Molecular Transitions
Absorption of electromagnetic (EM) radiaton: Transition to a higher energy level
Emission of EM radiation: Transition to a lower energy level
vibrational changes
Infrared (IR) radiation
rotational changes
Microwave radiation
Sample Problems
1. When O2 absorbs UV radiation in the wavelength 147 nm, by how many kJ per mole is the Energy increased?
2. How much is the energy of CO2 increased when it absorbs IR radiation with a wavenumber 2300 cm-?
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Petty 2004
Continuous Spectrum
Possible for molecules [solid/liquid] Strong interaction between the force fields of individual molecules Absorption and emission take place throughout a continuous spectrum of wavelengths
Spectrophotometer
The monochromatic light, with irradiance Po, strikes the sample of length b. Some of the Po may be absorbed by the sample. Thus, P Po
T- Transmittance
T=
P P0
% Transmittance = 100 x
= Log Log
P0 P
Beer-Lambert law
For molecules that absorb ultraviolet or visible light, the absorbance of a solution increases as attenuation of the beam increases. Beer's Law states that
A = abc
where a is a constant of proportionality, called the absorptivity.
Sample Problem
1. Find the absorbance and transmittance of a 0.0002040 M solution with a molar absorptivity of 313 M-cm- in a cell with 2.0 cm path length
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Spectroscopic Instruments
1. 2. 3. Spectroscope an instrument that is used to measure the amount of radiation absorbed or emitted by matter Spectrograph consists of a device that disperses the wavelength so that multichannel detection is possible Spectrometer a spectroscopic instrument that uses a monochromator or polychromator with a transducer to convert radiant intensities into electrical signals
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Spectroscopic Instruments
4. Spectrophotometer a spectrometer that allows measurement of the ratio of the radiant powers of two beams to give the absorbance. The wavelength can be varied continuously, making it possible to obtain an absorption spectrum Photometer uses a filter for wavelength selection in conjunction with a suitable radiation transducer. It is simpler and less expensive than spectrophotometers. It is also often used as detectors for HPLC, FIA, Electrophoresis and others
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5.
Single-Beam Spectrophotometer
Single-Beam Spectrophotometer
Double-Beam Spectrophotometer
Baseline Spectrum
2.0
3. Calculate the concentration of your sample using Beer Lambert Equation: A = abc
Wavelength (nm)
A C
1.0
x
0.5
4 2 3 Concentration (mg/ml)
0.8
0.4
Avoid very high or low absorbance when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line
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Light Sources
UV Spectrophotometer 1. Hydrogen Gas Lamp
2.
Mercury Lamp
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BEERS LAW
Applies to monochromatic radiation
Works well for dilute solutions (0.01 M)
Absorbance
1. Ideal range: 0.4 0.9
Sample Problems
1. Pure hexane has negligible UV absorbance above l = 200 nm. A solution prepared by dissolving 25.8 mg benzene (78.11) in hexane and diluting to 250.0 mL had an absorption peak at 256 nm and an absorbance of 0.266 in a 1.000-cm cell. Find the molar absorptivity of benzene. A sample of hexane contaminated with benzene had an absorbance of 0.070 at 256 nm in a cuvet with a 5.000-cm path length. Find the concentration of benzene in mg/L.
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2.
Answers
1. 201 M-cm-
2. 5.4 mg/L
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Chromophoric Structure
Group
Carbonyl
Structure
>C=O
nm
280
Azo
Nitro Thioketone Nitrite Conjugated Diene Conjugated Triene Conjugated Tetraene Benzene
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270 330 230 233 268 315 261
UV Spectrometer Application
Protein Amino Acids (aromatic) Pantothenic Acid Glucose Determination Enzyme Activity (Hexokinase)
Assignment
1. Calculate the Molar Extinction Coefficient at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 cm cell. Solution A C (M) 2.23 x 10-5 Po 100 P 27
1.90 x 10-5
100
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2. The molar extinction coefficient of compound riboflavin is 3 x 103 L/cm mole. If the absorbance reading at 350 nm is 0.90 using a cell of 1.00 cm, what is the concentration of riboflavin in the sample?
3. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 cm quartz cell was 0.40. What is the molar extinction coefficient of compound Y?
4. Calculate the molar extinction coefficient at 351 nm for aquocobalamin in 0.1 M phosphate buffer pH =7.0 from the following data which were obtained in 1 cm cell. Solution A C (M) 2.0 x 10-5 Po 100 P 30
5. A substance absorbs at 600 nm and 4000 nm. What type of energy transition most likely accounts for each of these absorption processes? 6. Complete the following table.
[X](M)
2.5 x 10-3 4.0 x 10-5 2.0 x 10-4
L(cm)
0.5 0.2
30
2000 2500
50
5000 150
7. The molar absorptivity of a pigment (molecular weight 300) is 30,000 at 550 nm. What is the absorptivity in L/g-cm? 8. The iron complex of o-phenanthroline (Molecular weight 236) has molar absorptivity of 10,000 at 525 nm. If the absorbance of 0.01 is the lowest detectable signal, what concentration in part per million can be detected in a 1-cm cell?