1. Application of UV/Vis Spectroscopy
Common Problems:
a) Mixtures:
- blank samples often contain multiple absorbing species.
- the absorbance is the sum of all the individual absorbencies
A= A1 + A2 +A3 + … = e1bc + e2bc + e3bc …
- substances in both the blank and sample which absorb can be “blanked out” in both double and single beam spectrometers.

2. But, If the blank absorbance is high, Po will decrease too much, the response will be slow and the results inaccurate
l scan of substance
Large blank
absorbance
If blank absorbance too high:
- dilute the sample
- use a different l where the analyte absorbs more relative to the interference.
- use a different method of separation

3. b) Instrumental Deviations from Beer’s Law:
- stray light (already discussed).
- polychromatic light (more then a single l)
‚ since all instruments have a finite band pass, a range of l’s are sent through the sample.
‚ e may be different for each l
Deviation’s from Beer’s law at high concentration

4. Illustration of Deviation from Beer’s law:
Let us say that exactly 2 wavelengths of light were entering the sample
l = 254 nm e254 =10,000
l = 255 nm e255 = 5,000
let Po = 1 at both l’s
What happens to the Beer’s law plot as c increases?
A = A254 + A255
A = log Po/P (total) = log (Po254 + Po255)/(P254 + P255)
At the individual l’s:
A254 = e254bc = log Po254/P254
10ebc = Po254/P254
P254 = Po/10ebc = Po10-ebc
For both together:
A = log (Po254 + Po255)/(Po25410-e254bc + Po25510-e255bc)

5. Since Po254 = Po255 =1:
A = 2.0/(Po25410-e254bc + Po25510-e255bc)
A = 2.0/(10-10,000x1.0xc x1.0xc) C
A (actual)
A(expected)
10-6M
0.0075
10-5M
0.074
0.075
10-4M
0.068
0.75
10-3M
5.3
7.5
The results are the same for more l’s of light. The situation is worse for greater differences in e’s (side of absorption peak, broad bandpass)
Always need to do calibration curve! Can not assume linearity outside the range of linearity curve!

6. b) Chemical Deviations from Beer’s Law:
- Molar absorptivity change in solutions more concentrated than 0.01M
‚ due to molecular interactions
‚ Beer’s law assumes species are independent
‚ electrolytes may also cause this problem
‚ e is also affected by the index of refraction
- association, dissociation, precipitation or reaction of analyte
‚ c in Beer’s law is the concentration of the absorbing species
‚ commonly use the analytical concentration – concentration of all forms of the species. Ka
phenolphthalein:
HIn
H In-
Red, l =600nm
colorless
If solution is buffered, then pH is constant and [HIn] is related to absorbance.

7. But, if unbuffered solution, equilibrium will shift depending on total analyte concentration
example: if Ka = 10-4
HIn Ka
H In-
CHIn
[HIn]
[In-]
[HIn]/[In-]
10-5
8.5x10-7
9.2x10-6
0.0924
10-4
3.8x10-5
6.2x10-5
0.613
10-3
7.3x10-4
2.7x10-4
2.70
“Apparent” deviation since can be accounted for by chemical equilibrium

8. At the isosbestic point in spectra: A = eb([HIn] + [In-])
But, if unbuffered solution, equilibrium will shift depending on total analyte concentration
example: if Ka = 10-4
HIn Ka
H In-
Isosbestic point
At the isosbestic point in spectra:
A = eb([HIn] + [In-])

9. A = log10 Po/P = ebc c) Non-constant b: - worse for round cuvettes
- use parallel cuvettes to help B2 P2 P0 B1 P1
A = log10 Po/P = ebc

10. d) Instrument noise:
noise - short term baseline fluctuations, which decrease the precision of the analysis
‚ can not measure A precisely
‚ the various sources of noise each cause some uncertainty
in the absorbance measurement and can be treated as individual standard deviations.
1) 0%T noise:
- noise when light beam is blocked
- seldom important
- typically ± 0.01%T
2) Readout Precision:
- especially with a meter
- typically ± 0.5%T  1-3% error in concentration
3) Shot Noise:
- occurs when e- transfers across a junction (like the space between cathode & anode in PMT).
- causes random fluctuations in current since individual e- arrive at random times
- increases with increase current (%T). Especially bad above 95%T.

11. Likely to be Important in
4) Flicker Noise:
- noise from the lamp due to intensity changes
- important at high transmittances.
5) Cell positioning uncertainty:
- not really noise, but affects precision
- minor imperfections, scratches or dirt change %T
- may be the major cause of imprecision
Category
Characterized by
Typical Sources
Likely to be Important in
Case I
ST = k1
Limited Readout resolution
Inexpensive photometers and spectrophotometers having small meter scales
Heat detector Johnson noise
IR and near-IR spectrophotometers and photometers
Dark current and amplifier noise
Regions where source intensity and detector are low
Case II
Photon detector shot noise
High-quality UV-visible spectrophotometers
Case III
ST = k3T
Cell positioning uncertainties
High-quality UV-visible and IR spectrophotometers
Source flicker
Inexpensive photometers and spectrophotometers

12. Taken together, these noise sources
indicate that the intermediate absorbance
and transmittance ranges should be used.
- at low %T, 0%T, noise and readout precision
are important
- at high %T, shot and flicker noise are large.
Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)

13. Applications: A) Molar Absorptivities (e) in UV-Vis Range: Name
e = 8.7 x 1019 PA
P – transition probability (ranges from 0.1 to 1, for likely transitions)
A – cross-section area of target molecule (cm2)
- ~10-15 cm2 for typical organics
- emax = 104 to 105 L/mol-cm
- e < 103 – low intensity (P £ 0.01)
Name
Structure
lmax e
but-1-en-3-yne
219
7,600
cyclohex-2-enone
225
10,300
toluene
206
7,000
3,4-dimethylpent-3-en-2-one
246
5,300

14. H2C=CHCH=CH2 lmax=217 emax = ~21,000
- For Compounds with Multiple Chromophores:
‚ If greater then one single bond apart
- e are additive
- l constant
CH3CH2CH2CH=CH2 lmax= emax = ~10,000
CH2=CHCH2CH2CH=CH2 lmax= emax = ~20,000
‚ If conjugated
- shifts to higher l’s (red shift)
H2C=CHCH=CH2 lmax=217 emax = ~21,000

15. Example 6: The equilibrium constant for the conjugate acid-base pair
HIn + H2O H3O+ + In-
Calculate the absorbance at 430 nm for an indicator concentration of 3.00x10-4 M
K = 8.00x10-5
e = 8.04x103
e = 0.755x103

16. Applications: B) Absorbing Species in UV/Vis:
1) Electronic transitions involving organic compounds, inorganic compounds, complexes, etc.
Basic process:
M + hn  M*
10-8 – 10-9s
M*  M + heat
(or fluorescence, light, or phosphorescence) or
M*  N
(new species, photochemical reaction)
Note: excited state (M*) is generally short and heat produced not generally measurable.
Thus, get minimal disturbance of systems (assuming no photochemical reaction)

17. - E(l) required differs with type of bonding electron.
2) Absorption occurs with bonding electrons.
- E(l) required differs with type of bonding electron.
- UV-Vis absorption gives some information on bonding electrons (functional groups in a compound.
- Most organic spectra are complex
‚ electronic and vibration transitions superimposed
‚ absorption bands usually broad
‚ detailed theoretical analysis not possible, but semi-quantitative or qualitative analysis of types of bonds is possible.
‚ effects of solvent & molecular details complicate comparison

18. Sigma (s) – single bond electron
- Single bonds usually too high excitation energy for most instruments (£ 185 nm)
‚ vacuum UV
‚ most compounds of atmosphere absorb in this range, so difficult to work with.
‚ usually concerned with functional groups with relatively low excitation energies (190 £ l £ 850 nm).
- Types of electron transitions:
i) s, p, n electrons
Sigma (s) – single bond electron
Low energy bonding orbital
High energy anti-bonding orbital

19. Pi (p) – double bond electron
Low energy bonding orbital
High energy anti-bonding orbital
Non-bonding electrons (n): don’t take part in any bonds,
neutral energy level.
Example: Formaldehyde

20. ‚ s  s* transition in vacuum UV
‚ n  s* saturated compounds with non-bonding electrons
l ~ nm
e ~ ( not strong)
‚ n  p*, p  p* requires unsaturated functional groups (eq. double bonds)
most commonly used, energy good range for UV/Vis
l ~ nm
n  p* : e ~
p  p*: e ~ 1000 – 10,000

21. Absorption Characteristics of Some Common Chromophores
Example
Solvent
lmax (nm)
emax
Type of transition
Alkene
n-Heptane
177
13,000
pp*
Alkyne
178
196
225
10,000
2,000
160 _
Carbonyl
n-Hexane
186
280
180
293
1,000 16
Large 12
ns*
np*
Carboxyl
Ethanol
204 41
Amido
Water
214 60
Azo
339 5
Nitro
CH3NO2
Isooctane 22
Nitroso
C4H9NO
Ethyl ether
300
665
100 20
Nitrate
C2H5ONO2
Dioxane
270

22. Other Examples of Some Common Chromophores

23. ii) d/f electrons (transition metal ions)
‚ Lanthanide and actinide series
- electronic transition of 4f & 5f electrons
- generally sharp, well-defined bands not affected by associated ligands
‚ 1st and 2nd transition metal series
- electronic transition of 3d & 4d electrons
- broad peaks
Crystal-Field Theory
In absence of external field d-orbitals are identical
Energies of d-orbitals in solution are not identical
Absorption involves e- transition between d-
orbitals
In complex, all orbitals increase in energy where
orbitals along bonding axis are destabilized

24. Magnitude of D depends on: - charge on metal ion
- position in periodic table
- ligand field strength :
I- < Br- < Cl- < F- < OH- < C2O42- ~ H2O < SCN- < NH3
< ethylenediamine < o-phenanthroline < NO2- < CN-
D increases with increasing field strength, so wavelength decreases

25. (colorless) (deep red color) iii) Charge Transfer Complexes
‚ Important analytically because of large e (> 10,000)
‚ Absorption of radiation involves transfer of e- from the donor to orbital associated with acceptor
- excited state is product of pseudo oxidation/reduction process
‚ Many inorganic complexes of electron donor (usually organic)
& electron acceptor (usually metal)
- examples: Iron III thiocyanate
Iron II phenanthroline
(colorless)
(deep red color)

26. C) Qualitative Analysis: 1) Limited since few resolved peaks
- unambiguous identification not usually possible.
2) Solvent can affect position and shape of curve.
- polar solvents broaden out peaks, eliminates fine structure.
Loss of fine structure for acetaldehyde when transfer to solvent from gas phase
Also need to consider absorbance of solvent.

27. 2) Solvent can affect position and shape of curve.
- polar solvents broaden out peaks, eliminates fine structure.
(a) Vapor
Loss of fine structure for 1,2,4,5-tetrazine as solvent polarity increases
(b) Hexane solution
(c) Aqueous

28. 3) Solvent can also absorb in UV-vis spectrum.

29. - 260 nm with some fine structure implies an aromatic ring.
3) Can obtain some functional group information for certain types of compounds..
- weak band at nm that is shifted to shorter l’s with an increase in polarity (solvent) implies a carbonyl group.
‚ acetone:
in hexane, max = 279 nm ( = 15)
in water, max = nm
- solvent effects due to stabilization or destabilization of ground or excited states, changing the energy gap.
‚ since most transitions result in an excited state that is more polar than the ground state
- 260 nm with some fine structure implies an aromatic ring.
Benzene in heptane
More complex ring systems shift to higher l’s (red shift) similar to conjugated alkenes

30. UV Spectral Nomenclature

31. C) Quantitative Analysis (Beer’s Law):
1) Widely used for Quantitative Analysis Characterization
- wide range of applications (organic & inorganic)
- limit of detection  10-4 to 10-5 M (10-6 to 10-7M; current)
- moderate to high selectivity
- typical accuracy of 1-3% ( can be ~0.1%)
- easy to perform, cheap
2) Strategies
a) absorbing species
- detect both organic and inorganic compounds containing any of these species (all the previous examples)

32. b) non- absorbing species
- react with reagent that forms colored product
- can also use for absorbing species to lower limit of detection
- items to consider:
l, pH, temperature, ionic strength
- prepare standard curve (match standards and samples as much as possible)
reagent
(colorless)
Complex
(red)
Non-absorbing
Species (colorless)
As Fe3+ continues to bind protein
red color and absorbance increases.
When all the protein is bound to Fe3+,
no further increase in absorbance.

33. Standard Addition Method (spiking the sample)
- used for analytes in a complex matrix where interferences in the UV/Vis for the analyte will occur: i.e. blood, sediment, human serum, etc..
- Method:
(1) Prepare several identical aliquots, Vx, of the unknown sample.
(2) Add a variable volume, Vs, of a standard solution of known concentration, cs, to each unknown aliquot.
Note: This method assumes a linear relationship between instrument response and sample concentration.

34. (3) Dilute each solution to an equal volume, Vt.
(4) Make instrumental measurements of each sample to get an instrument response, IR.

35. S = signal or instrument response k = proportionality constant
(5) Calculate unknown concentration, cx, from the following equation.
Note: assumes a linear relationship between instrument response and sample concentration.
Where:
S = signal or instrument response
k = proportionality constant
Vs = volume of standard added
cs = concentration of the standard
Vx = volume of the sample aliquot
cx = concentration of the sample
Vt = total volume of diluted solutions

36. c) Analysis of Mixtures
- use two different l’s with different e’s
A1 = e1MbcM + e1NbcN (l1)
A2 = e2MbcM + e2NbcN (l2)
Note: need to solve simultaneous equations

37. d) Photometric titration
- can measure titration with UV-vis spectroscopy.
- requires the analyte (A), titrant (T) or titration product (P) absorbs radiation

38. Absorbance (1.00 cm cell) Species 475 nm 700 nm A (7.50x10-5 M) 0.155
Example 7: Given:
Calculate the concentrations of A and B in solutions that yielded an absorbance of at 475 nm and at 700 nm in a 2.50-cm cell.
Absorbance (1.00 cm cell)
Species
475 nm
700 nm
A (7.50x10-5 M)
0.155
0.755
B (4.25x10-5 M)
0.702
0.091