PTT 202 Organic Chemistry for Biotechnology

1. Zulkarnain Mohamed Idris PTT 202 Organic Chemistry for Biotechnology zulkarnainidris@unimap.edu.my Lecture 2: Spectroscopy Semester 1 2013/2014

2. Introduction • Spectroscopy: the study of the adsorption and emission of radiation by matter. • Measurement of the intensity and wavelength of radiation (either absorbed or emitted) provide the basis for sensitive methods of detection and quantification. • Photometry: measurement of the intensity of radiation and most commonly used technique in biochemistry. • To use photometric instruments correctly and to develop or modify spectroscopic techniques, it is necessary to understand the principles of the interaction of radiation with matter.

3. Interaction of radiation with matter • Radiation: is a form of energy and shows both electrical and magnetic characteristics (electromagnetic radiation). Composed of a stream of separate groups of electromagnetic waves and energy associated with the radiation can be mathematically related to the waveform. • Wavelength (λ): the distance between successive peaks of a waveform. • Frequency of radiation (v): the number of successive peaks passing in a second (v). • Energy (E) associated with particular waveform can be calculated as following: • E = hv=hc/ λ , where h=Plank’s constant (6.626 x 10-34 J) and c=speed of light (3.0 x 108 ms-1) • Amplitude: related to intensity of the radiation.

4. Electromagnetic Spectrum

5. Interaction of radiation with matter • The adsorption or emission of radiation by matter involves the exchange of energy within an atom or molecule. • The internal energy of molecule due to: • The energy associated with electrons. • The energy associated with vibrations between the atoms. • The energy associated with the rotation of various groups of atoms within the molecule relative to other groups. • These energy levels may be altered by the adsorption or emission of energy as radiation. • The exact amount of energy required to produce a change in molecule from one energy level to another will be given by the photons of one particular frequency which will be selectively absorbed or emitted.

6. Interaction of radiation with matter • Qualitative spectroscopy: a study of the wavelength or frequency of radiation absorbed or emitted by atom or a molecule will give information about its identity. • Quantitative spectroscopy: measurement of the total amount of radiation will give information about the number of absorbing or emitting atoms or molecules.

7. Absorption of radiation • Atomic absorption: individual atoms cannot rotate or vibrate in the same manner as molecule and as a result the adsorption of energy is only associated with electronic transitions which involves the raising of an electron to higher energy level orbital as energy is absorbed. • Molecular absorption (UV and visible region): absorption of radiation in this region causes transitions of electrons from molecular bonding orbitals to the higher energy molecular antibonding orbitals. Molecules that show increasing degree of conjugation (alternate single & double bonds) require less energy for excitation and as a result absorb radiation of longer wavelengths (Table 2.1).

9. The bonding and antibonding orbitals of the hydrogen molecule. Electrons naturally occupy the bonding orbital, which is lower in energy.

10. Absorption of radiation • Molecular absorption (infrared region): not all organic compounds absorb radiation in the UV and visible regions but they do show the absorption of infrared radiation due to vibrational changes. Vibrational transitions (bending or stretching of bonds) achieved with energy level near infrared region while rotational changes in molecular energy associated with the far infrared region and microwave regions. An adsorption max. in infrared region can be demonstrated only when a vibration results in a change in the dipole (unequal distribution of charge molecule) of the molecules, a process where energy is required.

12. Emission of radiation • An atom or molecule is said to be excited when it has undergone a transition of some form either electronic or vibrational transitions. • Atom or molecule will return to its ground state by losing its energy in three ways: • As a result of a chemical reaction. • By dissipation as heat. • By emission as radiation. • If atom or molecule loses all or part of the energy as radiation, photon energy will be emitted which correspond to the difference between the energy levels involved that will be of specific frequencies and will show up as bright lines if the emitted light is dispersed as a spectrum.

13. Emission of radiation • If an excited electron of molecule return to its original state from singlet transition and radiation is emitted, the compound is said to be fluoresce. However, in a return to ground state from a triplet transitions, fluorescence will not occur and energy loss will probably be non- radiactive. • Some compounds do shows radiactive emission during triplet transitions and these compounds known to be phosphoresce. • Chemiluminescence is another form of molecular emission in which the initial electronic transition is caused by exergonic reaction rather than absorption of radiant energy.

14. Emission of radiation

15. Molecular absorptiometry • Photometric measurements provide the basis for majority of quantitative methods. • The amount of radiation absorbed by a substance cannot be measured directly and it is usually determined by measuring the difference in intensity between radiation falling on the sample (incident radiation, Io) and residual radiation emerges from the sample (transmitted radiation, I). • Beer-Lambert law expresses the amount of light absorbed by a sample in terms of the concentration of the sample and the length of path and this relationship can be expressed as an equation: where c is the conc.of substance (g mol l-1), l is light part (cm) and )

16. Molecular absorptiometry

17. Molecular absorptiometry • Transmittance (T): the amount of a light passing through a sample expressed as a percentage of the light incident on the sample. I/Io) x 100 • Absorbance (A): a measure of the amount of light absorbed by a sample expressed in term of logarithm of the ratio of the transmitted and incident radiation. Thus Beer-Lambert equation can be expressed as:

18. Molecular absorptiometry • The assumption that difference between incident and the transmitted radiation is a measure of the radiation absorbed by analyte is not completely true because certain amount of radiation will be reflected from surface of the sample holder or absorbed by the material of which the cell is composed, thus: Absorbed= Incident (Io) - Transmitted (I) - other losses • For this reason the radiation of transmitted by a blank sample is measured. This blank should be identical to the test sample in all aspects except the presence of the test substance. Hence: Absorbed= Blank - Transmitted (I) • For quantitative measurement, the radiation should monochromatic (single wavelength) and the absorbance measurement should be made at the absorption maximum.

19. Absorptiometry design • Spectrophotometer: instrument that is capable of producing radiation of defined wavelength characteristics from mixed source of radiation and subsequently measuring the intensity of that radiation. • Basic design (refer to Figure2.17 in Handout): • Light from lamp passes through a monochromating device • Selected that passed through the sample is measured by suitable photoelectric detector. • The intensity of the initial radiation is controlled either by attenuator (shutter system) or varying the voltage of the lamp. • The geometry of the light part is controlled by a series of lenses and mirrors.

21. Absorptiometry design • Radiation sources: • Tungsten lamps: for visible region of the spectrum. • Hydrogen or deuterium discharge lamps: ultraviolet radiation. • Black body radiators (e.g. Nerst glower consists of hallow rod made of the fused oxides of zirconium, yttrium and thorium): for infrared radiation. Monochromators (devices to produce of limited/single wavelength): • Coloured filters • Interference filters • Prisms • Diffraction gratings

22. Absorptiometry design • Detectors (produce a current which is proportional to the intensity of the light falling on them): • UV and visible regions: e.g. photodiodes, photoemissive tubes • Infrared regions: e.g photoconductive devices (such as lead sulphide), thermocouple • Optical materials (to transmit radiation): • Normal glass: visible and near infrared regions. • Quartz and fused silica: ultraviolet region. • Alkali metal halides: infrared region. • Optical systems: • Single-beam instrument • Double-beam instrument

24. Molecular fluorescence technique • Involves the emission of radiation as excited electrons return to its ground state. • Wavelengths of radiation emitted are different from those absorbed and useful for identification of molecules. • The intensity of the emitted radiation can be used in quantitative methods and the wavelength of maximum emission can be used qualitatively. • Provides the basis of very sensitive method of quantification. • Fluorescent compounds often contain multiple conjugated bond systems (delocalized pi electrons) and the presence of electron-donating groups such as amine and hydroxyl. • Most molecules that fluoresce have rigid, planar structures.

25. Instrument design