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Spectroscopy (continued)
• Last time we discussed what spectroscopy was, and how we could use the interaction of light with atoms and molecules to measure their concentrations.
• 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:
0.0
0.2
0.4
0.6
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1.0
0.0 0.2 0.4 0.6 0.8 1.0Distance through solution
(Proportional to concentration)
Ab
so
rba
nc
e
Beer’s Law
• We can write:
– this is the formal statement of Beer’s law• 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)
bcA
Beer’s Law• Molar absorptivity (ε) is constant for any one
substance at any one wavelength.– 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.).
– This should be constant for any experiment or spectrometer.
Molar absorptivity (ε)
Beer’s Law
• Since ε and b are constant, then we can express Beer’s Law as:
– 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).
bmxy
Beer’s Law• Limitations to applying Beer’s law
– At high concentrations absorbance is no longer proportional to concentration.
• Cannot produce a linear calibration curve.
– Beer’s Law only applies for monochromatic radiation since ε changes with wavelength.
• 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
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Light Source WavelengthSelector
(sometimes afterthe sample)
Sample(holder)
LightDetector
DataReadout
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• 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.– 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
• 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.
– Also cannot look at spectra.
• Flame potentially creates a high background (lowers sensitivity).
– 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
• 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:
– detector sensitivity,– Thermal noise,– interferences from other compounds.
Emission Spectroscopy
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Light Source WavelengthSelector
Sample(holder)
LightDetector
DataReadout
WavelengthSelector
Emission Spectroscopy
From: Willard, Merritt, Dean, and Settle, Instrumental Methods of Analysis 7 th Edition, Wadsworth Publishing, 1988.
A Closer Look atAbsorbance and Emission
From: Willard, Merritt, Dean, and Settle, Instrumental Methods of Analysis 7 th Edition, Wadsworth Publishing, 1988.
A Closer Look atAbsorbance 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 atAbsorbance 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):
• 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
ckPI o
Reading for Next Time
• pgs. 501 – 511
Problems to Work on
• Chap 18 (16)