1. Advanced Pharmaceutical Analysis UV spectroscopy
Lecture 1 6
Advanced Pharmaceutical Analysis UV spectroscopy
Dr. Baraa Ramzi

2. Pharmaceutical Analysis
Module Name
Pharmaceutical Analysis
Spectroscopy:
UV, IR, NMR
Separation:
HPLC, GC

3. Text Books
Douglas A. Skoog (2014). Fundamentals of Analytical Chemistry. Belmont: BROOKS.
Pavia (2009). Introduction to Spectroscopy. 4th ed. Belmont: BROOKS.

4. What Is spectroscopy
The interactions of radiation and matter are the subject of the science called spectroscopy.
Spectroscopic analytical methods are based on measuring the amount of radiation produced or absorbed by molecules or atoms.

5. The Spectrum

7. UV-VIS
X-Ray

8. Electronic Excitation
When radiation passes a material, a portion of the radiation may be absorbed.
As a result of energy absorption, atoms or molecules pass from a state of low energy (ground state) to a state of higher energy (the excited state).

9. Electronic Excitation
As a molecule absorbs energy, an electron is promoted from an occupied orbital to an unoccupied orbital of greater potential energy. Generally, the most probable transition is from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO).

11. Electronic Excitation
The lowest-energy occupied molecular orbitals are the σ orbitals, which correspond to σ bonds. The ∏ orbitals lie at somewhat higher energy levels, and orbitals that hold unshared pairs, the nonbonding (n) orbitals, lie at even higher energies.

14. Azo N=N

15. Instrumentation
The typical UV-VIS spectrophotometer consists of a light source, a monochromator, sample holder and a detector.
light source : deuterium lamp for UV and tungsten lamp for visible.
Monochromator :diffraction grating.
Sample holder : glass, plastic or quartz .
Detector : photodiodes.
The sample cell must be constructed of a material that is transparent to the electromagnetic radiation
deuterium contains one proton and one neutron.

16. Instrumentation

17. Instrumentation

18. Instrumentation
For spectra in the visible range of the spectrum, cells (sample holder AKA Cuvette) composed of glass or plastic are generally suitable.
For UV cells made of quartz must be used since quartz does not absorb radiation in this region.

19. A set of Cuvettes

20. UV instrument showing a cuvette

21. Finger prints and being wet can be problematic

22. Ultraviolet/Visible Photometers and Spectrophotometers
Spectrophotometers is a spectroscopic instrument that uses a monochromator.
Photometers use a filter for wavelength selection in conjunction with a suitable radiation source.
Spectrophotometers offer the considerable advantage that the wavelength used
can be varied continuously.
Photometers
have the advantages of simplicity, ruggedness, and low cost.

23. Single Beam vs Double-Beam

24. Qualitative vs Quantitative Analysis
Qualitative analysis : aims to determine the chemical and functional groups and eventually the structure of an analyte.
Quantitative analysis : aims to determine the amount (concentration) of the studied analyte.

25. Limited Qualitative Use
Most organic molecules and functional groups are transparent in the portions of the electromagnetic spectrum that we call the ultraviolet (UV) and visible (VIS) regions.
Consequently, absorption spectroscopy is of limited utility in this range of wavelengths.

26. Chromophore Vs Auxochrome
Chromophore : is a chemical group that absorbs light at a specific frequency giving color to a molecule.
Auxochrome : is a group of atoms attached to the chromophore which modifies the ability of the chromophore to absorb light, altering the wavelength or intensity of the absorption.

29. Lambda max ƛ
Lambda max ƛ : The wavelength at which maximum absorption occurs.

30. Lambda max ƛ

31. Lambda max ƛ

32. Auxochromes have four kinds of effects on the absorption :
1. Bathochromic shift (red shift): shift to lower energy or longer wavelength. 2. Hypsochromic shift (blue shift): shift to higher energy or shorter wavelength. 3. Hyperchromic effect : increase in intensity. 4. Hypochromic effect : decrease in intensity.
Drow it

33. Calculating Lambda max ƛ
Traditionally, analysts calculated Lambda max theoretically using prior studies and experiments to create co-relation tables with all possible Auxochromes for each Chromophore . (the next table). However, it is not used anymore as it has limited applications with minimum value.

35. Example

37. Example

40. Beer-Lambert law
It describes how the amount of light attenuation depends on the concentration of the absorbing molecules and the path length over which absorption occurs.

41. Light Attenuation

42. According to Beer’s law, absorbance is directly proportional to the concentration of the absorbing species, c, and to the path length, b, of the absorbing medium and a is a proportionality constant called the absorptivity.

43. When the concentration is in moles per liter and b in cm, the proportionality constant is called the molar absorptivity and is given the symbol Ɛ.

44. For an analyte solution of a given concentration, the longer the path length of the medium through which the light passes, the more absorption and the greater the attenuation.
The higher the concentration of absorbers, the stronger the attenuation (absorbance).

45. Typical absorption spectra of potassium permanganate at five
different concentrations.

46. Light Attenuation

47. The Transmittance T of the solution is the fraction of incident radiation transmitted by the solution.

50. What happens to the incident light
Reflection and scattering
losses with a solution contained in a
typical glass cell. Losses by reflection
can occur at all the boundaries that
separate the different materials. In this
example, the light passes through the
following boundaries, called interfaces:
air-glass, glass-solution, solution-glass,
and glass-air.

51. Example 1
Guanosine has a maximum absorbance at 275 nm. Ɛ at 275= 8400M−1cm−1 and the path length is 1 cm. Using a spectrophotometer, you find the that A at 275= ,  What is the concentration of guanosine?

52. Example 1

53. Example 2

54. Example 2

55. Example 2

56. Applying Beer’s Law to Mixtures
Beer’s law also applies to solutions containing more than one kind of absorbing substance.
absorbance for a multicomponent system at a single wavelength is the sum of the individual absorbances.

57. UV Spectra of substance X
UV Spectra of substance Y
Where A is absorbance and C is concentration.

58. UV Spectra of substances X and Y

59. Example 3
The concentrations of Fe3+ and Cu2+ in a mixture must be determined. Fe3+ (λmax = 550 nm) and Cu2+ (λmax = 396 nm). The molar absorptivities (M–1 cm–1) are as follows :
Ɛ 550 nm
Ɛ 396 nm
Fe3+
9970 84
Cu2+ 34
856

60. Example 3
When a sample containing Fe3+ and Cu2+ is analyzed in a cell with a path length of 1.00 cm, the absorbance at 550 nm is and the absorbance at 396 nm is What are the molar concentrations of Fe3+ and Cu2+ in the sample?

61. https://chem. libretexts

62. Limits of Beer’s Law
Deviations from the direct proportionality between absorbance and concentration can occur.
Some of these deviations, called real deviations, are fundamental and represent real limitations to the law.
Others are a result of methods (instrumental deviations) or from chemical changes (chemical deviations).
chemical deviations :deviations from Beer’s law appear when the absorbing
species undergoes association, dissociation, or reaction with the solvent to give
products that absorb differently from the analyte.

63. Real Limitations to Beer’s Law
Beer’s law describes the absorption behavior only of dilute solutions.
At concentrations exceeding about 0.01 M deviations from the linear relationship between absorbance and concentration occurs.
the average
distances between ions or molecules of the absorbing species are diminished to
the point where each particle affects the charge distribution and thus the extent
of absorption of its neighbors.

64. Deviation From linearity

65. Instrumental Deviations: Stray Light
Stray light is defined as radiation from the instrument that is outside the selected wavelength.
It is the result of scattering and reflection off the surfaces of gratings, lenses or mirrors, filters, and windows.

66. Stray Light Reflection and scattering
losses with a solution contained in a
typical glass cell. Losses by reflection
can occur at all the boundaries that
separate the different materials. In this
example, the light passes through the
following boundaries, called interfaces:
air-glass, glass-solution, solution-glass,
and glass-air.

67. Various Levels of Stray Light

68. Instrumental Deviations: Mismatched Cells
If the cells holding the analyte and blank solutions are not of equal path length and equivalent in optical characteristics , deviation would occur.
No calib , plastic cuvette , no match
Another way to avoid the mismatched-cell problem
with single beam instruments

69. Matched cuvettes