Spectroscopy (continued)
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Last time we discussed what
spectroscopy was, and how we could
use the interaction of light with atoms
and molecules to measure their
concentrations.
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Today we will expand on this and look at
specific types of spectroscopy.
Beer’s Law
We have already shown that Absorbance
is proportional to concentration:
1.0
0.8
Absorbance
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0.4
0.2
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0.4
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Distance through solution
(Proportional to concentration)
1.0
Beer’s Law
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We can write:
A  bc
– this is the formal statement of Beer’s law
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where A = absorbance (no units)
ε = molar absorptivity (M-1cm-1, or L µg-1 cm-1)
b = the pathlength (cm)
c = concentration (M, µg L-1)
Beer’s Law
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Molar absorptivity (ε) is constant for any one
substance at any one wavelength.
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It varies with substance and wavelength.
Gives an indication of how effective a substance is
at absorbing radiation at the specified wavelength.
The pathlength (b) is the distance that the
source radiation passes through the sample
(the length of the flame, the width of the
cuvette, etc.).
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This should be constant for any experiment or
spectrometer.
Molar absorptivity (ε)
Beer’s Law
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Since ε and b are constant, then we can
express Beer’s Law as:
y  mx  b
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where y = absorbance (A)
m = (εb)
x = concentration (c)
and b is the y-intecept (which should be
close to zero if we analyzed a blank).
Beer’s Law
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Limitations to applying Beer’s law
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At high concentrations absorbance is no longer
proportional to concentration.
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Beer’s Law only applies for monochromatic
radiation since ε changes with wavelength.
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–
Cannot produce a linear calibration curve.
Important to do quantitative analysis near the peak of molar
absorptivity where molar absorptivity does not change
much with wavelength.
Beware of shifts in chemical equilibrium for the
analyte of interest.
•
Changing concentrations or pH in solution may shift
equilibrium, thus shifting concentrations of analytes.
Absorbance Spectroscopy
Light Source
Wavelength
Selector
(sometimes after
the sample)
Sample
(holder)
Light
Detector
Data
Readout
0.365
Common types are:
• UV-VIS (ultraviolet – visible)
• Flame AA (atomic absorption)
• FTIR (Fourier transform infra-red)
UV-VIS Spectroscopy
From: http://icn2.umeche.maine.edu/genchemlabs/uv.html
UV-VIS Spectroscopy
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Can be used to identify compounds – but
many compounds’ spectra look alike.
Uses wavelengths from ~180 to 800nm.
Usually look at spectra to determine the best
wavelength for quantification.
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Then use the wavelength with the maximum
absorbance (molar absorptivity) for quantitative
analysis.
We will use UV-VIS spectroscopy to analyze
Co and Cr later in the semester.
UV-VIS is also the most common detector for
HPLC.
Flame AA Spectroscopy
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Excellent for the quantitative analysis of
elements.
Primarily uses ultraviolet wavelengths for
analysis.
Since we use a monochromatic light source
(hollow cathode lamp) there is virtually no
interferences from other elements.
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Also cannot look at spectra.
Flame potentially creates a high background
(lowers sensitivity).
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Using a graphite furnace can reduce this effect.
FTIR Spectroscopy
From: Silverstein, Bassler, and Morril, Spectrometric Identification of Organic Compounds,
John Wiley and Sons, 1991.
FTIR Spectroscopy
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Usually used for qualitative determination of
the identity of a compound.
Uses infra-red wavelengths (2000 – 25000nm)
Should have a purified sample for analysis.
Quantitative analysis is made difficult do to:
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detector sensitivity,
Thermal noise,
interferences from other compounds.
Emission Spectroscopy
Sample
(holder)
Light Source
Wavelength
Selector
Wavelength
Selector
Data
Readout
Light
Detector
0.365
Emission Spectroscopy
From: Willard, Merritt, Dean, and Settle, Instrumental Methods of Analysis 7th Edition,
Wadsworth Publishing, 1988.
A Closer Look at
Absorbance and Emission
From: Willard, Merritt, Dean, and Settle, Instrumental Methods of Analysis 7th Edition,
Wadsworth Publishing, 1988.
A Closer Look at
Absorbance and Emission
Absorption always occurs
at higher energies than
emission.
•Due to vibrational
transitions to the
ground vibrational
state within each
electronic state
A Closer Look at
Absorbance and Emission
• Fluorescence decay generally occurs 10-8
– 10-4 s after absorption.
• Phosphorescence decay generally occurs
10-4 – 102 seconds after absorption.
– Need an efficient chromaphore
• Usually large conjugated organic molecules.
• Fluorescence in molecules is somewhat
rare, and phosphorescence is very rare.
Emission Spectroscopy
• Emission occurs on a zero background.
– Since emission wavelength is always longer
than the excitation wavelength.
– Detection limits can be much lower than for
absorption.
• However, instrumentation is more
complicated and expensive.
• Limited to analyzing molecules that
fluoresce or phosphoresce.
– Or molecules that can be derivatized.
Emission Spectroscopy
• The emission intensity (I) is proportional to
concentration (c):
I  kPo c
• Where Po is the excitation irradiance, and
k is a proportionality constant (similar to ε).
– Therefore, we can still make a linear
calibration curve y = mx + b
– where y = emission intensity
– m = kPo
– and c = concentration of analyte
Reading for Next Time
• pgs. 501 – 511
Problems to Work on
• Chap 18 (16)