1. ULTRAVIOLET-VISIBLE SPECTROSCOPY AS AN ANLYTICAL TOOL
for
class B.Sc 5th semester By
Dr. S.B.MASHETTY
Department of Chemistry
KARNATAKA ARTS ,SCIENCE AND COMMERCE
Bidar

2. ULTRAVIOLET-VISIBLE SPECTROSCOPY

5. THE ELECTROMAGNETIC SPECTRUM
The wavelength (λ, the length of 1 cycle in meters) times the frequency (ν, the number of cycles per second) equals the speed of light (c, a constant that equals 3.0 x 108 meters/second). That is,
c = λν = 3.0 x 108 meters/second
If λ increases, then ν must decrease so that c remains constant.
If λ decreases, then ν must increase so that c remains constant.

6. THE ELECTROMAGNETIC SPECTRUM
Electromagnetic radiation is also a stream of energy packets called photons.
The energy of a single photon (E, in joules) equals Planck’s constant (h, x joule second) times the frequency (ν, the number of cycles per second). That is,
E = hν = hc/λ
If the frequency (ν) increases, the energy (E) .
If the wavelength (λ) decreases, the energy (E) .
increases
increases

7. THE ELECTROMAGNETIC SPECTRUM
The ultraviolet (UV) region of the electromagnetic spectrum includes all wavelengths from 10 nanometers (nm) to 380 nm. The vacuum-ultraviolet region goes from 10 nm to 200 nm because air absorbs strongly at these wavelengths so instruments must be operated under a vacuum in this region. The near-ultraviolet region goes from 200 nm to 380 nm.
The visible (Vis) region goes from 380 nm to 780 nm and can be seen by the human eye.
The infrared (IR) region goes from 0.78 micrometers (μm) or 780 nm to 300 μm. However, the near-infrared (0.8 μm to 2.5 μm) and the NaCl-infrared regions (2.5 μm to 16 μm) are the most commonly used by analytical chemists.

8. THE ELECTROMAGNETIC SPECTRUM
The ultraviolet (UV) region of the electromagnetic spectrum includes all wavelengths from 10 nanometers (nm) to 380 nm. The vacuum-ultraviolet region goes from 10 nm to 200 nm because air absorbs strongly at these wavelengths so instruments must be operated under a vacuum in this region. The near-ultraviolet region goes from 200 nm to 380 nm.
The visible (Vis) region goes from 380 nm to 780 nm and can be seen by the human eye.
The infrared (IR) region goes from 0.78 micrometers (μm) or 780 nm to 300 μm. However, the near-infrared (0.8 μm to 2.5 μm) and the NaCl-infrared regions (2.5 μm to 16 μm) are the most commonly used by analytical chemists.

9. THE ABSORPTION OF ELECTROMAGNETIC RADIATION BY MOLECULES
Humans see color when an object transmits or reflects visible light.
More specifically, an object may absorb specific wavelengths of electromagnetic radiation. The unabsorbed wavelengths from the visible region are transmitted and seen as color.
For example, leaves are green because the pigment chlorophyll absorbs violet, blue, and red light.
Why is my car blue?
It’s blue because it absorbs yellow.

10. THE ABSORPTION OF ELECTROMAGNETIC RADIATION BY MOLECULES
There are 3 ways that a molecule can absorb electromagnetic radiation. All 3 ways raise the molecule to a higher internal energy level. All these changes in energy are quantized; that is, they occur at discrete levels.
Rotational Transitions: The molecule rotates around various axes. Rotational transitions require the least amount of energy. Purely rotational transitions can occur in the far-infrared and microwave regions.
Vibrational Transitions: Atoms or groups of atoms within a molecule vibrate relative to each other. Vibrational transitions require an intermediate amount of energy and typically begin to occur in the mid-infrared and far-infrared regions. Therefore, as energy is increased (or wavelength is decreased) vibrational transitions occur in addition to rotational transitions.
Electronic Transitions: An electron within a molecule is typically promoted from its ground state to an excited state. Electronic transitions require the most amount of energy and typically begin to occur in the visible and ultraviolet regions. Therefore, as energy is increased (or wavelength is decreased) electronic transitions occur in addition to vibrational and rotational transitions.

11. THE ABSORPTION OF ELECTROMAGNETIC RADIATION BY MOLECULES

12. SCHEMATIC OF A SPECTROPHOTOMETER
The most common light source for the visible region spectrophotometry is a tungsten filament incandescent lamp. A tungsten lamp emits useful light from approximately 325 nm to 3,000 nm.
A monochromator uses a prism or a diffraction grating to separate polychromatic (many wavelengths) light into monochromatic (single wavelength) light.
A cell or cuvette is used to hold the sample during analysis.
The detector uses a phototube or a photomultiplier tube to convert light into an electrical signal that is sent to a recorder or computer.

13. Ultraviolet (UV) Spectroscopy – Use and Analysis
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When continuous wave radiation is passed through a prism a diffraction pattern is produced (called a spectrum) made up of all the wavelengths associated with the incident radiation.
When continuous wave radiation passes through a transparent material (solid or liquid) some of the radiation might be absorbed by that material.
Spectrum with ‘gaps’ in it
Spectrum
Transparent material that absorbs some radiation
Diffraction prism
Radiation source
If, having passed through the material, the beam is diffracted by passing through a prism it will produce a light spectrum that has gaps in it (caused by the absorption of radiation by the transparent material through which is passed).
The effect of absorption of radiation on the transparent material is to change is from a low energy state (called the ground state) to a higher energy state (called the excited state).
The difference between all the spectroscopic techniques is that they use different wavelength radiation that has different associated energy which can cause different modes of excitation in a molecule.
For instance, with infra red spectroscopy the low energy radiation simply causes bonds to bend and stretch when a molecule absorbs the radiation. With high energy UV radiation the absorption of energy causes transition of bonding electrons from a low energy orbital to a higher energy orbital.
The energy of the ‘missing’ parts of the spectrum corresponds exactly to the energy difference between the orbitals involved in the transition.

14. Ultraviolet (UV) Spectroscopy – Use and Analysis
The bonding orbitals with which you are familiar are the -bonding orbitals typified by simple alkanes. These are low energy (that is, stable). *
Increasing energy
Unoccupied Energy Levels
Next (in terms of increasing energy) are the -bonding orbitals present in all functional groups that contain double and triple bonds (e.g. carbonyl groups and alkenes). *
Higher energy still are the non-bonding orbitals present on atoms that have lone pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds).
All of the above 3 kinds of orbitals may be occupied in the ground state.
Occupied Energy Levels n
Two other sort of orbitals, called antibonding orbitals, can only be occupied by an electron in an excited state (having absorbed UV for instance). These are the * and * orbitals (the * denotes antibonding). Although you are not too familiar with the concept of an antibonding orbital just remember the following – whilst electron density in a bonding orbital is a stabilising influence it is a destabilising influence (bond weakening) in an antibonding orbital.
Antibonding orbitals are unoccupied in the ground state   UV
A transition of an electron from occupied to an unoccupied energy level can be caused by UV radiation. Not all transitions are allowed but the definition of which are and which are not are beyond the scope of this tutorial. For the time being be aware that commonly seen transitions are  to * which correctly implies that UV is useful with compounds containing double bonds.
A schematic of the transition of an electron from  to * is shown on the left.

15. Ultraviolet (UV) Spectroscopy – The Instrumentation
The instrumentation used to run a UV is shown below. It involves two lamps (one for visible light and one for UV light) and a series of mirrors and prisms as well as an appropriate detector. The spectrometer effectively varies the wavelength of the light directed through a sample from high wavelength (low energy) to low wavelength (high energy).
As it does so any chemical dissolved in a sample cell through which the light is passing may undergo electronic transitions from the ground state to the excited state when the incident radiation energy is exactly the same as the energy difference between these two states. A recorder is then used to record, on a suitable scale, the absorption of energy that occurs at each of the wavelengths through which the spectrometer scans.
The recorder assembly
The spectrometer itself – this houses the lamps, mirrors, prisms and detector. The spectrometer splits the beam of radiation into two and passes one through a sample and one through a reference solution (that is always made up of the solvent in which you have dissolved the sample). The detector measures the difference between the sample and reference readings and communicates this to the recorder.
The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes. The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are normally designed to allow the radiation to pass through the sample over a distance of 1cm.

16. LEARNING OBJECTIVES
Describe the dependence of transmitted light intensity for a light absorbing medium on sample depth and concentration.
Define the term max (lambda max).
Define ‘absorbance’ in terms of transmittance, pathlength and concentration (the Beer-Lambert law).
Define the term ‘molar absorption coefficient’.
Apply the Beer-Lambert law to problems in UV-VIS spectroscopy.
Define the term ‘conjugation’.
READING
Atkins & Jones, Chapter 7.
Clayden et. al., ‘Organic Chemistry’, Chapter 7

21. Ultraviolet (UV) Spectroscopy – The Output
The output from a UV scanning spectrometer is not the most informative looking piece of data!! It looks like a series of broad humps of varying height. An example is shown below.
Increasing absorbance *
*Absorbance has no units – it is actually the logarithm of the ratio of light intensity incident on the sample divided by the light intensity leaving the sample.
Beer Lambert Law
A = .c.l
Decreasing wavelength in nm
There are two particular strengths of UV (i) it is very sensitive (ii) it is very useful in determining the quantity of a known compound in a solution of unknown concentration. It is not so useful in determining structure although it has been used in this way in the past.
The concentration of a sample is related to the absorbance according to the Beer Lambert Law which is described above.
A = absorbance; c = concentration in moles l-1; l = pathlength in cm ;  = molar absorptivity (also known as extinction coefficient) which has units of moles-1 L cm -1.

22. THE BEER-LAMBERT LAW
For a light absorbing medium, the light intensity falls exponentially with sample depth.
T = transmittance. Io
light intensity (I) Io It l
cuvette It l
Sample depth

23. THE BEER-LAMBERT LAW
For a light absorbing medium, the light intensity falls exponentially with increasing sample concentration. Io It l
cuvette Io
light intensity (I)
It(i) Io It l
It(ii)
(i)
(ii)
Sample concentration

24. THE BEER-LAMBERT LAW
The negative logarithm of T is called the absorbance (A) and this is directly proportional to sample depth (called pathlength, l) and sample concentration (c). The equation is called the Beer-Lambert law.
Absorbance
Concentration

25. THE BEER-LAMBERT LAW
A plot of A versus c is called a Beer-Lambert plot.
 is called the molar absorption coefficient and has units of dm3 mol-1 cm-1
In the UV-VIS region (electronic spectroscopy) the maximum value of  observed is ~105 dm3 mol-1 cm-1, which is found in many natural pigments (e.g.chlorophyll) and in sunscreens for example.
max~45,000
Mexoryl® SX

26. THE BEER-LAMBERT LAW

27. THE BEER-LAMBERT LAW A
Cocaine

28. Handling samples of known concentration
Ultraviolet (UV) Spectroscopy – Analysing the Output
Beer Lambert Law
A = .c.l
Handling samples of known concentration
If you know the structure of your compound X and you wish to acquire UV data you would do the following.
Prepare a known concentration solution of your sample.
Run a UV spectrum (typically from 500 down to 220 nm).
From the spectrum read off the wavelength values for each of the maxima of the spectra (see left)
Read off the absorbance values of each of the maxima (see left).
Then using the known concentration (in moles L-1 ) and the known pathlength (1 cm) calculate the molar absorptivity () for each of the maxima.
Finally quote the data as follows (for instance for the largest peak in the spectrum to the left and assuming a concentration of moles L-1 ).
max = 487nm A= 0.75
 = /(0.001 x 1.0) = 7500 moles-1 L cm -1
Determining concentration of samples with known molar absorptivity ().
Having used the calculation in the yellow box to work out the molar absorptivity of a compound you can now use UV to determine the concentration of compound X in other samples (provided that these sample only contain pure X).
Simply run the UV of the unknown and take the absorbance reading at the maxima for which you have a known value of . In the case above this is at the peak with the highest wavelength (see above).
Having found the absorbance value and knowing  and l you can calculate c.
This is the basis of your calculation in Experiment 4 of CH199 and also the principle used in many experiments to determine the concentration of a known compound in a particular test sample – for instance monitoring of drug metabolites in the urine of drug takers; monitoring biomolecules produced in the body during particular disease states

44. HIGHLY ‘CONJUGATED’ MOLECULES ARE COLOURED
PARACETAMOL
LYCOPENE
-CAROTENE

56. CLASS PROBLEMS
The following spectrum was recorded for a 2.0 x 10-4 mol dm-3 aqueous solution of paracetamol in a cuvette of pathlength 0.5 cm. State max and estimate the molar absorption coefficient () of paracetamol at max. Also determine the transmittance (T) of the solution at max and at 270 nm..
The following data were recorded at 450 nm using solutions of -carotene in cyclohexane in a cuvette of pathlength 0.5 cm. Determine the molar absorption coefficient, .
c/10-5 M
450 nm
0.22
0.38
0.35
0.72
0.53
1.08
0.74
1.62
1.0
1.95

57. CLASS PROBLEMS
The following spectrum was recorded for a 2.0 x 10-4 mol dm-3 aqueous solution of paracetamol in a cuvette of pathlength 0.5 cm. State max and estimate the molar absorption coefficient () of paracetamol at max. Also determine the transmittance (T) of the solution at max and at 270 nm..
The following data were recorded at 450 nm using solutions of -carotene in cyclohexane in a cuvette of pathlength 0.5 cm. Determine the molar absorption coefficient, .
c/10-5 M
450 nm
0.22
0.38
0.35
0.72
0.53
1.08
0.74
1.62
1.0
1.95

58. CLASS PROBLEMS
In a UV-VIS study of commercial alcoholic drinks, it was found that an alcohol sample had an absorbance of 0.21 at its max (630 nm) in a 1 cm cuvette. Given that the molar absorption coefficient of the dye is 50,000 dm3 mol-1 cm-1, what is the dye concentration and what colour is the dye likely to be?

59. Applications of U.V. Spectroscopy:
1. Detection of Impurities
UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected.

60. U.V. Spectra of Paracetamol (PCM)

61. 2. Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms.
From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc.

62. 3. Quantitative analysis
UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. This determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc 
Where :
ε -is extinction co-efficient,
c- is concentration, and
b- is the length of the cell that is used in UV spectrophotometer.

63. Beer’s law

64. 4. Qualitative analysis
UV absorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds.

65. U.V. Spectra's of Ibuprofen

66. 5. Chemical kinetics
Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed.

67. 6. Detection of Functional Groups
This technique is used to detect the presence or absence of functional group in the compound
Absence of a band at particular wavelength regarded as an evidence for absence of particular group

68. Benzene
Tolune

69. 7. Quantitative analysis of pharmaceutical substances
Many drugs are either in the form of raw material or in the form of formulation. They can be assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm.

70. 8. Examination of Polynuclear Hydrocarbons
Benzene and Polynuclear hydrocarbons have characteristic spectra in ultraviolet and visible region. Thus identification of Polynuclear hydrocarbons can be made by comparison with the spectra of known Polynuclear compounds.
Polynuclear hydrocarbons are the Hydrocarbon molecule with two or more closed rings; examples are naphthalene, C10H8, with two benzene rings side by side, or diphenyl, (C6H5)2, with two bond-connected benzene rings. Also known as polycyclic hydrocarbon. 

71. Naphthalene
Diphenyl

72. 9. Molecular weight determination
Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.
For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate. Then known concentration of amine picrate is dissolved in a litre of solution and its optical density is measured at λmax 380 nm.

73. After this the concentration of the solution in gm moles per litre can be calculated by using the following formula.
"c" can be calculated using above equation, the weight "w" of amine picrate is known. From "c" and "w", molecular weight of amine picrate can be calculated. And the molecular weight of picrate can be calculated using the molecular weight of amine picrate.

74. 10. HPLC detector
A UV/Vis spectrophotometer may be used as a detector for HPLC.