ch 103: spectrophotometry the electromagnetic spectrum

• Electromagnetic radiation is a self-propagating wave with Electromagnetic radiation is a self-propagating wave with an electric component and a magnetic component. These an electric component and a magnetic component. These 2 components oscillate at right angles to each other and 2 components oscillate at right angles to each other and are in phase with each other.are in phase with each other.

• Electromagnetic radiation travels at the speed of light. In Electromagnetic radiation travels at the speed of light. In fact, the “light” in this room is electromagnetic radiation.fact, the “light” in this room is electromagnetic radiation.

CH 103: SPECTROPHOTOMETRYCH 103: SPECTROPHOTOMETRY

THE ELECTROMAGNETIC SPECTRUMTHE ELECTROMAGNETIC SPECTRUM

decreasedecrease

increaseincrease

• The wavelength (λ, the length of 1 cycle in meters) times the The wavelength (λ, the length of 1 cycle in meters) times the frequency (ν, the number of cycles per second) equals the frequency (ν, the number of cycles per second) equals the speed of light (c, a constant that equals 3.0 x 10speed of light (c, a constant that equals 3.0 x 1088 meters/second). That is,meters/second). That is,

c = λν = 3.0 x 10c = λν = 3.0 x 1088 meters/second meters/second

• If λ increases, then ν must If λ increases, then ν must so that c remains so that c remains constant.constant.

• If λ decreases, then ν must If λ decreases, then ν must so that c remains so that c remains constant.constant.


• Electromagnetic radiation is also a stream of energy Electromagnetic radiation is also a stream of energy packets called photons.packets called photons.

• The energy of a single photon (E, in joules) equals Planck’s The energy of a single photon (E, in joules) equals Planck’s constant (h, 6.626 x 10constant (h, 6.626 x 10-34-34 joule second) times the frequency joule second) times the frequency (ν, the number of cycles per second). That is,(ν, the number of cycles per second). That is,

E = hν = hc/λE = hν = hc/λ

• If the frequency (ν) increases, the energy (E)If the frequency (ν) increases, the energy (E) ..

• If the wavelength (λ) decreases, the energy (E)If the wavelength (λ) decreases, the energy (E) . .


increasesincreases

increasesincreases

• The The ultraviolet (UV)ultraviolet (UV) region of the electromagnetic spectrum region of the electromagnetic spectrum includes all wavelengths from 10 nanometers (nm) to 380 includes all wavelengths from 10 nanometers (nm) to 380 nm. The nm. The vacuum-ultravioletvacuum-ultraviolet region goes from 10 nm to 200 region goes from 10 nm to 200 nm because air absorbs strongly at these wavelengths so nm because air absorbs strongly at these wavelengths so instruments must be operated under a vacuum in this instruments must be operated under a vacuum in this region. The region. The near-ultravioletnear-ultraviolet region goes from 200 nm to 380 region goes from 200 nm to 380 nm.nm.

• The The visible (Vis)visible (Vis) region goes from 380 nm to 780 nm and region goes from 380 nm to 780 nm and can be seen by the human eye.can be seen by the human eye.

• The The infrared (IR)infrared (IR) region goes from 0.78 micrometers (μm) or region goes from 0.78 micrometers (μm) or 780 nm to 300 μm. However, the 780 nm to 300 μm. However, the near-infrarednear-infrared (0.8 μm to 2.5 (0.8 μm to 2.5 μm) and the μm) and the NaCl-infraredNaCl-infrared regions (2.5 μm to 16 μm) are the regions (2.5 μm to 16 μm) are the most commonly used by analytical chemists.most commonly used by analytical chemists.


• Humans see color when an object transmits or reflects visible light.Humans see color when an object transmits or reflects visible light.• More specifically, an object may absorb specific wavelengths of More specifically, an object may absorb specific wavelengths of

electromagnetic radiation. The unabsorbed wavelengths from the electromagnetic radiation. The unabsorbed wavelengths from the visible region are transmitted and seen as color.visible region are transmitted and seen as color.

• For example, leaves are green because the pigment chlorophyll For example, leaves are green because the pigment chlorophyll absorbs violet, blue, and red light.absorbs violet, blue, and red light.

• Why is my car blue?Why is my car blue?• It’s blue because it absorbs yellow.It’s blue because it absorbs yellow.

THE ABSORPTION OF ELECTROMAGNETIC RADIATION BY MOLECULESTHE ABSORPTION OF ELECTROMAGNETIC RADIATION BY MOLECULES

• There are 3 ways that a molecule can absorb electromagnetic radiation. There are 3 ways that a molecule can absorb electromagnetic radiation. All 3 ways raise the molecule to a higher internal energy level. All these All 3 ways raise the molecule to a higher internal energy level. All these changes in energy are changes in energy are quantizedquantized; that is, they occur at discrete levels.; that is, they occur at discrete levels.

• Rotational Transitions:Rotational Transitions: The molecule rotates around various axes. The molecule rotates around various axes. Rotational transitions require the least amount of energy. Purely Rotational transitions require the least amount of energy. Purely rotational transitions can occur in the far-infrared and microwave rotational transitions can occur in the far-infrared and microwave regions.regions.

• Vibrational Transitions:Vibrational Transitions: Atoms or groups of atoms within a molecule Atoms or groups of atoms within a molecule vibrate relative to each other. Vibrational transitions require an vibrate relative to each other. Vibrational transitions require an intermediate amount of energy and typically begin to occur in the mid-intermediate amount of energy and typically begin to occur in the mid-infrared and far-infrared regions. Therefore, as energy is increased (or infrared and far-infrared regions. Therefore, as energy is increased (or wavelength is decreased) vibrational transitions occur in addition to wavelength is decreased) vibrational transitions occur in addition to rotational transitions.rotational transitions.

• Electronic Transitions:Electronic Transitions: An electron within a molecule is typically An electron within a molecule is typically promoted from its ground state to an excited state. Electronic promoted from its ground state to an excited state. Electronic transitions require the most amount of energy and typically begin to transitions require the most amount of energy and typically begin to occur in the visible and ultraviolet regions. Therefore, as energy is occur in the visible and ultraviolet regions. Therefore, as energy is increased (or wavelength is decreased) electronic transitions occur in increased (or wavelength is decreased) electronic transitions occur in addition to vibrational and rotational transitions.addition to vibrational and rotational transitions.


SCHEMATIC OF A SPECTROPHOTOMETERSCHEMATIC OF A SPECTROPHOTOMETER

• The most common The most common light sourcelight source for the visible region for the visible region spectrophotometry is a tungsten filament incandescent lamp. A spectrophotometry is a tungsten filament incandescent lamp. A tungsten lamp emits useful light from approximately 325 nm to 3,000 tungsten lamp emits useful light from approximately 325 nm to 3,000 nm.nm.

• A A monochromatormonochromator uses a prism or a diffraction grating to separate uses a prism or a diffraction grating to separate polychromatic (many wavelengths) light into monochromatic (single polychromatic (many wavelengths) light into monochromatic (single wavelength) light.wavelength) light.

• A A cellcell or or cuvettecuvette is used to hold the sample during analysis. is used to hold the sample during analysis.• The The detectordetector uses a phototube or a photomultiplier tube to convert light uses a phototube or a photomultiplier tube to convert light

into an electrical signal that is sent to a recorder or computer.into an electrical signal that is sent to a recorder or computer.

THE SPECTRONIC 20D SPECTROPHOTOMETERTHE SPECTRONIC 20D SPECTROPHOTOMETER

The controls.The controls. Loading a sample.Loading a sample.THE HACH DR2010 SPECTROPHOTOMETERTHE HACH DR2010 SPECTROPHOTOMETER

A sample in a cell or cuvette during spectrophotometric analysis.A sample in a cell or cuvette during spectrophotometric analysis.

• PPoo = the power of monochromatic = the power of monochromatic light entering the sample.light entering the sample.• PP = the power of monochromatic = the power of monochromatic light leaving the sample.light leaving the sample.• aa = the absorptivity constant, which depends on the wavelength and the = the absorptivity constant, which depends on the wavelength and the

nature of the absorbing compound.nature of the absorbing compound.• bb = the path length through the absorbing compound. = the path length through the absorbing compound.• cc = the concentration of absorbing compound in the cuvette. = the concentration of absorbing compound in the cuvette.

TRANSMITTANCE AND PERCENT TRANSMITTANCETRANSMITTANCE AND PERCENT TRANSMITTANCE

• The Beer-Bouguer-Lambert law, more commonly called The Beer-Bouguer-Lambert law, more commonly called Beer’s lawBeer’s law, is , is

A = A = abcabc

ABSORBANCEABSORBANCE

• This is the absorption spectrum for Co(HThis is the absorption spectrum for Co(H22O)O)663+3+. The wavelength at the . The wavelength at the

absorbance maximum is called λabsorbance maximum is called λmaxmax. What is the λ. What is the λmaxmax for Co(H for Co(H22O)O)663+3+??

• 510 nm.510 nm.• Why does λWhy does λmaxmax give the most sensitive measurement? give the most sensitive measurement?• It gives the largest response per mole of analyte.It gives the largest response per mole of analyte.

SELECTING λSELECTING λmaxmax FOR SOLUTIONS WITH 1 ABSORBING FOR SOLUTIONS WITH 1 ABSORBING

COMPOUNDCOMPOUND

• Where is λWhere is λmaxmax for substance x? Where is λ for substance x? Where is λmaxmax for substance y? for substance y?• The total absorbance (AThe total absorbance (Atotaltotal) at a given wavelength equals the sum of the ) at a given wavelength equals the sum of the

absorbances for all compounds at this wavelength. That is, absorbances for all compounds at this wavelength. That is, at λ1: Aat λ1: Atotaltotal = A = Axλ1xλ1 + A + Ayλ1yλ1 = a = axλ1xλ1bcbcxx + a + ayλ1yλ1bcbcyy

at λ2: Aat λ2: Atotaltotal = A = Axλ2xλ2 + A + Ayλ2yλ2 = a = axλ2xλ2bcbcxx + a + ayλ2yλ2bcbcyy

MIXTURES OF 2 ABSORBING COMPOUNDSMIXTURES OF 2 ABSORBING COMPOUNDS

• In 1988 over 30,000 fish died suddenly and unexpectedly in the Fox In 1988 over 30,000 fish died suddenly and unexpectedly in the Fox River at Oshkosh, Wisconsin. Such “fish kills” are often caused by a River at Oshkosh, Wisconsin. Such “fish kills” are often caused by a lack of dissolved oxygen (Olack of dissolved oxygen (O22), or a release of pesticides, organic ), or a release of pesticides, organic

compounds, chlorine (Clcompounds, chlorine (Cl22), or heavy metals into the environment. ), or heavy metals into the environment.

However, none of these caused the Fox River fish kill.However, none of these caused the Fox River fish kill.

CASE STUDY: UV/Vis SPECTROSCOPY AND THE FOX RIVER CASE STUDY: UV/Vis SPECTROSCOPY AND THE FOX RIVER MYSTERYMYSTERY

• Finally, it was suggested that carbon monoxide (CO) gas from outboard Finally, it was suggested that carbon monoxide (CO) gas from outboard motor exhaust at a testing facility might be causing this fish kill. motor exhaust at a testing facility might be causing this fish kill. Normally, ONormally, O22 weakly bonds to the iron (Fe) atom in fish hemoglobin weakly bonds to the iron (Fe) atom in fish hemoglobin

during respiration. However, CO tightly bonds to this Fe atom and as a during respiration. However, CO tightly bonds to this Fe atom and as a result stops respiration.result stops respiration.


• The hypothesis that CO was The hypothesis that CO was causing this fish kill was tested by causing this fish kill was tested by UV/Vis spectroscopy.UV/Vis spectroscopy.

• In review, UV/Vis spectroscopy In review, UV/Vis spectroscopy measures the absorption of measures the absorption of electromagnetic radiation caused by electromagnetic radiation caused by electronic transitions within atoms electronic transitions within atoms and molecules. Different atoms and and molecules. Different atoms and molecules will have different UV/Vis molecules will have different UV/Vis spectra.spectra.


• Work in groups of 4.Work in groups of 4.• Every student does 1 quantitative analysis.Every student does 1 quantitative analysis.

• Every student in each group gets a different color (blue, purple, red, Every student in each group gets a different color (blue, purple, red, or yellow) of crepe paper.or yellow) of crepe paper.

• Each student selects λEach student selects λmaxmax from an absorption spectrum of their from an absorption spectrum of their

crepe paper.crepe paper.• Each student makes 4 standard solutions by extracting different Each student makes 4 standard solutions by extracting different

amounts of dye from their crepe paper.amounts of dye from their crepe paper.• Each student uses these solutions and Beer’s law to make a Each student uses these solutions and Beer’s law to make a

calibration curve.calibration curve.• Each student uses this calibration curve to measure the Each student uses this calibration curve to measure the

concentration of an unknown solution that was made from their concentration of an unknown solution that was made from their assigned color of crepe paper.assigned color of crepe paper.

• Every group does 1 qualitative analysis.Every group does 1 qualitative analysis.• Each group analyses the absorption spectrum from another Each group analyses the absorption spectrum from another

unknown solution that was made by extracting the dye from 2 unknown solution that was made by extracting the dye from 2 different colors of crepe paper. They will determine which 2 colors different colors of crepe paper. They will determine which 2 colors were used to make this solution.were used to make this solution.

TODAY’S EXPERIMENTTODAY’S EXPERIMENT

SOURCESSOURCES

• Aquanic. 2006. Fishkill. Available: Aquanic. 2006. Fishkill. Available: http://aquanic.org/images/photos/ill-in/fishkill.jpg [accessed 2 September [accessed 2 September 2006].2006].

• Beck, J. 2006. Unit 3 Spectrophotometry. Available: Beck, J. 2006. Unit 3 Spectrophotometry. Available: http://iws.ccccd.edu/jbeck/Spectrophotometryweb/Page.html [accessed 2 [accessed 2 October 2006].October 2006].

• Christian, G.D. 1986. Analytical Chemistry, 3rd ed. New York, NY: John Christian, G.D. 1986. Analytical Chemistry, 3rd ed. New York, NY: John Wiley & Sons, Inc.Wiley & Sons, Inc.

• Harris, D.C. 1999. Quantitative Chemical Analysis, 5th ed. New York, Harris, D.C. 1999. Quantitative Chemical Analysis, 5th ed. New York, NY: W.H. Freeman Company.NY: W.H. Freeman Company.

• Raven, P.H., R.F. Evert, H. Curtis. 1981. Biology of Plants, 3rd ed. New Raven, P.H., R.F. Evert, H. Curtis. 1981. Biology of Plants, 3rd ed. New York, NY: Worth Publishers, Inc.York, NY: Worth Publishers, Inc.

• Spencer, J.N., G.M. Bodner, L.H. Rickard. 2006. Chemistry Structure Spencer, J.N., G.M. Bodner, L.H. Rickard. 2006. Chemistry Structure and Dynamics, 3rd ed. New York, NY: John Wiley & Sons, Inc.and Dynamics, 3rd ed. New York, NY: John Wiley & Sons, Inc.

• Wikipedia. 2006. Image:Light-wave.png. Available: Wikipedia. 2006. Image:Light-wave.png. Available: http://en.wikipedia.org/wiki/Image:Light-wave.png [accessed 2 September [accessed 2 September 2006].2006].

http://aquanic.org/images/photos/ill-in/fishkill.jpg

http://iws.ccccd.edu/jbeck/Spectrophotometryweb/Page.html

http://iws.ccccd.edu/jbeck/Spectrophotometryweb/Page.html

http://en.wikipedia.org/wiki/Image:Light-wave.png

ch 103: spectrophotometry the electromagnetic spectrum

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