100 mg of Paracetamol was weighed and transferred to a ... 100 mg of Caffeine was weighed and ... 100 mg of Aspirin was weighed and transferred to a 100.
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
Electronic Excitation by UV/Vis Spectroscopy X-ray: core electron excitation
UV: valance electronic excitation
IR: molecular vibrations
Radio waves: Nuclear spin states (in a magnetic field)
Dr.Samer HOUSHEH
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
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
Molecules have quantized energy levels.
Bonding orbitals are lower in energy than antibonding 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
The Important Transitions are: from pi bonding orbitals to pi anti-bonding orbitals.
from non-bonding orbitals to pi anti-bonding orbitals.
from
Groups in a molecule which absorb light are known as chromophores.
non-bonding bonding orbitals.
orbitals
to
sigma
Dr.Samer HOUSHEH
anti-
s* (anti-bonding) p* (anti-bonding)
Four types of transitions ss*
n (non-bonding)
pp* ns*
p (bonding)
np*
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
Still rather high in energy. between 150 and 250 nm. Not many molecules with ns* 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
Most UV/vis spectra involve these transitions. pp* are generally more intense than np*. max
emax
C6H13CH=CH2
177
13000
pp*
C5H11CC–CH3
178
10000
pp*
186
1000
ns*
CH3COH
204
41
np*
CH3NO2
280
22
np*
CH3N=NCH3
339
5
np*
type
O CH3CCH3 O
Dr.Samer HOUSHEH
Absorption Characteristics of Some Common Chromophores Chromophore Alkene
Example
Solvent
Type of transition
177
13,000
pp*
n-Heptane
178 196 225
10,000 2,000 160
pp* _ _
O
n-Hexane
186 280
1,000 16
CH3CCH3 O
ns* np*
n-Hexane
180 293
Large 12
Ethanol
204
41
np*
Water
214
60
np*
Ethanol
339
5
np*
C5H11C Carbonyl
Amido
emax
n-Heptane
C6H13HC
CH2
Alkyne
Carboxyl
max (nm)
C
CH3
CH3CH O CH3COH O
ns* np*
CH3CNH2 Azo
H3CN
NCH3
Nitro
CH3NO2
Isooctane
280
22
np*
Nitroso
C4H9NO
Ethyl ether
300 665
100 20
_
np*
270
12
np*
Nitrate
C2H5ONO2
Dioxane
Dr.Samer HOUSHEH
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
Dr.Samer HOUSHEH
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
There are two types of Chromophore:
chromogen.
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
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
Red Shift (Bathochromic) Peaks shift to longer wavelength.
Blue Shift (Hypsochromic) Peaks shift to shorter wavelength.
Dr.Samer HOUSHEH
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
For Compounds with Multiple Chromophores
Dr.Samer HOUSHEH
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
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
max
emax
Hexane
260
2000
Chloroform
263
4500
Ethanol
260
4000
Water
260
4000
Ethanol - HCl (1:1)
262
5200
Solvent
Dr.Samer HOUSHEH
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 Phenol
+ H2O
H3O+
+
O
Phenoxide ion
Dr.Samer HOUSHEH
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 Aniline
OH-
+
NH4+ Aniliniumion
Dr.Samer HOUSHEH
Dr.Samer HOUSHEH
Scanning of UV Spectrum in different pH for some drugs Paracetamol (Acetaminophen) Caffeine
Dr.Samer HOUSHEH
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
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
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
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
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
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
Quartz Cell
Thanks for Paying Attention
Dr.Samer HOUSHEH
