Instrumental Analysis

1. Instrumental Analysis Spectrometric Instrumentations UV-VIS Spectrophotometry Fluorometry Polarometry Atomic Absorption Spectrometry Flame Photometry Infra-Red Spectrometry (IR) Mass Spectrometry (MS) Nuclear Magnetic Resonance Spectrometry (1H-NMR/13C-NMR)

2. UV/VIS SPECTROPHOTOMETRY Contents Light and radiation Electromagnetic spectrum Interaction of Photons with matter Absorption spectrum, Characteristics and shifts Chromophores, and auxochromes Factors affecting absorption spectrum (pH, solvent) Beer’s law and quantitative analysis Instrumentation (Basic component of spectrophotometer) Analysis of some pharmaceutical drugs

3. Introduction Definition of Spectrometry: The underlying principle of spectrophotometry is to shine light on a sample and to analyze how the sample affects the light. Principle of Spectrophotometry: Absorption spectrophotometry: is the measurement of an interaction between electromagnetic radiation and the molecules, or atoms, of a chemical substance Colorimetry: Spectrophotometric measurement in the visible region was formerly referred to as colorimetry; however, it is more precise to use the term “colorimetry” only when considering human perception of color. Fluorescence spectrophotometryis the measurement of the emission of light from a chemical substance while it is being exposed to ultraviolet, visible, or other electromagnetic radiation. In general, the light emitted by a fluorescent solution is of maximum intensity at a wavelength longer than that of the exciting radiation, usually by some 20 to 30 nm.

4. light spectrum Analyst prism spectrometer What is spectroscopy ? Spectroscopyis basically an experimental subject and is concerned with the absorption, emission, or scattering of electromagnetic radiation by atoms and molecules. Spectroscopy is the study of the interaction of electromagnetic radiation with matter. molecules, or atoms, of a chemical substance

5. A Light and radiation Light can be described as a wave. This wave has an electric component and a magnetic component which are perpendicular to each other E Electric field Velocity = c Direction of propagation H Magnetic field Wavelength ()

6.   x-ray UV visible IR Rf 390 450 520 590 620 780 Wavelength (nm) IncreasingEnergy UV IR Increasing Wavelength Light and radiation Electric magnetic spectrum Wavelength (, cm) 10-11 10-9 10-6 10-5 10-4 10-2 102 1021 1019 1016 1015 1014 1012 108 Frequency (, Hz)

7. Interaction of Photons with matter When a light wave encounters a particle, or molecule, it can be either scattered (i.e., direction changed) or absorbed (energy transferred). Molecules can only absorb discreet packets of energy, or quanta. Absorption occurs when the energy of the photon corresponds to differences between energy levels in that particular molecule. Absorption of the energy from the photon elevates the molecule to an excited state. A molecule or substance that absorbs light is known as a chromophore. Chromophores exhibit unique absorption spectra and can be defined by a wavelength of maximum absorption, or λmax. Scattering losses in solution Reflection losses at interfaces I (transmitted intensity) I0 (radiant intensity) Reflection and scattering losses

8. Interaction of Photons with matter A molecule may absorb light energy in three ways: [1] by raising an electron to a higher energy level (transitional energy) when molecule absorb visible and UV light. [2] by increasing the vibration of constituent nuclei (vibrational), when molecule absorb IR irradiation. [3] by increasing the rotation of molecule about its axis (rotational) when molecule absorb FT-IR irradiation. When a molecule interact with 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.

9. max Absorbance max Spectrum absorption spectrum, UV Scan, UV-VIS scan spectrum Absorption spectrum is: Characteristic to substance, and the wavelength at which the maximum absorption is recorded is used to trace the substance strength to enhance the sensitivity.

10. 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 have unsaturated bonds such as C=C, -C=O, -N=N, and -C≡N (etc π 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. Ex: -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 due substitution or solvent effects. Hyperchromic effect: enhancement of molecule absorptivity (or absorption intensity). Hypochromic effect: decrease of molecule absorptivity (or absorption intensity).

11. Hyperchromic Bathochromic Hypsochromic APEX Absorbance, mAU Hyporchromic Wavelength, nm Some important terms

12. Factors affecting absorption spectrum Affect what? Intensity Maximum wavelength Effect of pH on absorption spectra Effect of solvent on absorption spectra Effect of redox reaction on the absorption spectrum

13. Phenol in acid medium max = 270 nm Phenol in alkaline medium max = 290 nm Aniline in alkaline medium max = 280 nm Anilinium ion in acid medium max = 254 nm [1] Effect of pH Ex 1: 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 pai electrons in resonance with the π-electrons of the benzene ring, thus increasing thedelocalization of the π-electrons. Ex 2: On the other hand, 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).

14. detector monochromator lens Sample cuvette amplifier readout lamp