Structure Determination by Spectro

scopy
• Spectroscopy: study EMV and matter
• Spectroscopy method for determination of organic molec
ular structure

• Mass spectroscopy
• Ultraviolet-visible spectroscopy
• Infrared spectroscopy
• Nuclear magnetic resonance spectroscopy

1
Interaction of electromagnetic radia
tion energy and matter
• When EMR is directed at a substance, the radiatio
n can be:

– Absorbed
– Transmitted
– Reflected

depending on the frequency (or wavelength or energy) of the

radiation and the structure of the substance.

2
Interaction of electromagnetic radia
tion energy and matter
• When EMR is directed at a substance, the radiatio
n can be:

– Absorbed
– Transmitted
– Reflected

depending on the frequency (or wavelength or energy) of the

radiation and the structure of the substance.

3
Electromagnetic Radiation

4
 Mathematical Relationships

c =  E = h E = hc / 

 = Frequency (Hz) c = Velocity of Light

(3 x 1010 cm/sec)

 = Wavelength (cm) h = Planck’s Constant

(6.62 x 10-27 erg-sec)

6
Interaction of electromagnetic radia
tion energy and matter
• Molecules exist only in discrete states that
correspond to discrete energy content.
• The EMR energy that is absorbed is quanti
zed and brings about certain specific chan
ges in the molecule.
– electronic transitions (UV-vis)
– vibrations (IR)
– rotations (IR)

7
Interaction of electromagnetic radia
tion energy and matter
• Exact energies absorbed by a molecule ar
e highly characteristic of the structure and
are unique for each compound.
– spectroscopic “fingerprint”

• Similar functional groups absorb similar en

ergies regardless of the structure of the re
st of the compound.
8
ULTRAVIOLET VISIBEL (UV-VIS)
Spectrophotometer
INSTRUMENT
INSTRUMENT
 UV-visible Spectroscopy
• Ultraviolet: 200 nm – 400 nm Visible: 400 nm – 800 nm
• Most organic molecules and functional groups do not absorb
energy in the UV-visible part of the EMR spectrum and thus, a
bsorption spectroscopy in the ultraviolet-visible range is of limi
ted utility.
• When a molecule does absorb in the UV-vis, the energy transi
tions that occur are between electronic energy levels of valen
ce electrons, that is, electrons in orbitals of lower energy are e
xcited to orbitals of higher energy.
• Energy differences generally of 30 –150 kcal/mole

12
Electronic molecular orbital energies
 s  s* transition in vacuum UV (<180 nm) not use in organic C-C 135 nm
 n  s* saturated compounds with non-bonding electrons (N, O, S, hal)
 ~ 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
 ~ 200 - 700 nm
n  p* : e ~ 10-100
p  p*: e ~ 1000 – 10,000
 RELATION OF STRUCTURE AND
e
Name Structure max 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

- 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    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
 UV-visible Spectroscopy
• The ground state of an organic molecule can contain valence ele
ctrons in three principal types of molecular orbitals:

s (sigma)
C:H

p (pi) C::C

n (non-bonding)

18
Difference transition of n to pi* and pi to pi*
The effect of transition to λ
Absorption Characteristics of Some Common Chromophores
Chromophore Example Solvent max (nm) emax Type of tra
nsition
Alkene C
H
61
3H
CC
H2 n-Heptane 177 13,000 pp*
Alkyne n-Heptane 178 10,000 pp*
C
H
5C
1
1 CC
H
3 196 2,000 _
225 160
_
Carbonyl O n-Hexane 186 1,000 ns*
280 16 np*
CH3CCH3
O n-Hexane 180 Large
293 12
ns*
CH 3 CH np*
Carboxyl O Ethanol 204 41 np*
CH3COH
Amido O Water 214 60 np*
CH3CNH2
Azo H
CNN
CH Ethanol 339 5 np*
3 3
Nitro CH3NO2 Isooctane 280 22 np*
Nitroso C4H9NO Ethyl ether 300 100 _
665 20 np*
Nitrate C2H5ONO2 Dioxane 270 12 np*
Other Examples of Some Com
mon Chromophores
 UV-visible Spectroscopy
• Electrons in sigma bonds (single bonds) are too tightly bound
to be promoted to a higher energy level by UV-visible radiatio
n.
– alkanes, alcohols, alkyl halides, simple alkenes do not absorb in the UV

• Electrons in pi bonds and non-bonding orbitals are more loose

ly held and can be more easily promoted.
– Conjugation of pi bonds lowers the energy of the radiation that is absorb
ed by a molecule.
– Conjugated unsaturated systems are molecules with two or more doubl
e or triple bonds each alternating with a single bond.
– If a molecule does not absorb in the UV, then it does not contain a conju
gated system of alternating double bonds or a carbonyl group.

23
 1). For Compounds with Multiple Chromophores:
 If greater then one single bond apart
- e are additive
-  constant

CH3CH2CH2CH=CH2 max= 184 emax = ~10,000

CH2=CHCH2CH2CH=CH2 max=185 emax = ~20,000

 If conjugated
- shifts to higher ’s (red shift)

H2C=CHCH=CH2 max=217 emax = ~21,000

2) Absorption occurs with bonding electrons.
- E() 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
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 ab

sorbance 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 1,2,4,5-tetrazi

ne 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 ’s with an increase
in polarity (solvent) implies a carbonyl group.
 acetone:
in hexane, max = 279 nm (e = 15)
in water, max = 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
’s (red shift) similar to conjugated alkenes
 The effect of pH to λ
Borax buffer: 0.05M (pH 9.2 ~ 239 nm) and A 452
NaOH : pH 13 ~ 254 and A = 342
The effect of conjugation bond to absorption
PIGMENTS ON VISIBLE LIGHT
UV Spectral Nomenclature
Λ max SOME ORGANIC COMPOUNDS
The Applications of UV for determining of Λ max
of organic compounds
EMPIRICAL RULES FOR ABSORPTION WAVELENGTHS
OF CONJUGATED SYSTEMS
SOME EXAMPLES THAT ILLUSTRATE THESE RULES FOLLOW
SOME EXAMPLES TO PREDICT THE Λ max
SOME WORKED EXAMPLES TO PREDICT THE Λ max
2. Woodward-Fieser rules for calculating π to π* of conjugated
Carbonyl compounds
Similar for enones
Similar for acid and ester
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:
, pH, temperature, ionic strength
- prepare standard curve (match standards and samples as much as
possible)

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 concen
tration.
(3) Dilute each solution to an equal volume, Vt.

(4) Make instrumental measurements of each sample to get an

instrument response, IR.
 (5) Calculate unknown concentration, cx, from the following equation.

m =  y/  x
Instrument Response ( S )

kV
scs kVc
b = y-intercept S  x
x
V V
S s
mVb t t
(V s ) 0
SV
1 c
x
C s s
Vs (S2S)
1V x
Where:
S = signal or instrument response
k = proportionality constant
Vs = volume of standard added
bcs cs = concentration of the standard
cx  Vx = volume of the sample aliquot
mVx cx = concentration of the sample
Vt = total volume of diluted solutions

Note: assumes a linear relationship between instrument response and sample concentration.
c) Analysis of Mixtures
- use two different ’s with different e’s

A1 = e1MbcM + e1NbcN (1)

A2 = e2MbcM + e2NbcN (2)

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 a
nd 1.025 at 700 nm in a 2.50-cm cell.