Transcript Chapters 13-14: UV/Visible Molecular Absorption Spectroscopy

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.
But, If the blank absorbance is high, Po will decrease too much, the
response will be slow and the results inaccurate
Large blank
absorbance
l scan of substance
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
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
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)
Since Po254 = Po255 =1:
A = 2.0/(Po25410-e254bc + Po25510-e255bc)
8
A = 2.0/(10-10,000x1.0xc + 10-5000x1.0xc)
7
6
C
A (actual)
A(expected)
5
10-6M
0.0075
0.0075
4
10-5M
0.074
0.075
3
10-4M
0.068
0.75
A = e bc
Negative Deviation
2
1
10-3M
5.3
7.5
0
2 10-4
4 10-4
6 10-4
8 10-4
1 10-3
Concentration (M)
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!
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
HIn
H+ + In-
Red, l =600nm
colorless
phenolphthalein:
If solution is buffered, then pH is constant and [HIn] is related to absorbance.
But, if unbuffered solution, equilibrium will shift depending on total analyte concentration
example: if Ka =
10-4
Ka
HIn
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
H+ + In-
Expected
Actual
C
HIn
“Apparent” deviation since can be accounted for by chemical equilibrium
But, if unbuffered solution, equilibrium will shift depending on total analyte concentration
example: if Ka =
10-4
Ka
HIn
Isosbestic point
H+ + In-
At the isosbestic point in spectra:
A = eb([HIn] + [In-])
c) Non-constant b:
- worse for round cuvettes
- use parallel cuvettes to help
B2
P2
P0
B1
P1
A = log10 Po/P = ebc
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.
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
Case I
Typical Sources
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
Photon detector shot noise
High-quality UV-visible spectrophotometers
Cell positioning uncertainties
High-quality UV-visible and IR
spectrophotometers
Source flicker
Inexpensive photometers and
spectrophotometers
S T = k1
Case II
Case III
ST  k 2 T 2  T
ST = k3T
Likely to be Important in
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)
Applications:
A) Molar Absorptivities (e) in UV-Vis Range:
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)
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
Name
Structure
- For Compounds with Multiple Chromophores:
 If greater then one single bond apart
- e are additive
- l constant
CH3CH2CH2CH=CH2
lmax= 184
emax = ~10,000
CH2=CHCH2CH2CH=CH2
lmax=185
emax = ~20,000
 If conjugated
- shifts to higher l’s (red shift)
H2C=CHCH=CH2
lmax=217 emax = ~21,000
Example 6: The equilibrium constant for the conjugate acid-base pair
K = 8.00x10-5
HIn + H2O
H3O+ + In-
e = 8.04x103
e = 0.755x103
Calculate the absorbance at 430 nm for an indicator concentration of 3.00x10 -4 M
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
10-8 – 10-9s
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)
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
- 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
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
 s  s* transition in vacuum UV
 n  s* saturated compounds with non-bonding electrons
l~ 150-250 nm
e ~ 100-3000 ( not strong)
 n  p*, p  p* requires unsaturated functional groups (eq. double bonds)
most commonly used, energy good range for UV/Vis
l ~ 200 - 700 nm
n  p* : e ~ 10-100
p  p*: e ~ 1000 – 10,000
Absorption Characteristics of Some Common Chromophores
Chromophore
Alkene
Example
C6H13HC
Solvent
CH2
Alkyne
C5H11C
C
Type of
transition
177
13,000
pp*
n-Heptane
178
196
225
10,000
2,000
160
pp*
_
_
n-Hexane
186
280
1,000
16
ns*
np*
n-Hexane
180
293
Large
12
Ethanol
204
41
np*
Water
214
60
np*
Ethanol
339
5
np*
CH3CCH3
O
emax
n-Heptane
CH3
O
Carbonyl
lmax (nm)
CH3CH
O
Carboxyl
Amido
CH3COH
O
ns*
np*
CH3CNH2
Azo
H3CN
NCH3
Nitro
CH3NO2
Isooctane
280
22
np*
Nitroso
C4H9NO
Ethyl ether
300
665
100
20
_
np*
270
12
np*
Nitrate
C2H5ONO2
Dioxane
Other Examples of Some
Common Chromophores
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 dorbitals
- In complex, all orbitals increase in energy where
orbitals along bonding axis are destabilized
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
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)
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.
2) Solvent can affect position and shape of curve.
- polar solvents broaden out peaks, eliminates fine structure.
(a) Vapor
Loss of fine structure for 1,2,4,5tetrazine as solvent polarity increases
(b) Hexane solution
(c) Aqueous
3) Solvent can also absorb in UV-vis spectrum.
3) Can obtain some functional group information for certain types of compounds..
- weak band at 280-290 nm that is shifted to shorter l’s with an increase
in polarity (solvent) implies a carbonyl group.
 acetone:
in hexane, lmax = 279 nm (e = 15)
in water, lmax = 264.5 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
UV Spectral Nomenclature
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)
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)
Non-absorbing
Species (colorless)
Complex
(red)
When all the protein is bound to Fe3+,
no further increase in absorbance.
As Fe3+ continues to bind protein
red color and absorbance increases.
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.
(3) Dilute each solution to an equal volume, Vt.
(4) Make instrumental measurements of each sample to get an
instrument response, IR.
Instrument Response ( S )
(5) Calculate unknown concentration, cx, from the following equation.
m = D y/ D x
b = y-intercept
S  mVs  b
(V s ) 0
Vs
kVscs kVxcx
S

Vt
Vt
S 1Vs cs
Cx 
( S 2  S 1 )Vx
Where:
cx 
bcs
m Vx
S =
k =
Vs =
cs =
Vx =
cx =
Vt =
signal or instrument response
proportionality constant
volume of standard added
concentration of the standard
volume of the sample aliquot
concentration of the sample
total volume of diluted solutions
Note: assumes a linear relationship between instrument response and sample concentration.
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
d) Photometric titration
- can measure titration with UV-vis spectroscopy.
- requires the analyte (A), titrant (T) or titration product (P) absorbs radiation
Example 7: Given:
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
Calculate the concentrations of A and B in solutions that yielded an absorbance of 0.439 at 475 nm
and 1.025 at 700 nm in a 2.50-cm cell.