitted as electron moves from higher E orbit (excited
state) to lower E orbit (ground state) and light absorbed as
electron moves from lower E orbit to higher E orbit.
Chapter 18
Absorption of light
Irradiance Energy per second per unit
Irradiance:
area, P
Transmittance Fraction of the original
Transmittance:
light energy that passes through the
sample, T
T=
P
P0
Absorbance The negative logarithm value A = − log(T ) = log⎛⎜ 0 ⎞⎟
Absorbance:
⎝P⎠
of the transmittance
P
1
Chapter 18
Absorption of light
A = εbc
Concentration
of the analyte
Molar absorptivity
Path length
or,
extinction coefficient
Absorbance
Beer-Lambert Law and Its Limitation
Absorbance
Absorption is proportional to the concentration
It works really well for
a) monochromatic radiation
b) dilute solution ( ≤ 0.01 M)
Concentration
Chapter 18
Absorption of light
Absorbance (arbitrarry unit)
A green leaf absorbs all the colored light rays, except for
those corresponding to green
Chapter 18
Absorption of light
Highly conjugated molecules are colored
LYCOPENE
β-CAROTENE
2
Chapter 18
Absorption of light
What Happens when a Molecule Absorbs Light?
Excited
state
∆E=hν
Ground
state
How are the electrons arranged at the ground and
excited states?
Chapter 18
Absorption of light
Combined Electronic, Vibrational and Rotational
Transitions
Chapter 18
Absorption and Emission
3
Chapter 18
Absorption and Emission
Electron Spins and Exited states
Franck–Condon Principle
Franck–Condon principle is an approximation that states that an
electronic transition is most likely to occur without changes in the positions
of the nuclei in the molecular entity and its environment. The resulting
state is called a Franck–Condon state, and the transition involved, a vertical
transition.
Chapter 20
Spectrophotometers
Basically composed of two parts
A device for tapping light to be analyzed from
an optical system (Analytes) under study.
1.
‰
2
2.
Composed of an optical part (a lamp, a slit and a grating)
A spectrophotometric transducer (ST)
Composed of an opto-electrical part (an array
photodetector and an analog-to-digital converter)
‰ ST is converting input light into numerical data
‰
Diffraction Grating
A diffraction grating is the tool of choice for
separating the colors in incident light.
4
Chapter 20
Spectrophotometers
Chapter 20
The Components of a Spec-20D
Light source
- white light of constant intensity
slits
filter
occluder
Grating
Phototube
detects light &
measures intensity
slits
Sample
When blank is the sample
Po is determined
otherwise P is measured
Separates white light
into various colors
Rotating the grating
changes the wavelength going
through the sample
Chapter 20
Spectrophotometers
5
Chapter 20
Spectrophotometric Calibration
Calibration
‰ A study of the detector response as a function of analyte
concentration
Beer-Lambert Law (sometime referred as Beer’s Law)
A = ε bc
Absorbance
Molar absorptivity
or,
extinction coefficient
Concentration of the analyte
Path length
Spectrophotometric Calibration
concentration 2
with sample
concentration 1
P < Po
blank where Po = P
light
source
detector
Po
P
With increasing
concentration of the
analyte, less light
Cell with
was transmitted
Pathlength, b,
(more light
containing solution absorbed).
⎛P ⎞
A = − log(T ) = log⎜ 0 ⎟
⎝P⎠
b
Chapter 20
Spectrophotometric Calibration
Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm (detector
range) to produce an absorption spectrum (A vs. λ)
Crystal Violet Absorption Spectrum
1.4
Absorbance
1.2
1
0.8
0.6
λmax
0.4
0.2
0
200
250
300
350
400
450
500
wavelength, nm
550
600
650
700
750
λmax- wavelength where maximum absorbance occurs
6
Chapter 18
Various Energy States
Combined Electronic, Vibrational and Rotational
Transitions
Chapter 18
Absorption Between Various Levels
Combined Electronic, Vibrational and Rotational
Transitions
Chapter 20
Spectrophotometric Calibration
0.6
Blank solution: All reagents and
solvent; the analyte omitted
0.5
Absorbance
Standard solution: Analyte with
known concentration
0.4
0.3
0.2
0.1
0
0
10
20
30
Amount of the analyte
Amount of
analyte
0
5.0
10.0
15.0
20.0
25.0
Absorbance of
independent samples
0.099
0.185
0.282
0.345
0.425
0.483
0.099
0.187
0.272
0.347
0.425
0.488
0.100
0.188
0.272
0.352
0.430
0.496
Average
0.099
0.187
0.275
0.348
0.427
0.489
7
Chapter 20
Application of Spectrophotometry
Beer’s Law in Chemical Analysis : Serum
Iron Determination
Absorption Maxima, λmax = 562 nm
Iron is transported in bloodstream
by a a protein called transferrin
λmax
Visible spectrum of the purple complex [Fe(II) (ferrozine)3]4-
Chapter 18
Application of Spectrophotometry
Beer’s Law in Chemical Analysis : Serum Iron
Determination
Step 1
Reduction with
thioglycolic acid
Fe3+
Fe2+
Step 2
trichloroacetic acid
protein (aq)
protein (s)
Step 3
Fe2+ + 3 ferrozine–
[Fe(II) (ferrozine)3]4-
Chapter 20
Application of Spectrophotometry
Analysis of hydrogen peroxide with potassium
permanganate in an acidic solution
absorbancce
5H2O2 + 2MnO4- + 6H+ Æ 5O2 (g) +
purple
2Mn+2 + 8H2O
Equivalence point
MnO4-
reacting,
color disappears
excess MnO4accumulates
Volume of titrant (mL KMnO4)
8
Chapter 19
Application of Spectrophotometry
Monitoring a reaction: Isobestic point
Methyl red
An isobestic point gives evidence that there are only two principal
chromophore species are present
Chapter 19
Advanced Studies
FRET: The fluorescence resonance energy
transfer depends on the donor-acceptor distance
Chapter 19
Advanced Studies
Monitoring small molecule binding to macromolecules
+
Ruthenium complex is strongly luminescent only when bound to DNA.
So the binding can be monitored by observing change in the
luminescence
9