Quality Control

1. ULTRAVIOLET/VISIBLE
ABSORPTION SPECTROSCOPY
Widely used in chemistry.
Perhaps the most widely used in Biological Chemistry.
Easy to do.
Very easy to do wrong.
Dr.Samer HOUSHEH

2. Electronic Excitation by UV/Vis Spectroscopy
UV: Radio waves:
X-ray: IR:
valance Nuclear spin states
core electron molecular
excitation electronic (in a magnetic field)
vibrations
excitation
Dr.Samer HOUSHEH

3.  Used to study molecules and their electronic
transitions.
 Principle: The energy absorbed corresponds to
the amount necessary to promote an electron
from one orbital to another.
 Commonly used to determine the concentration
of an absorbing species in solution (Quantitative
Analysis)using Beer-Lambert law:
Dr.Samer HOUSHEH

4. The wavelength and amount of light that a compound absorbs depends on
its molecular structure and the concentration of the compound used.
The concentration dependence follows Beer’s Law.
A=ebc = log I/I0
Where A is absorbance
e is the molar absorptivity with units of L mol-1 cm-1
b is the path length of the sample (typically in cm).
c is the concentration of the compound in solution, expressed in mol L-1
Dr.Samer HOUSHEH

5.  Molecules have quantized energy levels.
 Bonding orbitals are lower in energy than anti-
bonding orbitals.
 Non-bonding orbitals contains lone pair of
electrons.
 As light absorbs electrons „jumps“ from bonding
or non-bonding orbital to the anti-bonding
orbitals.
Dr.Samer HOUSHEH

6. The Important Transitions are:
 from pi bonding orbitals to pi anti-bonding
orbitals.
 from non-bonding orbitals to pi anti-bonding
orbitals.
 from non-bonding orbitals to sigma anti-
bonding orbitals.
 Groups in a molecule which absorb light are
known as chromophores.
Dr.Samer HOUSHEH

7. s* (anti-bonding)
p* (anti-bonding)
Four types of transitions
ss*
n (non-bonding) pp*
ns*
p (bonding)
np*
s (bonding)
s  s* transition in vacuum UV
n  s* saturated compounds with non-bonding electrons
n ~ 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
n ~ 200 - 700 nm
n  p* : e ~ 10-100
p  p*: e ~ 1000 – 10,000
Dr.Samer HOUSHEH

8. Still rather high in energy.  between 150 and 250 nm.
Not many molecules with ns* transitions in UV/vis region
max emax
H2O 167 1480
CH3OH 184 150
CH3Cl 173 200
CH3I 258 365
(CH3)2S 229 140
(CH3)2O 184 2520
CH3NH2 215 600
(CH3)3N 227 900
Dr.Samer HOUSHEH

9. Most UV/vis spectra involve these transitions. pp* are
generally more intense than np*.
max emax type
C6H13CH=CH2 177 13000 pp*
C5H11CC–CH3 178 10000 pp*
O
CH3CCH3 186 1000 ns*
O
CH3COH 204 41 np*
CH3NO2 280 22 np*
CH3N=NCH3 339 5 np*
Dr.Samer HOUSHEH

10. Absorption Characteristics of Some Common Chromophores
Chromophore Example Solvent max (nm) emax Type of
transition
Alkene C6H13HC CH2 n-Heptane 177 13,000 pp*
Alkyne n-Heptane 178 10,000 pp*
C5H11C C CH3 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*
CH3CH np*
Carboxyl O Ethanol 204 41 np*
CH3COH
Amido O Water 214 60 np*
CH3CNH2
Azo H3CN NCH3 Ethanol 339 5 np*
Nitro CH3NO2 Isooctane 280 22 np*
Nitroso C4H9NO Ethyl ether 300 100 _
665 20 np*
Nitrate C2H5ONO2 Dioxane 270 12 np*
Dr.Samer HOUSHEH

11.  Has four π molecular orbitals
 Bonding orbitals are occupied
 Anti-bonding orbitals are unoccupied
 The interaction of the two double bonds with each other to
produce a delocalized system of pi electrons over all four
atoms is known as conjugation.
Dr.Samer HOUSHEH

12. Dr.Samer HOUSHEH

13.  Chromophore: A covalently unsaturated group
responsible for electronic absorption. or Any group of
atoms that absorbs light whether or not a color is
thereby produced. e.g. C=C, C=O, NO2 etc.
 A compound containing Chromophore is called
chromogen.
 There are two types of Chromophore:
 Independent Chromophore: single Chromophore is sufficient
to import color to the compound e.g. Azo group
 Dependent Chromophore: When more than one
Chromophore is required to produce color. e.g. acetone
having one ketone group is colorless where as diacetyl
having two ketone group is yellow.
Dr.Samer HOUSHEH

14.  Auxochrome: A saturated group with non-bonding electron when
attached to Chromophore alters both wavelengths as well as
intensity of absorption. e.g. OH, NH2, NHR etc.
 Bathochromic group: The group which deepens the color of
Chromophore is called bathochromic group. e.g. Primary,
secondary and tertiary amino groups.
 Terminology: Auxochrome
 Bathochromic shift: (Red shift) shift of lambda max (λmax)to longer
side or less energy is called bathochromic shift or read shift. This
is due to substitution or solvent effect.
 Hypsochromic shift:(Blue shift)shift of lambda max (λmax)to shorter
side and higher energy is called hypsochromic or blue shift. e.g
solvent effect.
 Hyperchromic effect: an increase in absorption intensity
 Hypochromic effect: a decrease in absorption intensity
Dr.Samer HOUSHEH

15. Blue Shift Red Shift
(Hypsochromic) (Bathochromic)
Peaks shift to Peaks shift to longer
shorter wavelength. wavelength.
Dr.Samer HOUSHEH

16. For Compounds with Multiple Chromophores:
If isolated (more than 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)
1,3 butadiene: max= 217 nm ; emax= 21,000
1,3,5-hexatriene max= 258 nm ; emax= 35,000
Dr.Samer HOUSHEH

17. For Compounds with Multiple Chromophores
Dr.Samer HOUSHEH

18.  Different compounds may have very different absorption
maxima and absorbances.
 Intensely absorbing compounds must be examined in dilute
solution, so that significant light energy is received by the
detector, and this requires the use of completely
transparent(non-absorbing) solvents.
 Typical solvents are water, ethanol, hexane and cyclohexane.
 Solvents having double or triple bonds, or heavy atoms (e.g. S,
Br & I) are generally avoided.
 Because the absorbance of a sample will be proportional to its
molar concentration in the sample cuvette, a corrected
absorption value known as the molar absorptivity is used when
comparing the spectra of different compounds.
Dr.Samer HOUSHEH

19. Solvents can induce significant changes in the intensity of
peaks.
Hyperchromic – Increase in absorption intensity.
Hypochromic – Decrease in absorption intensity.
Absorption characteristics of 2-methylpyridine
Solvent max emax
Hexane 260 2000
Chloroform 263 4500
Ethanol 260 4000
Water 260 4000
Ethanol - HCl (1:1) 262 5200
Dr.Samer HOUSHEH

20.  Increasing pH shifts equilibrium to right
 More non-bonding electrons in phenoxide ion
 higher extinction coefficient
 greater delocalization  bathochromic shift
(,e)=(270,1450) (287,2600)
OH + H2O H3O+ + O
Phenol Phenoxide ion
Dr.Samer HOUSHEH

21.  Decreasing pH shifts equilibrium to right
 No non-bonding electrons in anilinium ion
 lower extinction coefficient
 less delocalization  hypsochromic shift
(,e)=(280,1430) (254,169)
NH3 + H2O OH- + NH4+
Aniline Aniliniumion
Dr.Samer HOUSHEH

22. Dr.Samer HOUSHEH

23.  Scanning of UV Spectrum in different pH for
some drugs
 Paracetamol (Acetaminophen)
 Caffeine
Dr.Samer HOUSHEH

24.  100 mg of Paracetamol was weighed and
transferred to a 100 ml volumetric flask,
sonicated with MeOH (or EtOH) made up to volume
with same solvent. From this solution, appropriate
volume of 25 ml was transferred to 100 ml
volumetric flask and volume was adjusted up to the
mark with same solvent.
Dr.Samer HOUSHEH

25.  100 mg of Paracetamol was weighed and transferred to a
100 ml volumetric flask, sonicated with MeOH (or EtOH)
made up to volume with same solvent. From this solution,
appropriate volume of 25 ml was transferred to 100 ml
volumetric flask and volume was adjusted up to the mark
with NaOH 0.1N.
 100 mg of Paracetamol was weighed and transferred to a
100 ml volumetric flask, sonicated with MeOH (or EtOH)
made up to volume with same solvent. From this solution,
appropriate volume of 25 ml was transferred to 100 ml
volumetric flask and volume was adjusted up to the mark
with HCl 0.1N.
Dr.Samer HOUSHEH

26.  Make a scan for the three previous solutions in the
UV spectroscopy and determine λmax of the three
solutions.
 Compare the three spectra and record your notes.
 Explain the presence or differences.
Dr.Samer HOUSHEH

27.  100 mg of Caffeine was weighed and
transferred to a 100 ml volumetric
flask, sonicated with hot water (or EtOH)
made up to volume with same solvent. From
this solution, appropriate volume of 25 ml
was transferred to 100 ml volumetric flask
and volume was adjusted up to the mark with
same solvent.
Dr.Samer HOUSHEH

28.  100 mg of Aspirin was weighed and transferred to a 100
ml volumetric flask, sonicated with hot water (or EtOH)
made up to volume with same solvent. From this solution,
appropriate volume of 25 ml was transferred to 100 ml
volumetric flask and volume was adjusted up to the mark
with NaOH 0.1N.
 100 mg of Aspirin was weighed and transferred to a 100
ml volumetric flask, sonicated with hot water (or EtOH)
made up to volume with same solvent. From this solution,
appropriate volume of 25 ml was transferred to 100 ml
volumetric flask and volume was adjusted up to the mark
with HCl 0.1N.
Dr.Samer HOUSHEH

29.  Make a scan for the three previous solutions in the
UV spectroscopy and determine λmax of the three
solutions.
 Compare the three spectra and record your notes.
 Explain the presence or differences.
Dr.Samer HOUSHEH

30. Quartz Cell
Thanks for Paying Attention
Dr.Samer HOUSHEH