CEE 772: Instrumental Methods in Environmental Analysis€¦ · · 2014-09-187 CEE 772 #5 David Reckhow ... 8 CEE 772 #5 David Reckhow ... information for qualitative or quantitative
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Line sources Discrete wavelengths Used for AA and will be discussed later
Continuous Sources Used for molecular absorption and fluorescence Visible: tungsten lamp UV: deuterium lamp
Tungsten Lamp
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Black body radiation High temp (2870 oC)
Therefore good voltage control is quite important
4
4
~
~
etemperatur
voltageoutput
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Deuterium Arc Lamp
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Deuterium provides more power than hydrogen
Where
About 1000 hr half-life
hHHHEH e *22
HHe EEEh
Reflection Gratings
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Each groove behaves as a point source
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Reflection Gratings 1
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Each groove behaves as a point source
Reflection Gratings 2
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Full destructive interference occurs when: The difference in length of adjacent light paths is exactly equal to
the wavelength of that light or some multiple of the wavelength.
And since
Then
ban
sin
sin
db
da
sinsin dn
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Grating Monochromator
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Sample Cells
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Window Wavelength Range (nm)
Letter Code
Lot #s Color Code
Optical Glass 360 - 1000 OG yellow Near-UV Glass or Special Optical Glass
300 - 1000 OS or SG
180's green
Standard Silica 220 - 2500 Supracil Quartz or Quartz UV
165 - 2600, 2850 - 3600
QS or UV
280's blue
Infracil Quartz or Quartz IR
220 - 3600 QI or IR
300's red
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Detectors 1
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Photomultipliers consists of a photocathode and a series of dynodes in an evacuated glass enclosure.
When a photon of sufficient energy strikes the photocathode, it ejects a photoelectron due to the photoelectric effect. The photocathode material is usually a mixture of alkali metals, which make the PMT sensitive to photons throughout the visible region of the electromagnetic spectrum. The photocathode is at a high negative voltage, typically -500 to -1500 volts. The photoelectron is accelerated towards a series of additional electrodes called dynodes. These electrodes are each maintained at successively less negative potentials. Additional electrons are generated at each dynode.
This cascading effect creates 105 to 107 electrons for each photoelectron that is ejected from the photocathode. The amplification depends on the number of dynodes and the accelerating voltage. This amplified electrical signal is collected at an anode at ground potential, which can be measured.
Detectors 2
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Phototubes similar to PMTs, but consist of only a photocathode and anode. Since phototubes do not have a dynode chain to provide internal
amplification, they are used in less sensitive applications such as absorption spectrometers
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Simple Spectrophotometer
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Spectronic 20
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Turner 350
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Double Beam Spectrophotometer
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UV-Vis, Perkin Elmer 57 series
Errors
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Bandwidth and linearity
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Errors
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Bandwidth and linearity
Errors
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Band width and resolution
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Errors
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Effect of stray light on absorbance spectra Example of stray light in a
spectrophotometer with glass optics (A) compared to quartz optics (B)
Impacts of Stray light
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Causes increase in signal to detector that is not really “transmitted light”
Thomas & Burgess, 2007
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Errors, cont.
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Effect of stray light on linearity
The full picture
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Exact area of greatest accuracy depends on instrument
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Errors
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Precision vs. Abs
Optrodes
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Optical Fiber construction
Generic Instrument Design Probe type photometer Called an optrode when combined with chemical detectors that
produce an absorbing product with analyte Fig 7.9, pg 198
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Optrodes (cont.)
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Fiber sensor used for measuring penicillin in blood Harris pg. 597
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Fluorescence & Luminescence
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• Three types of Luminescence methods are: – (i) molecular fluorescence– (ii) phosphorescence– (iii) chemiluminescence
• In each, molecules of the analyte are excited to give a species whose emission spectrum provides information for qualitative or quantitative analysis.
Description
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Fluorescence: absorption of photon, short-lived excited state (singlet), emission of photon.
Phosphorescence: absorption of photon, long-lived excited state (triplet), emission of photon.
Chemiluminescence: no excitation source – chemical reaction provides energy to excite molecule, emission of photon.
Luminescence: High sensitivity strong signal against a dark background.
Used as detectors for HPLC.
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Energy Diagram
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Quantum Yield
Quantum Yield, = total # luminescing molecules
total # of excited molecules
k = rate constant]
k
k k k k k k
f
f i ec ic pd d[
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Structure effects
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Low–energy * (aromatic): most intense fluorescence.
Heterocycles do not fluoresce; heterocycles fused to other rings fluoresce. Heteroatom increases ISC then f decreases.
Conjugated double bond structures exhibit fluorescence.
Structural rigidity (e.g., naphthalene or fluorene vs biphenyl). Flexibility increases then f decreases.
Temperature: increase fluorescence intensity with decreasing T (reduce number of deactivating collisions).
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Solvent: increase fluorescence with increased viscosity (decreased likelihood of external conversion – radiationless deactivation)
Heavy atoms such as I, Br, Th increases ISC as a consequence f
decreases
pH: Increased resonance structures (protonation or deprotonation) stable excited state and greater quantum yield
pH can also influence emission wavelength (changes in acid dissociation constant with excitation)
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Protein fluorescence
H2N CH C
CH2
OH
O
H2N CH C
CH2
OH
O
H2N CH C
CH2
OH
O
HN
H2N CH C
CH2
OH
O
HN
H2N CH C
CH2
OH
O
OH
H2N CH C
CH2
OH
O
OH
TRP PHE TYR
Protein-labeling Probes
Absorption and fluorescence emission spectra of dansyl cadaverine in methanol.
= 10 -15 ns
= 0.1 - 0.3
340 nm540 nm
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Protein-labeling Probes
Absorption and fluorescence emission spectra of goat anti-mouse IgG labeled with fluorescein-5-isothio-cyanate in pH 8.0 buffer.
•Probes localize in different regions–Experience different electrical
environments
Fluor. excitation and emission spectra
N O O
CF3
ON
CH3
O
N O O
O-/Na+
O
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Fluorescence - EEMs
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Contour plots of 7 components identified from the complete F-EEMs dataset.
Baghoth et al., 2011
Terrestrial Humic
Marine & Terrestrial Humic
Terrestrial Humic
Amino acid
Terrestrial/ Anthro-pogenic Humic
Amino Acid
Marine & Terrestrial Humic
Excitation-Emission Matrices: Fluorescence intensity across the range of emission wavelengths while also scanning across excitation wavelengths
Correlates well with some NOM properties, but fundamental understanding is still not good
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Assignment of EEM Regions
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Location of EEM peaks (symbols) based on literature reports and operationally defined excitation and emission wavelength boundaries (dashed lines) for five EEM regions Chen et al., 2003