1. UV- Visible Spectrophotometry
Wrya O. Karim, PhD

2. Outlines
1. Electromagnetic Radiation (EMR) 2. Principle of UV- Visible 3. Beer’s Law 4. Limitations and solutions 5. Calibration Curve 6. Standard Addition method

3. 1. Electromagnetic Radiation (EMR) Radiation is a form of energy that its presence can be verified via our sense of sight and ability to feel radiant heat. It may be considered in terms of a wave motion where the wavelength, λ, is the distance between two successive peaks. The frequency, ν, is the number of peaks passing a given point per second. These terms are related to each other: c=νλ Where, c is the velocity of light in a vacuum.

5. Note 1/ Besides the sun, the most conveniently available source of visible radiation is the tungsten lamp.
Note 2/ The human eye is only sensitive to a tiny proportion of the total electromagnetic spectrum between approximately 400 and 750 nm.
Note 3/ The quartz cell transmits UV radiation whereas glass cell absorbs. For the UV region itself, the most common source is the deuterium lamp and a UV-Visible spectrometer will usually have both lamp types to cover the entire wavelength range.

6. 2. Principle of UV- Visible Radiation
The energy of a photon absorbed or emitted during a transition from one molecular energy level to another is given by the equation E=hν Where, h is known as Planck's constant and ν is the frequency of the photon. We have already seen that c= νλ , Thus, E= hc/λ

7. When white light falls upon a sample, the light may be totally reflected or absorbed: a. Substance appears white or the light may be totally absorbed. b. The color of the sample is determined by the reflected light.

8. Colour absorbed
Absorbed Radiation (nm)
Violet
Blue
Green-blue
Blue-green
Green
Yellow-green
Yellow
Orange
Red

9. Potentially, three types of ground state orbitals may be involved:
i) σ (bonding) molecular orbital. ii) π (bonding) molecular orbital. iii) n (non-bonding) atomic orbital. In addition, two types of antibonding orbitals may be involved in the transition: i) σ* (sigma star) molecular orbital. ii) π* (pi star) molecular orbital.

10. Types of Transition
σ to σ*
2. n to σ*
3. n to π*
4. π to π*

11. Correlation of Molecular Structure and Spectra Conjugation
Moleucle
Transition, λmax (nm)
Ethane
σ σ, 135
Methanol
σ σ,
η σ,
Ethylene
π π , 175
Benzene
π π , 254
Acetone
η π , 290

12. Note 3/ The characteristic energy of a transition and hence the wavelength of absorption is a property of a group of atoms rather than the electrons themselves. When such absorption occurs, two types of groups can influence the resulting absorption spectrum of the molecule: chromophores and auxochromes. a.Chromophores: A chromophore (literally color-bearing) group is a functional group, responsible for compound’s colour, which exhibits a characteristic absorption spectrum in the ultraviolet or visible region. Some of the more important chromophoric groups are: Nitro, Nitroso, Azo, Carbonyl and Thiocarbonyl.

13. Note 4 / If any of the simple chromophores are conjugated with another (of the same type or different type) a multiple chromophore is formed having a new absorption band which is more intense and at a longer wavelength that the strong bands of the simple chromophore. Note 5/ This displacement of an absorption maximum towards a longer wavelength (i.e. from blue to red) is termed a bathochromic shift. The displacement of an absorption maximum from the red to violet is termed a hypsochromic shift.

14. b. Auxochromes
The color of a molecule may be intensified by groups called auxochromes which generally do not absorb significantly in the nm region, but will affect the spectrum of the chromophore to which it is attached. The most important auxochromic groups are OH, NH2, CH3 and NO2 and their properties are acidic (phenolic) or basic. The actual impact of an auxochrome on a chromophore relies upon the polarity of the auxochrome, e.g. CH3-, CH3CH2- and Cl- have very little effect, usually a small red shift of 5-10 nm. Other groups such as -NH2 and -NO2 are very popular and completely alter the spectra of chromophores.

15. Note 6/ The ability to complex many metals, particularly the transition elements, with organic and inorganic molecules which absorb in the visible region provides the basis for their quantitative spectrometric determination. The absorptions are owing to electron transition within energy levels of the organo-metal complex. The most common reagents are: dithizone, azo (PAN, thorin, zincon), dithiocarbamate, 8-hydroxyquinoline, formaldoxime and thiocyanate. In addition, many inorganic ions in solution also absorb in the visible region e.g. salts of Ni, Co, Cu, V…etc.

16. 3.Beer-LambertLaw The Beer-Lambert Law states that the concentration of a substance in solution is directly proportional to the 'absorbance ', A, of the solution. A=εbc Absorbance A: absorbance, ε: molar absorptivity, c: concentration b: cell length -The law is only true for monochromatic light, that is light of a single wavelength or narrow band of wavelengths. When monochromatic radiation passes through a homogeneous solution in a cell, the intensity of the emitted radiation depends upon the thickness (b) and the concentration (c) of the solution.

17. 4. Limitations and solutions
The law is applicable at low concentration, otherwise a deviation in linearity occurs in the calibration curve.
Stray radiations: these radiations come from outside of the instrument.
Instrumental imperfection: this comprises allowing polychromatic to pass through the solution.
Interferences (Selectivity): samples with multi colour components, absorption occurs by all components.
Sensitivity: at relatively very low conc., linearity cannot be obtained.

18. Typical absorption spectra

19. 5. Calibration Curve
A typical calibration curve

20. 6. Standard addition method
Whenever the desired components in a sample is very low in concentration, standard addition is obeyed.
In this methodology the standard solution with different concentration of the analyte is added into a series of sample solution portions with the same volume.