PMC 205 L 1.PDF

Pharmaceutical analytical Chemistry 2 Dr. Mahmoud Mohamed Abbas Lecturer of Pharmaceutical Analytical Chemistry Faculty of Pharmacy, Galala University gu.edu.eg  Volumetric Quantitative Gravime...

Pharmaceutical analytical Chemistry 2 Dr. Mahmoud Mohamed Abbas Lecturer of Pharmaceutical Analytical Chemistry Faculty of Pharmacy, Galala University gu.edu.eg  Volumetric Quantitative Gravimetric Electrochemistry Analytical Chemistry Qualitative Instrumental Spectroscopy Separation techniques Advantages of volumetric & gravimetric analysis: 1. Easily applied 2. Highly accurate results Limitations of volumetric & gravimetric analysis: 1. Old techniques 2. Limited applications 3. Can’t achieve high degree of sensitivity Instrumental (physicochemical) analysis depends on measuring a physical property (optical or electrical) that is quantitatively related to the concentration of the analyte Introduction to spectrophotometry Electromagnetic radiation UV-Vis Spectrophotometric absorption UV-Vis absorption spectrum Shifts to Absorption Spectrum Factors affecting Absorption spectrum Introduction to spectrophotometry Spectroscopy: The science which studies the interaction of radiant energy (light energy) with matter. When a substance is subjected to radiant energy, some energy changes occur in nuclei, molecules or atoms. NUCLEI ATOMS MOLECULES Nuclear magnetic Atomic spectroscopy Molecular resonance (NMR) AAS spectroscopy UV-VIS NIR/FTIR Fluorescence Spectrophotometry: instrumental method of analysis in which the absorption and/or emission of electromagnetic radiation [light] by matter is studied and measured to give qualitative information about the nature of the substance i.e. to identify it as well as to determine it i.e. quantitative analysis. Introduction to spectrophotometry Electromagnetic radiation (EMR) is a type of energy that is transmitted through space at enormous velocity (C= .= 3 x 1010 cm/sec). It has both particle and wave properties (Dualism). A) Electromagnetic radiation as “particle” EMR is a stream of discrete particles of energy called photons which move in the form of wave. The energy of a photon is given by E = hν = hc/λ where h is Plank's constant (6.63 x 10-34 Joule⋅Hz−1 ) Introduction to spectrophotometry B) Electromagnetic radiation as “wave” -EMR travels in straight lines and has 2 fields (electric & magnetic) which are perpendicular on the direction of radiation propagation -EMR can be reflected, refracted and diffracted -EMR has wave properties: Wavelength (): is the linear distance between two successive maxima or minima of a wave (nm). Wave number (`): the number of waves per 1 cm (cm-1). `= 1/  Frequency (): is the number of waves (or cycles) passing a fixed point per second Hertz (Hz). = C/ ….. cm/sec.cm….sec-1 Introduction to spectrophotometry  Electromagnetic radiations Ultraviolet (UV) region extends from 100 - 400 nm, 100 - 200 nm is called far ultraviolet region but the most useful region for analysis is from 200 - 400 nm, called near ultraviolet region. The visible region is actually a very small part of the electromagnetic spectrum, and it is the region of wavelengths that can be seen by the human eye, that is where the light appears as a color. Visible region extend from about 400 - 800 nm. The infrared region extends from about 0.8 - 1000 µm. Electromagnetic radiations The white visible light is a mixture of the well known seven colors “VIBGYOR” discovered by Newton. (Violet–Indigo– Blue–Green–Yellow–Orange–Red) Electromagnetic radiations The color of any substance apparent to the human eye is the complementary color of the one absorbed by the substance. Color wheel: colors directly opposite each other on the color wheel are said to be complementary colors.  Electromagnetic radiations Absorption of Radiation: It is a process in which chemical species (atom, ion or molecule) selectively gain certain frequencies of EMR. M + EMR → M* (excitation) After a brief period (10-6 - 10-9 sec), M* relaxes to its ground state. M* → M + heat or radiation (relaxation) In relaxation process, M loses the same amount of the previously gained energy either as heat (collisions) or sometimes emits radiation of specific wavelength i.e. light (fluorescence or phosphorescence) Electromagnetic radiations A variety of energy absorption is possible depending upon the nature of the bonds within a molecule. For instance, the outermost electrons in organic molecules may be strong  bonds, or weaker π bonds or nonbonding (n). When energy is absorbed, all of these types of electrons can be elevated to excited antibonding states * and π* which can be represented diagramatically as below: UV-vis Spectrophotometric absorption UV-vis Spectrophotometric absorption Spectra- structure correlations The absorption of electromagnetic radiation in the near UV- Vis regions depends primarily upon the number and the arrangement of the electrons in the absorbing molecules (i.e. structure). Therefore, it is necessary to define certain terms that are frequently used in discussing electronic spectra.  Chromophores Auxochromes - Unsaturated groups responsible - Saturated groups which contain atoms with unshared for π → π* and n → π* electronic electron pairs transitions in near UV /visible light - They do not absorb radiation longer than 200 nm but (200 - 800 nm) and imparts color to when attached to a given chromophore they alter both the molecules the wavelength and the intensity of absorption maxima - The absorption of the molecule is of chromophores present in the molecule. the sum of absorption of all its - Auxochromes function by entering into resonance chromophores. interaction with nearby chromophore, thus increase the extent of conjugation and shift the max to longer wavelength and also increases the intensity of absorption. e.g. C=C, C=O, N=N, N=O e.g. hydroxyl group (-OH), amino group (-NH2) UV-vis Spectrophotometric absorption The more the conjugation of double bonds, the higher the wavelength of maximum absorption (λmax ) UV-vis Spectrophotometric absorption Auxochrome UV-Vis Absorption spectrum Absorption spectrum (curve): It is a plot of the absorbance (A) of a solution (the amount of light absorbed by a sample) as a function of wavelength λ. Each substance has its characteristic absorption spectrum which depends on its structure. It may be flat, with no prominent peak, it may show a single peak with a wavelength of maximum absorption (λmax), it may show more than one peak i.e. multipeak it may show a shoulder (side projection of the curve) or even may not show any absorption λmax is used in quantitative measurement in order to increase the sensitivity of the analytical method. Shifts to Absorption spectrum Bathochromic shift (Red shift) The shift of λmax to a longer wavelength due to substitution or solvent effect. Hypsochromic shift (Blue shift) The shift of λmax to shorter wavelength due to substitution or solvent effect Shifts to Absorption spectrum Hyperchromic effect An increase in the intensity of absorption. Hypochromic effect A decrease in the intensity of absorption Factors affecting Absorption Spectrum 1- Effect of pH: The spectra of a compound containing acidic or basic groups are pH dependent. In case of acidic groups (phenol), in acid medium the predominant species is the undissociated form (benzenoid) while in alkaline medium the predominant species is the phenate form (quinonoid) with delocalizaion of the π electrons i.e. conjugation, resulting in absorption of lower energy, i.e. longer λ (bathochromic shift). Factors affecting Absorption Spectrum In case of alkaline group (aniline), in acid medium, the amino group is protonated and thus the lone pair of electrons is no longer available for the quinonoid conjugated structure which is formed in alkaline medium. The UV spectrum of aniline in acid medium shows hypsochromic shift. Factors affecting Absorption Spectrum Thus solutions must be buffered at specific pH or measurements are carried out at the isosbestic point. An isosbestic point is a specific wavelength at which two chemical species have the same absorbance An isosbestic plot is constructed by the superposition of the absorption spectra of the substance at different pH values (the isosbestic point corresponds to a wavelength at which these spectra intersect each other. Factors affecting Absorption Spectrum 2- Effect of dilution: An example of this effect is the change of color of dichromate solution upon dilution with water Cr2O72- + H₂O ↔ 2 HCrO4 ↔ 2H+ + CrO42- Orange max 440 nm yellow max 390 nm 3- Effect of temperature: Change in the temperature may shift ionic equilibrium. An increase in temperature may cause bathochromic effect on some ions e.g. on boiling solution of ferric chloride in HCI changes from yellow to red. Thus, temperature should be the same for all measurements.  Factors affecting Absorption Spectrum 4- Effect of solvent: The same substance usually has different spectrum in different solvents, the spectrum in polar solvent varies than in non polar solvent because polar solvent tends to interact with functional groups by hydrogen bonding or Vander wal forces. For n → π* transition: increase in polarity of solvent shifts the transition to shorter wavelength (blue shift). For π → π* transition: increase in polarity of solvent shifts the transitions to longer wavelength (red shift). For measurements, the solvent used should be specified. Thank You For any questions, feel free to contact me by email [email protected] gu.edu.eg