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UV-VISIBLEUV-VISIBLE
SPECTROPHOTOMETRYSPECTROPHOTOMETRY
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ObjectivesObjectives To understand the process of absorption of light by molecules To understand the relationships between Absorbance,
Transmittance, path length and analyte concentration (Beer-Lambert Law).
To be familiar with the quantitative applications of the Beer-LambertLaw in the multi-component analysis of mixtures, and in standard
addition. To be aware of the chemical and instrumental limitations of the
Beer-lambert Law.
To be aware of the major components of the instruments used for
measurement of absorbance. To understand the experimental requirements for
spectrophotometric measurements.
To be familiar with typical applications of spectrophotometric
analysis.
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1. Introduction1. IntroductionSpectrophotometry - measurement ofSpectrophotometry - measurement of
light absorption to quantifylight absorption to quantifyconcentration ofconcentration of analyteanalyte
1.1 Light Absorption1.1 Light Absorption
Absorption of a photon by a molecule results in an excited stateexcited state
Different wavelengths of light can be absorbed, which cause
different forms of excitation
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1. Introduction1. Introduction Absorption of
UV and visible
light by a
molecule
causes
electronic
excitation1020 1018 1016 1014 1012 108
Cosmic
rays -rays X-rays UV
Visible
Infrared MicrowaveR
adio
Electronicexcitation
Bond breakingand ionization Vibration Rotation
Visible Spectrum
400 500 600 700
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1.1. IntroductionIntroduction
Bonding in organic molecules is based on overlap betweenand
atomic orbitals.Electrons inElectrons inandandorbitalsorbitalscan be excited to thecan be excited to theantibonding** andand**orbitalsorbitals- involves absorption of different energies/wavelengths.- involves absorption of different energies/wavelengths.
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Electronic energy levels ofpolyatomic molecules
* (antibonding)
* (antibonding)
n (non-bonding)
(bonding) (bonding)
1.1. IntroductionIntroduction * and * transitions aremore likely than n * and n *
Stronger absorptions occur forthese transitions.
Wavelength and intensity ofabsorption is affected by:
molecular geometry ofmolecule
types of substituent groups
natureof solvent (polarity)
The part of the molecule whichabsorbs light is called thechromophorechromophore..
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1. Introduction1. Introduction1.2 Complementary Colours1.2 Complementary Colours
When white light is absorbed by a chromophore, the eye detect
the colours that are notnot absorbed. This is called the
complementary colour to the colour absorbed.
Determination of concentration depends on detection of change
in colour intensity (absorption) at a particular wavelength.
ofmaximumabsorption
Colour Absorbed Colour Observed
380-440 violet-blue green-yellow440-500 blue-green orange-red
500-580 green-yellow violet-blue
580-680 orange-red blue-green
680-780 purple green
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2. Colorimetric Analysis2. Colorimetric Analysis2.1 Photometric measurement2.1 Photometric measurement
(a)visual comparison using colour standards
P Po
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2. Colorimetric Analysis2. Colorimetric Analysis(b) Colorimeter/Photometer
Filters used to select narrow wavelength
Detection with photosensing device
PPo
Filter
wheel
Photodetector
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2. Colorimetric Analysis2. Colorimetric Analysis2.22.2 SpectrophotometricSpectrophotometricAnalysisAnalysis
Spectral bandwidth 1 nm, i.e very monochromatic light.
can operate in visible and UV ranges
Colorimetry and spectrophotometry provide sensitivesensitivemethods ofanalysis, i.e. ppm to ppb ranges.
PPo
PhotodetectorMonochromator
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3. Quantifying Light Absorption3. Quantifying Light Absorption
PPo
b
Absorbing solution
of concentration,c.
Reflected
beam
Pr
Pa
3.1 Incident Light3.1 Incident Light
P0 = Pr + Pa + P (1)
Pr 4% for air-glass interface;I.e.use blank to zero P0= Pa+ P (2)
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3. Quantifying Light Absorption3. Quantifying Light Absorption3.2 Lamberts Law3.2 Lamberts Law
Formonochromaticlight passing
though a transparent medium:
3.3 Beers Law3.3 Beers LawThe intensity of a monochromatic
light beam decreaseswithconcentrationincrease.
A = logP0
P
dPP
= - k' dc
Pa
P
-dPP
= k' 0
c
dc
lnP0
P = k'c or,
logP0P
= k"c
A = k"c
A = kb
-dP = - k P db
P=Po
P=P
-dP
P
=k
b=0
b=b
db
lnP0
P = kb
Absorbance, A, defined as:
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3. Quantifying Light Absorption3. Quantifying Light Absorption3.4 Beer-Lambert Law3.4 Beer-Lambert Law
Combining these gives:
A = a b c Where: a is the absorptivity,absorptivity,b is thepath lengthpath length, and c is the
concentration.concentration.
When c is in mol/L, and b is in cm, A = b c
Where is the molar absorptivitymolar absorptivity.
TransmittanceTransmittancedefined as: T = P/Po Hence:
A = log (1/T) = log(100/%T) = 2 - log(%T)
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4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law4.14.1 Analysis of a singleAnalysis of a single analyteanalyte
At fixed and b, is constant for a given chromophore.
Add chromogenic reagentschromogenic reagents to sample and standards, and measureabsorbance.
Assumes that the chemical matrix of standard is the same as thesample.
Ax
Cx
4
3
2
1
0 2 3
A
A
A
A
A0
C C 1 C C C 4
Concentration
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4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law
6005004003000.0
0.1
0.2
0.3
0.4
0.5
0.6
MN
M+N
Wavelength (nm)
1111 2222
4.2 Analysis of mixtures4.2 Analysis of mixtures
Beer-Lambert Law applies to mixtures of of non-interacting components.
At1: A1 = 1,M bcM + 1,N bcN,at and 2: A2 = 2,M bcM + 2,N bcN
1,M, 1,N, 2,M, 2,N are found from separate calibrations at1and 2. b is aconstant.
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4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law Measure A1and A2 at 1and 2, and solve for cMand cN.
Most accurate when differences between values are the greatest,
i.e choose appropriate values.
Accuracy decreases as the number of components increases.
See Problem 5
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4.34.3 Standard AdditionStandard Addition
-Multi Point Method -Multi Point Method Used for samples with complex
matrix chemicalinterference if
calibration method applied.
Measure A of sample+ chromogenicreagent.
Repeat with added increments of
standard to same vol. of sample.
4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law
Vol. Std added (mL)0 Vs
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4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law
Sample Conc = Cx, Vol sample = Vx , Vol std added =Vs. Std Conc =Cs, Total vol = Vt.
Plot A vs Cs, gives straight line: A = + Vs
Where = bCxVx/Vt, and = bCs/ Vt Hence:
Cx= Cs / Vx
Before addition: A =b VxCx
Vt
After addition: A =b VsCs
Vt+
b VxCx
Vt
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4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law4.44.4 Standard Addition-Two Point MethodStandard Addition-Two Point Method
See problem 4
Before addition: A1=b VxCx
Vt
After addition: A2 =b VsCs
Vt +b VxCx
Vt
and hence: Cx =A1Cs Vs(A2- A1)Vx
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5. Limitations of the Beer-Lambert Law5. Limitations of the Beer-Lambert Law5.1 Concentration effects5.1 Concentration effects
B-L law applies to dilute
solutions (negligible interactionbetween solute ions).
Higher concentrations ofanalyte (i.e. > 10-2M) or high
electrolyte concentrations,molecular/ionic interactions
reduced light absorption atsome's.
= f(n, refr. index of soln). Ifchanges in n large, deviation
from B-L observed.
Replace with n/(n2+ 2)2inB-L equation
Concentration
Deviation from B-L law
(loss of sensitivity)
Adherence to B-L law
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5. Limitations of the Beer-Lambert Law5. Limitations of the Beer-Lambert Law5.2 Instrumental deviations5.2 Instrumental deviations
Occur whenpolychromaticpolychromatic rather than monochromatic light is
used.
For a light beam consisting of 'and , B-L Law becomes:
A = log(P0'+ P0") - log(P0'10-'bc + P0"10
-"bc)
Only when ' " is B-L law obeyed, i.e A = e'bc
Concentration
' "Band A
' < "Band B
'
Wavelength
Band B
Band A
' "
"
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6. Experimental Considerations6. Experimental Considerations6.1 Wavelength selection6.1 Wavelength selection
Choose where A is large to obtain best sensitivity.
Choose where dA/d = 0 or is small.
Wavelength
?
Ab
sor
bance
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6. Experimental Considerations6. Experimental Considerations6.2 Chromogenic reagents for colorimetric analysis6.2 Chromogenic reagents for colorimetric analysis
Should be stable and pure,
Should not absorb at of measurement, but.
Should react rapidly with analyte to give a stable chromophore.
Absorptivity,,should not be sensitive to minor pH, T, electrolytechanges, etc.
Should be selective.
For UV measurements, often measure particular chromophoredirectly, I.e. no reagent necessary (e.g. nitrate in GDR expt).
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7. Instrumentation7. Instrumentation7.17.1 PrinciplePrinciple
7.2 Photometer/Colorimeter7.2 Photometer/ColorimeterSource: Generally visible light (Tungsten)
selector: Filters, simple monochromator
Sample blank: Single beam, no ref.
Detector: Photocell or photodiode.
Output: Microammeter/galvanometer/digital
Advantages / Disadvantages:
Lower cost
Light source fluctuations during measurement.
Light poorly monochromated.
Light
Source
Wavelength
selection
Sample/
Blank
Signal
detection
Data/
Output
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7. Instrumentation7. Instrumentation
Single beam spectrometerSingle beam spectrometer
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7. Instrumentation7. Instrumentation
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7. Instrumentation7. Instrumentation
7.37.3 UV-visible SpectrophotometerUV-visible Spectrophotometer
Source: UV-deuterium,visible-quartz halogen
selector: Grating or Prism monochromator, scanning.
Sample/blank:Dual beam, can reference source fluctuations
Detector: Phototube, photodiode, photomultiplier.
Output: Digital, VDU,
Advantages: More sensitive,
UV and visible spectra, Scanning or diode array simultaneous spectral aquisition
better resolution (narrow spectral bandwidth)
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7. Instrumentation7. Instrumentation
Scanning, double beam UV-vis spectrometerScanning, double beam UV-vis spectrometer
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7. Instrumentation7. Instrumentation
Diode array spectrometerDiode array spectrometer
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7. Instrumentation7. Instrumentation
7.4 Probe photometers7.4 Probe photometers
To Detector
Optical fibre bundle
Optical filter
Mirror
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7. Instrumentation7. Instrumentation
7.5 Optical Sensing Devices (7.5 Optical Sensing Devices (OptrodesOptrodes,, OptodesOptodes)) Reflectance spectroscopy
Used for miniaturized in-situmeasurement of glucose, pH
To Detector
Optical fibre bundle
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8.Applications8.Applications
Clinical,environmental, industrial and forensic Screening of drugs
e.g. UV spectral identification and quantitation of amphetamines
Determination of cyanide e.g. in Tylenol tablets
Use of UV-vis for detection after liquid, paper or thin-layerchromatographic separation
Collect,
extract,
analyseby UV-VisS
td
1
Std2
Sample
Elution
Flow-through
UV-vis spectro-
photometerPump
Time/Distance
Concentration
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7. Instrumentation7. Instrumentation
7.6 Flow Injection Analysis7.6 Flow Injection Analysis
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8. Applications8. Applications
Rapid ScreeningRapid Screening White powder is seized in a police
raid. Is it heroin? UV spectrum of heroin shows maxat
278 nm gives tentative identification.
Allows elimination of other materials
(e.g. starch, sugar, which are commondiluents used in heroin).
Allows thousands of other substancesto be eliminated.
Need to do confirmatory test, eg gaschromatography, or mass spec.
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8.Applications8.Applications
Determination ofDetermination of
ethanol in expiredethanol in expired
breath (Thebreath (TheBreathalyserBreathalyser))
2K2Cr2O7+ 3C2H5OH 8H2SO4
2Cr2(SO4)3 + 2K2SO4+
3CH3COOH + 11 H2O
Measure K2Cr2O7 at 420nm
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8.Applications8.Applications
Screening (Fuel cell) breath testerScreening (Fuel cell) breath testerAbsorbed methanol reacts giving small current.
Approx 3000 tests,