2. UV-VISIBLE SPECTROPHOTOMETRY
Ashraf M. Mahmoud, Associate professor

3. Contents Light and radiation Electromagnetic spectrum Light as energy
Interaction of photons with matter
Absorption spectrum, characteristics and shifts
Types of electronic transitions
Chromophores, and auxochromes
Factors affecting absorption spectrum (pH, solvent)
Instrumentation (basic component of spectrophotometer)

4. Light and Radiation E = h u E = h c/
Light can be described as a wave. This wave has an electric component and a magnetic component which are perpendicular to each other
Electric Field
Magnetic Field
Wavelength ()
E = Energy in joules (J)
u = Frequency (Hz)
h = Plank’s constant (6.63x erg. s)
 = Wavelength (m) = C/ u
c = Speed of light (3 x 108 m/s in vacuum) =  u
E = h u
E = h c/

5. Electromagnetic Spectrum
-ray
x-ray
Visible IR w Rw
10-9
10-7
10-5
10-3
10-1
102
Wavelength (, cm )
Frequency ( , Hz )
108
1012
1014
1016
1018
1020 UV
Wavelength (nm)
400
500
600
700
800

6. Interaction of Photons with Matter
A molecule may absorb light energy in three ways:
[1] Increasing the rotation of molecule around its axis (rotational)
when molecule absorb F-IR irradiation.
[2] Increasing the vibration of constituent nuclei (vibrational)
when molecule absorb IR irradiation.
[3] Raising an electron to a higher energy level (transitional energy)
when molecule absorb visible and UV light.
E total = E transitional + E vibrational + E rotational

7. Interaction of Photons with Matter
When a molecule absorbs photons in the UV-VIS region, the absorption of energy results in displacing an outer electron (valence electron) in the molecule. The molecule is said to undergo transition from the ground state of energy level to an excited state of energy level.

8. Interaction of Photons with Matter
An excited molecule losses energy and returns to the ground state.
The energy is released in the form of:
Heat: electrons return directly to ground state.
Light (fluorescence): electrons return via a second excited state.
Molecular collisions.

9. Absorption Spectrum
Is a plot of absorption intensity versus the wavelength of the absorbed light
Line spectrum (for atoms) and band spectrum (for molecules)
Line Spectra for some elements
Wavelength, nm
400
450
600
800
500 K Na

10. Absorption Spectrum Absorption band spectrum for some molecules
max
Amax
Absorption spectrum: it is characteristic to substance, and the wavelength at which the maximum absorption is recorded and used to trace the substance strength to enhance the sensitivity.

11. Types of Electronic Transitions
 Anti-bonding
  Anti-bonding
 -  
 - 
n -  
 -  
 - 
n - 
n Non-bonding
 Bonding
 Bonding
200
300
Wavelength, nm
150
250

12. Types of Electronic Transitions
  Anti-bonding
 Anti-bonding
n Non-bonding
 Bonding
 Bonding
150
200
250
300
Wavelength, nm
Solvent
, nm
Water
190
Benzene
280
Ether
205
Acetone
331
Ethanol
207
Methanol
210
Cut-off wavelengths of some common solvents

13. Some Important Terms Chromophores: (Chrom = color, phore = carrier)
They are functional groups which confer color on substances capable of absorbing UV and/or visible light. They are conjugated unsaturated bonds.
Examples: C = C, - C = O, - N = N, and – C ≡ N ( electrons).
Auxochromes: They are functional groups which can not confer colors on substances but have the ability to increase the coloring power of chromophores, they does not absorb radiations longer than 200 nm, but when attached to a given chromophore, cause a shift to a longer wavelength with increase in absorption intensity.
Examples: - OH, - NH2.
Bathochromic shift ( red shift ): it is the shift of max to a longer wavelength due to substitution or solvent effects.
Hypsochromic shift ( blue shift ): it is the shift of max to a shorter wavelength.
Hyperchromic effect: enhancement of molecule absorptivity (or absorption intensity).
Hypochromic effect: decrease of molecule absorptivity (or absorption intensity).

14. Some Important Terms Absorbance Wavelength, nm Hyperchromic
APEX
Hypsochromic
Bathochromic
Hyporchromic

15. Absorption Characteristics of Chromophores
[1] Ethylenic chromophores.
[2] Carbon-heteroatom chromophores.
[3] Conjugated chromophores.
[4] Aromatic system:
(a) Benzene ring.
(b) Monosubstituted benzenes.

16. Absorption Characteristics of Chromophores
[1] Ethylenic chromophores:
Their bands are difficult to be observed in near UV region, so they are not useful analytically. Substitution and certain structural features may cause red shift rendering the band observable:
Alkyl substitution: due to hyper-conjugation and stabilization of excited state.
Exocyclic nature: due to relaxation of strain upon excitation.
(c) Attachment to auxochromes: due to extension of conjugation.

17. Absorption Characteristics of Chromophores
[2] Carbon-heteratom chromophores:
Such as: - C = O, - C = N, - C = S, ………etc.
They exhibit some common characteristics:
n -  transition: undergoes a blue shift on increasing solvent polarity
(b) Alkyl substitution: causes red shift
(c) Attachment of hetero atom or group to –C=O: attachment of Cl, -NH2, or –OH causes blue shift

18. Absorption Characteristics of Chromophores
[3] Conjugated chromophores:
Separated ethylenic chromophores (by two or more single bonds) have little additive effect because there is a little or no electronic interaction between separated double bonds.
Compound
Position of
band (), nm
Intensity (),
L cm-1 mol-1
CH2 = CH - (CH)2 - CH3
180
10,000
CH2 = CH - CH2 - CH2 - CH = CH2
20,000
Absorption characteristics of separated ethylenic chromophores
CH3 - CH = CH - CH = CH – CH3
227
25,000

19. Absorption Characteristics of Chromophores
Conjugated systems: when double bonds are separated by only one single bond. This results in red shift by nm in the max.
Position of
band (), nm
Intensity (),
L cm-1 mol-1
Acetic acid
197 60
Crotonic acid
208
12,500
Effect of conjugation on electronic absorption spectra
Sorbic acid
261
25,600
2,4,6-Octatrienoic acid
303
36,500
2,4,6,8-Decatetraenoic acid
332
50,000
CH3 – (CH = CH) n - COOH n 1 2 3 4

20. C C Absorption Characteristics of Chromophores (X)
Ethylene: CH2 = CH2
max = 190 nm 
(X) C C 
Energy Level diagram of ethylene

21. Absorption Characteristics of Chromophores
butadiene: CH2 = CH-CH=CH2
max = 215 nm C  
(X)
(Y)
LEMO
HOMO 1 2
1 
2 
Energy Level diagram of ethylene and butadiene

22. Absorption Characteristics of Chromophores
[4] Aromatic systems:
Wavelength, nm
Absorbance
(a) Benzene ring:
184
Ethylenic band (E-band):  - 
204
Not useful
Useful
254
Benzenoid structure (B-band)
, nm
Origin
Usefulness

23. Absorption Characteristics of Chromophores
(b) Monosubstituted benzenes: R
Substitution with electron-donating or electron with drawing groups causes red shift for both E- and B-bands with hyperchromic effect and loss for most of the fine structure of B-band:
-----
254 OH
270
215
1,450
NH2
280
1,430 R
, nm
, L cm-1 mol-1 Cl
265
240
CH3
261
300 SH
269
700
Effect of auxochromes on absorption spectrum of benzene

24. Absorption Characteristics of Chromophores
Calculation of max of conjugated polyenes by Kuhn and Häusser rule:
n = number of conjugated
double bonds
max (nm) = 134  n
max (nm) = 134  n
max (nm) = 134  =  331 nm
Calculation of approximate number of double bonds from max :
n = [ max ) / 134 ]2
If max of 433 nm:
n = [ ) / 134 ]2 = 9

25. Factors Affecting Absorption Spectrum
Effect of pH on absorption spectra
Effect of solvent on absorption spectra
Affect what ?
Maximum wavelength ( max )
Intensity (  )

26. Factors Affecting Absorption Spectrum
Effect of pH on absorption spectra
Phenol
270 nm
1,450
Phenolate
290 nm
2,600 • Na +
max
intensity
The UV spectrum of phenol in acidic medium is completely different from its spectrum in alkaline medium (using same concentration). The spectrum in alkaline medium exhibits bathochromic shift with hyperchromic effect. The red shift is due to the participation of the pair electrons in resonance with the  -electrons of the benzene ring, thus increasing the delocalization of the -electrons.

27. Factors Affecting Absorption Spectrum
Effect of pH on absorption spectra
HCl • + Cl
Aniline
280 nm
1,430
Anilinium
254 nm
160
max 
The UV spectrum of aniline in acid medium shows:
hypsochromic shift with hypochromic effect.
This blue shift is due to the protonation of the amino group, hence the pair of electrons is no longer available and the spectrum in this case is similar to that of benzene (thus called benzenoid spectrum).

28. Factors Affecting Absorption Spectrum
Effect of solvent on absorption spectra
In general: less polar solvents (e.g. hydrocarbons) interact less strongly than the polar solvents (e.g. water and alcohols) do.
 -  * transitions;
Red shift occurs on increasing solvent polarity due to stabilization of excited state by dipole-dipole solvent interaction. . 
1 *
2 *
n-  transitions:
blue shift occurs with increasing solvent polarity due to stabilization of the ground state by hydrogen bonding:  1 2

29. Interaction of Photons with Matter
When a monochromatic light passes through a cell containing an absorbing substance, then the effects occurring will include:
Reflection, refraction, scattering, absorption, and transmission.
Incident
intensity (I0)
Transmitted
intensity (It)
Scattering from solution (Is)
Reflection at interfaces (Ir)
Refraction from solution (If)
Light source
Io (incidence light) = Ia + Ir + It + If + Is

30. Interaction of Photons with Matter
For Clear Solution
scattering (Is) = 0
light reflection (Ir)
refraction (If),
absorption (Ia), and
transmitting of light (It).
Cancelled by Blank
Transmitted
intensity (It)
Incident
intensity (I0)
Light source
Io (incidence light) = Ia + It + Is + If + Ir

31. Laws of Light Absorption
Lambert’s Law:
It relates absorption capacity to the thickness of an absorbing solute (path length of light).
Io Incident light
It Transmitted light
K Proportionality constant
b Light path length
Log Io / It = K b
Beer’s Law:
It relates absorption capacity to the concentration of an absorbing solute.
Io Incident light
It Transmitted light
K Proportionality constant C Concentration
Log Io / It = K C

32. Laws of Light Absorption
Beer’s - Lambert’s Law:
It relates absorption capacity to the thickness of an absorbing solute (path length of light) and the concentration. It is a combination between Beer’s law and lambert’s law.
Io Incident light
It Transmitted light
a Absorpitivity
b Light path length (in cm)
C Concentration (in g/L)
Log Io / It = a b C
Log Io / It = A (absorbance)
A = a b C
Usually b = 1 cm
A = a C

33. Laws of Light Absorption
Beer’s - Lambert’s Law:
A = a b C
Concentration
Absorbance

34. Laws of Light Absorption
Beer’s - Lambert’s Law:
A = a b C
Expressions of a
a absorptivity,
if concentration (c) expressed as gram / Liter.
A = a b c
ε (Epsilon), Molar absorptivity,
if concentration (c) expressed as molar solution.
A = ε b c
A one percent one centimeter
if c is expressed in g/100 mL
A1%
1cm
b c
A =
ε at max it is called εmax.
= ε x 10 / molecular weight
A1%
1cm

35. Laws of Light Absorption
Deviations from Beer’s - Lambert’s Law:
Concentration
Absorbance
[1] Real deviation:
In high concentration due to crowding, molecules interaction & association.
[2] Instrumental deviation:
Irregular deviation due to unmatched cells, unclean handling, and unclean optics.
(b) Regular deviation due to slit width control, stray light is indefinite wavelength, also any light reaches the detector without passes through the sample.
(c) Other errors as non linear response of photo cells, radio and TV interfaces, and unstabilized power supply.
[3] Chemical deviation:
pH effect, solvent interaction, temperature effects, dipole interactions due to high concentrations, and time factor affecting oxidation, reduction, or hydrolysis.

36. UV-VIS Instrumentation
Methodology and Instrumentation
UV-VIS Instrumentation
Colorimetry
UV Spectrophotometry nm nm R e d V i o l t B u G r n Y w O a g
Complimentary colors:
Wavelength (nm)
400
500
600
620
450
570
Absorbed colors

37. Quantitative Colorimetry
Colorimetry: It is a technique used for measuring the visible radiation.
A compound can be analyzed colorimetrically if:
[1] The substance is colored (e.g. CuSO4, KMnO4, methylene blue,….…etc.)
[2] The substance give colored product when treated with special reagent (e.g. Fe 2+ when treated with 1,10-phenanthroline in presence of phosphate buffer).
[3] The substance can be converted into some derivative that can react with special reagent to give a colored product.

38. Standard Series Method
The color intensity is observed by eye.
Depend on matching the colored standard series with the unknown concentration.
Example: Assay of Fe(III) as thiocyanate complex using standard series method.
Exact concentration is not required; Limit test of iron by USP/BP.
Standard solutions
Unknown

39. Photoelectric Colorimeters & Spectrophotometers
- Used for electric measuring the light absorbed by the sample.
- Monochromatic light is used instead of polychromatic light frequently used in visual methods.
Components of Instruments:
1. Light source
2. Monochromator
3. Sample cell
4. Detector
5. Recorder (meter)
Types of Instruments:
Single-beam spectrophotometers.
Double-beam spectrophotometers.

40. Components of Spectrophotometers
Light source:
- UV measurement: hydrogen or deuterium discharge lamp (190 – 375 nm)
- Visible measurement: Tungsten filament or Tungestin halogen lamp
(350 – 1000 nm)
Tungsten Lamp
Monochromator:
Function: To select light beam of certain wavelength.
- Filter
- Prisms
- Grating

41. Components of Spectrophotometers
Monochromators:
Filters: function via selective absorption of unwanted wavelength and transmitting the complementary color. It consists of colored glass, or dye suspended in gelatin and sandwiched between two glass plates.
Prisms: function via refraction of light.

42. Components of Spectrophotometers
Monochromators:
Gratings: Consist of large number of parallel ruled very close to each other on a highly polished surface, e.g. aluminium, or aluminized glass (600 groove/mm). Each ruled groove functions as a scattering center for light falling on its edge and through diffraction and interference the grating disperses the light beam into almost single wavelength.
Incident
light
Diffracted

43. Components of Spectrophotometers
Sample Cell (Quvette) :
Transparent
Quartz for UV measurements
Glass or Quartz cell for VIS measurements
Pathlength: usually 0.5, 1 or  1 cm
Sample cuvette

44. Components of Spectrophotometers
Detectors :
Receive light emerged from the sample, which excite electrons and generate an electric current that proportional to the received light intensity.
Photomultiplier tube
Anode
(iron)
Electrons
Cathode
(selenium)
Light
beam
Photocell

45. Types of Spectrophotometers
Single-Beam Spectrophotometers:
Sample
cuvette
Detector
Light source
Monochromator
Amplifier
Meter

46. Types of Spectrophotometers
Double-Beam Spectrophotometers:
Blank cuvette
Sample
cuvette
Detector 1
Detector 2
Monochromator
Amplifier
Beam splitter
Light source
Meter

47. APPLICATIONS OF SPECTROPHOTOMETRY
Qualitative Analysis by Spectrophotometry
1- Identification with absorption spectrum :
1-It is used for the identification of new drugs and natural products.
2- UV-Visible spectrum give useful information about substance via examination of its max and emax, which could be correlated with the structural features (See the following table).
3- The spectrum is a physical constant, which along with melting & boiling points, refractive index and other properties may be used for characterization of compounds

48. Qualitative Analysis by Spectrophotometry
4- An absorption band at 254 nm with characteristic vibrational fine structures may be an evidence for existence of aromatic structure.
5- Three characteristic bands at 278, 361 &550 nm with absorbance ratio of 2:3:1 is very characteristic for cyanocobalamin.

49. Qualitative Analysis by Spectrophotometry
Identification of morphine:
since morphine is a phenolic compound, the observation of bathochromic shift with hyperchromic effect in KOH is consistent with, but not definite proof of, the presence of morphine in the sample.
Since other phenolic compounds show similar behavior, this test is a definite proof of the absence of morphine in the sample.
For definite proof for the presence of morphine, better method (e.g. infrared spectroscopy) should be used.

50. Qualitative Analysis by Spectrophotometry
Identification of phenolphthalein:
Benzenoid (colorless, pH 7)
Quinonoid (pink, pH 9)
max in UV region
max of nm

51. Qualitative Analysis by Spectrophotometry
Identification of barbiturates (toxicological analysis):
Up to pH = 8
pH = 10
pH = 12
Lactam form
Lactin forms

52. Qualitative Analysis by Spectrophotometry
pH = up to 8
pH = 10
pH = 12
230
250
220
240
260
270
210
Wavelength, nm
Absorption spectra of barbiturates at different pH values

53. Quantitative Analysis by Spectrophotometry
General Procedure:
1. Determination of proper max :
450
550
650
350
Wavelength, nm

54. Quantitative Analysis by Spectrophotometry
2. Generating the calibration curve at max :
Unknown
Standard solutions
Concentration
Absorbance
Linear equation: A = a + b C
Unknown conc. Is determined by:
Calibration curve: graphically

55. Quantitative Analysis by Spectrophotometry
1. Analysis of inorganic compounds:
Determination of copper via ammine complex.
Cu NH [ Cu(NH3)4 ]2+ ( Blue color)
Determination of ferric via thiocyanate complex.
Fe SCN [ Fe(SCN)]2+ ( Blood red color)
+ Fe2+ Fe 3
Determination of ferrous via 1,10-phenanthroline complex.
1,10-phenanthroline
Dense red color 2+

56. Quantitative Analysis by Spectrophotometry
1. Analysis of inorganic compounds:
Determination of ferrous via 2,2’-bipyridyl complex complex.
Red color 3 Fe 2+
+ Fe2+
2,2’-Bipyridyl

57. Quantitative Analysis by Spectrophotometry
2. Analysis of organic compounds:
- Analysis of aromatic amines by diazotization and coupling : N H 2
Amine
NaNO2
HCl C l - + N H - ( C 2 ) N = H - ( C 2 )
Red color, max of nm

58. Quantitative Analysis by Spectrophotometry
2. Analysis of organic compounds:
- Analysis of carbonyl compounds by conversion into phenyl hydrazones via reaction with 2,4-dinitrphenylhydrazine:
Carbonyl compound
2,4-dinitrophenyl hydrazine
phenyl hydrazone derivative
Orange color, max of nm

59. Quantitative Analysis by Spectrophotometry
- Analysis of phenols via coupling with diazotized primary aromatic amines: + C l -
Diazotized
amine
Orange-red color
Phenolic
compound

60. Quantitative Analysis by Spectrophotometry
3. Determination of dissociation constant (pKa) of an indicator:
Methyl red
OH- H+
Acidic form (HMR ), pH = 4
red ( max = 520 nm )
Basic form ( MR- ), pH = 6
yellow ( max = 430 nm )
pH = pKa + log [ MR- ] / [ HMR ]

61. Quantitative Analysis by Spectrophotometry
3. Determination of dissociation constant (pKa) of an indicator:
MR-
HMR
450
550
400
500
600
650
Wavelength, nm
Absorbance
at 430 nm
at 520 nm 5 4 6 pH

62. Thank You
Ashraf M Mahmoud