Pharmaceutical Analytical Chemistry III (PA 303) Lecture Notes PDF

Made with Xodo PDF Reader and Editor Pharmaceutical Analytical Chemistry III (PA 303) Level 2 PharmD (Spectroscopy) (Lecture 1) Dr. Bassam Shaaban 10/10/2023 2 ...

Made with Xodo PDF Reader and Editor Pharmaceutical Analytical Chemistry III (PA 303) Level 2 PharmD (Spectroscopy) (Lecture 1) Dr. Bassam Shaaban 10/10/2023 2  Made with Xodo PDF Reader and Editor Contents ▪ Principles of spectrophotometry ▪ Light and radiation ▪ Electromagnetic spectrum ▪ Light as energy ▪ Interaction of photons with matter ▪ Absorption spectrum, characteristics and shifts ▪ Types of electronic transitions ▪ Chromophores, and auxochromes ▪ Factors affecting absorption spectrum (pH, solvent) ▪ Quantitative spectrophotometry (laws of light absorption) ▪ Instrumentation (basic component of spectrophotometer) ▪ Applications of spectrophotometry  Made with Xodo PDF Reader and Editor Analytical chemistry Qualitative Quantitative Volumetric Instrumental Spectroscopy Electrochemical Chromatography 4  Made with Xodo PDF Reader and Editor Instrumental Methods of Analysis (Physicochemical) ▪ It depends on measuring some physical properties that is quantitatively related to the concentration of the constituent to be analyzed. ▪ It requires certain instruments, that is why it is called instrumental analysis. Assoc. Prof. Dr. Hytham M. 10/10/2023 5 Ahmed  Made with Xodo PDF Reader and Editor Spectroscopy Why we see color of solution or matter? 6 10/10/2023  Made with Xodo PDF Reader and Editor From the earliest times, color has been used to identify chemical substances and spectrometry is an extensive of this primitive technique. Historically, spectroscopy referred to a branch of science in which visible light was used for theoretical studies on the structure of matter and for qualitative and quantitative analyses. Spectroscopy: is based on the absorption of light by analyte. Also, defined as the study of the interaction between light and matter. 7 10/10/2023  Made with Xodo PDF Reader and Editor What is Light? Light is an electromagnetic radiation (EMR), composed of both electric and magnetic components. EMR is stream of discrete particles or wave packets of energy called photons or quanta. It displays the property of continuous waves and can be described by the characteristics of wave motion 8 10/10/2023  Made with Xodo PDF Reader and Editor Wave Properties of Electromagnetic Radiation Light exhibits wave property during its propagation and energy particle during its interaction with matter. Light waves, propagate at the highest known velocity of 3 x 1010cm/sec. or 3 x 108 m/sec. Such wave motion may be classified according to the wavelength (λ lambda) 9 10/10/2023  Made with Xodo PDF Reader and Editor Wavelength is the linear distance measured along the line of propagation, between the crest of one wave to that of the next wave. 10 10/10/2023  Made with Xodo PDF Reader and Editor Different units of length are used to express wavelengths in different parts of the EM-spectrum. Metre (m) = 100 cm = 1000 mm = 1000000 (106 µm) = 10 9 nm = 1010 A˚ The following units are in common use: 1 angstrom =1 A˚ =10-10m = 10-8 cm 1 nanometre = 1 nm = 10 A˚ =10-7 cm 1 micrometer = 1 um =104 A˚ =10-4 cm 11 10/10/2023  Made with Xodo PDF Reader and Editor 12 10/10/2023  Made with Xodo PDF Reader and Editor Some other terms are also used such as: Frequency (ν), which is the number of waves occurring per second and is expressed in cycles/sec(CPS) or Hertz(Hz) Wavelength inversely proportional to the frequency, Wavenumber(ύ) is number of waves per centimetre, which is expressed in cm-1. wavenumber (ύ ) =. 1/ λ cm-1 13 10/10/2023  Made with Xodo PDF Reader and Editor Relations between λ, ν and ύ are given by the following equations: 1 = wave number (ύ) Frequency (ν ) = Wavelength (λ) velocity of light (C) Where C is the velocity of light in vacuum = 3 x 1010 cm/sec Note that, the longer the wavelength, the lower the frequency and the smaller the wave number and14 vice versa. 10/10/2023 Example:  Made with Xodo PDF Reader and Editor Particle properties of electromagnetic radiation When a sample absorbs electromagnetic radiation it undergoes a change in energy, if we assume that electromagnetic radiation consists of a beam of energetic particles called photons. Photon energy is directly proportional to the frequency E=hν Where h is the Planck’s constant (6.63x10-34J.s) The energy of a photon is [in joules] We can also relate the energy of radiation to wavelength and wave- number: E = hc / λ = hc ύ Note that the wave-number in common with frequency is directly proportion to energy. 15 10/10/2023  Made with Xodo PDF Reader and Editor This electromagnetic spectrum ranges from very short wavelengths (including gamma and x-rays) to very long wavelengths (including microwaves and broadcast radio waves). 16 10/10/2023  Made with Xodo PDF Reader and Editor 17 10/10/2023  Made with Xodo PDF Reader and Editor 18 10/10/2023  Made with Xodo PDF Reader and Editor Light and radiation 19 10/10/2023  Made with Xodo PDF Reader and Editor Wavelength/Energy Practice Problems 1. The yellow light given off by a sodium vapor lamp used for public lighting has a wavelength of 589 nm. What is the frequency of this radiation? Solution: From our equation, we know that c=λv. (speed of light (meters) = wavelength (meters) times frequency (hertz)) c = 3.00 × 108 m/s 3.00 × 108 m/s = 589nm x v We must convert nanometers to meters. This is a very commonly missed step! We must have all units when dividing be equal. 589 nanometers= 5.89 × 10-7 meters 3.00 × 108 m/s / 5.89 × 10-7 m = v v = 5.093378608 × 1014 Hz 10/10/2023 20  Made with Xodo PDF Reader and Editor 2. A certain microwave has a wavelength of 0.032 meters. Calculate the frequency of this microwave. Solution: From our equation, we know that c=λv. c = 3.00 × 108 m. 3.00 × 108 m/s =.032m x v 3.00 × 108 m/s /.032m=v v=9.375 × 109 Hz 10/10/2023 21  Made with Xodo PDF Reader and Editor 3. A radio station broadcasts at a frequency of 590 KHz. What is the wavelength of the radio waves? Solution: 10/10/2023 22  Made with Xodo PDF Reader and Editor 5. Calculate the energy of one photon of yellow light that has a wavelength of 589nm. Solution: 10/10/2023 23  Made with Xodo PDF Reader and Editor Ultraviolet -Visible Spectrum  UV-visible absorption spectra can be used to help identify compounds and to measure the concentrations of solutions  Visible spectrum: The human eye is only sensitive to a tiny proportion of the total electromagnetic spectrum between approximately 400 and 800 nm and within this area we perceive the colors of the rainbow from violet through to red 10/10/2023 24  Made with Xodo PDF Reader and Editor It comes in colors as seen when white light is passed through a prism and broken into a rainbow. 10/10/2023 25  Made with Xodo PDF Reader and Editor Color Wheel 10/10/2023 26  Made with Xodo PDF Reader and Editor The visible spectrum forms only a small part [very tiny fraction ] of the complete EMR radiation spectrum. It extends from 400 to 800 nm and is further subdivided into colors according to their wavelengths. Violet: 400 - 420 nm Indigo: 420 - 440 nm Blue: 440 - 490 nm Green: 490 - 570 nm Yellow: 570 - 585 nm Orange: 585 - 620 nm Red: 620 - 780 nm 10/10/2023 27  Made with Xodo PDF Reader and Editor When polychromatic light (white light), which contains the whole spectrum of wavelength in the visible region is passed through an object, the object will absorb certain of the wavelengths, leaving the unabsorbed wavelengths to be transmitted. These residual transmitted wavelengths will be seen as color. 10/10/2023 28  Made with Xodo PDF Reader and Editor Interactions of photons with matter: There are three basic process by which a molecule can absorb radiation all involve raising the molecule to a higher internal energy level 1) By increasing the rotation of molecule about various axes so the molecule may absorb radiation and be raised to higher rotational energy level. (rotational transition). 2) Atoms or groups of atoms within molecule vibrate relative to each other (vibrational transition). 3)By raising an electron of molecule to a higher electron energy level (electronic transition). E total = E electronic + E vibrational + E rotational 10/10/2023 29  Made with Xodo PDF Reader and Editor The molecule at room temperature is usually in its lowest electronic energy state called ground state When a molecule interacts with photons in the UV-VIS region, the absorption of the 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 (Eg) to an excited state of energy level (Es). Energy of transition is given by the equation: ∆E= Es - Eg = hν = h C/ λ Excited state Es ∆E ground state Eg 10/10/2023 30  Made with Xodo PDF Reader and Editor Lecture 2 Pharmaceutical Analytical Chemistry iii  SPECTROSCOPY Dr. Bassam Shaaban, PhD 2 12-Oct-24  Made with Xodo PDF Reader and Editor UV-VIS Luminescence Spectroscopy  Visible spectrum:  The human eye is only sensitive to a tiny proportion of the total electromagnetic spectrum between approximately 400 and 800 nm and within this area we perceive the colors of the rainbow from violet through to red [It comes in colors as seen when white light is passed through a prism and broken into a rainbow]  UV-visible absorption spectra can be used to help identify compounds and to measure the concentrations of colored solutions 12-Oct-24 3  Made with Xodo PDF Reader and Editor The visible spectrum forms only a small part [very tiny fraction ] of the complete EMR radiation spectrum. It extends from 400 to 800 nm and is further subdivided into colors according to their wavelengths. Violet: 400 - 420 nm  Indigo: 420 - 440 nm  Blue: 440 - 490 nm  Green: 490 - 570 nm  Yellow: 570 - 585 nm  Orange: 585 - 620 nm  Red: 620 - 780 nm  12-Oct-24 4  Made with Xodo PDF Reader and Editor When polychromatic light (white light), which contains the whole spectrum of wavelength in the visible region is passed through an object, the object will absorb certain of the wavelengths, leaving the unabsorbed wavelengths to be transmitted. These residual transmitted wavelengths will be seen as color. 12-Oct-24 5  Made with Xodo PDF Reader and Editor Interactions of photons with matter 12-Oct-24 6  Made with Xodo PDF Reader and Editor Interactions of photons with matter: There are three basic process by which a molecule can absorb radiation all involve raising the molecule to a higher internal energy level 1) By increasing the rotation of molecule about various axes so the molecule may absorb radiation and be raised to higher rotational energy level. (rotational transition). 2) Atoms or groups of atoms within molecule vibrate relative to each other (vibrational transition). 3) By raising an electron of molecule to a higher electron energy level (electronic transition). E total = E electronic + E vibrational + E rotational 12-Oct-24 7  Made with Xodo PDF Reader and Editor The molecule at room temperature is usually in its lowest electronic energy state called ground state When a molecule interacts with photons in the UV-VIS region, the absorption of the 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 (Eg) to an excited state of energy level (Es). Energy of transition is given by the equation: ∆E= Es - Eg = hν = h C/ λ Excited state Es ∆E ground state Eg 12-Oct-24 8  Made with Xodo PDF Reader and Editor  Electronic Spectra &Molecular Structure  The electronic transitions that take place in the UV- Vis regions of the spectrum are due to the absorption of radiation by specific types of groups (functional groups ) & bonds within the molecule.  When light passes through a compound, some of the energy in the light kicks an electron from one of the bonding or non-bonding orbitals into one of the anti-bonding ones.  The energy gaps between these levels determine the frequency (or wavelength) of the light absorbed, and those gaps will be different in different compounds.  Electrons in molecule can be classified into different types 12-Oct-24 9  Made with Xodo PDF Reader and Editor Types of electrons:  (1) Closed shell – electrons that are not involved in bonding (very high excitation energies and don’t contribute to absorption in Vis or UV regions).  (2) Covalent single –bond electrons (σ or sigma, electrons) These also posses too high excitation energy to contribute to absorption of Vis or UV radiation (eg.-CH2-CH2- ).  (3) electrons in π ( pi) orbital for example in double or triple bonds.  (4) Paired non –bonding outer shell electrons (n electrons) such as those on N,O, P, S and halogen absorb UV radiation. 12-Oct-24 10  Made with Xodo PDF Reader and Editor Accordingly, there are three types of Transitions: a) σ -electrons; They are bonding electrons, represent valence bonds and possess the lowest energy level ( the most stable) b) π -electrons; They are bonding electrons, forming the π-bonds (double bounds), and possess higher energy than σ - electrons. c) n -electrons; They are nonbonding electrons, present in atomic orbital of hetero atoms (N, O, P, S or halogens). They usually occupy the highest energy level of the ground state. A molecule also possesses normally unoccupied orbitals called antibonding orbitals. 12-Oct-24 11  Made with Xodo PDF Reader and Editor σ-Electrons: Compounds containing only σ-electrons are the saturated hydrocarbons which absorb below 170nm (in the far UV region). They are transparent in the near UV region (200 - 400 nm) and this make them ideal solvents for other compounds studied in this range. They characterized by σ - σ* transition only. 12-Oct-24 12  Made with Xodo PDF Reader and Editor n-Electrons : Saturated organic compounds containing hetero-atoms, possess n-electrons in addition to σ -electrons. So they characterized by the σ - σ * and n - σ * transitions. The majority of these compounds show no absorption in the near UV region.  They are useful also as common solvents in the near UV region. However, their intense absorption usually extends to the edge of the near UV producing what is called end absorption (cut-off wavelength) mostly in the 200 – 250 nm region. UV cut off is defined as the wave length where solvent also absorbs light (UV or Visible). In that region, the measurement should be avoided. It is difficult to determine the absorbance comes from your analyte or your solvent. 12-Oct-24 13  Made with Xodo PDF Reader and Editor Cut-off wavelengths of some common solvents Solvent , nm Solvent , nm Solvent , nm Water 190 Ethanol 207 Benzene 280 Ether 205 Methanol 210 Acetone 331  Made with Xodo PDF Reader and Editor Types of Electronic Transitions   Anti-bonding  Anti-bonding  -  n- -  -  n -  - n Non-bonding  Bonding  Bonding 150 200 250 300 Wavelength, nm  Made with Xodo PDF Reader and Editor Look again at the possible jumps. This time, the important jumps are shown in black, and a less important one in grey. The grey dotted arrows show jumps which absorb light outside the region of the spectrum we are working in. 12-Oct-24 17  Made with Xodo PDF Reader and Editor Absorption spectrum  The spectrum itself is a plot of absorbance vs wavelength and is characterized by the wavelength (λmax) at which the absorbance is the greatest. a) Line spectrum: Occur with atomic spectra such as sodium metal which has a sharp line of λ at 589nm b) Band spectrum: Occurs with molecules due to the presence of different vibration and rotation sub-levels which the molecules may occupy on transition to excited state. That is, rotational and vibration modes will be found combined with electronic transition result in band rather than line spectra 12-Oct-24 18  Made with Xodo PDF Reader and Editor Absorption band spectrum for some molecules Amax max Absorption spectrum: it is characteristic to substance, and the wavelength at which the maximum absorption is recorded and used to trace the substance strength to enhance the sensitivity. 12-Oct-24 19  Made with Xodo PDF Reader and Editor Absorption Spectrum Is a plot of absorption intensity versus the wavelength of the absorbed light Line spectrum (for atoms) ) Line Spectra for some elements K Na 400 500 450 600 800 Wavelength, nm  Made with Xodo PDF Reader and Editor  According to the electronic transition that occur in each organic molecule, absorption spectrum is obtained by plotting A as a function of λ.  It has characteristic shape which show λ of maximum absorbance [λmax].  λ max characteristic for each molecule according to its structure [Number & arrangement of electrons] and consequently types of transitions. Therefore it is used for; - identification of a chemical substance - Used for quantitative measurement in order to increase sensitivity and to minimize error of analytical method. 12-Oct-24 21  Made with Xodo PDF Reader and Editor There are two parameters which define an absorption band: 1) Its position (λmax) on the wavelength scale. 2) Its intensity on the absorbance scale. An excited molecule return to the ground state in about 10-8 sec. Energy must be released in the form of: 1- Heat: When excited electron returns directly to the ground state. 2- fluorescence or phosphorescence ‫وميض‬ 3- Molecular collisions. 12-Oct-24 22  Made with Xodo PDF Reader and Editor 12-Oct-24 23  Made with Xodo PDF Reader and Editor Spectral changes 12-Oct-24 24  Made with Xodo PDF Reader and Editor SPECTRAL CHANGES & CONJUGATED SYSTEM Bathochromic (Red) shift Shift of absorption to longer wavelength due to substitution and solvent effects. Hypsochromic (Blue) shift It is shift of absorption to shorter wavelength. Hyperchromic & hypochromic effects: It is the increase and decrease in absorption intensity respectively. 12-Oct-24 25  Made with Xodo PDF Reader and Editor Some Important Terms Hyperchromic Hypsochromic Bathochromic Absorbance APEX Hyporchromic Wavelength, nm  Made with Xodo PDF Reader and Editor Terminology for Absorption Shifts Nature of Shift Descriptive Term To Longer Wavelength Bathochromic To Shorter Wavelength Hypsochromic To Greater Absorbance Hyperchromic To Lower Absorbance Hypochromic 12-Oct-24 27  Made with Xodo PDF Reader and Editor Relationship between the number of fused rings and wavelength 12-Oct-24 28 Made with Xodo PDF Reader and Editor 12-Oct-24 29  Made with Xodo PDF Reader and Editor Some important terms: spectra-structure correlation The absorbance of EMR depends primarily upon the number and arrangement of electrons in organic molecule of absorbing substance. Chromophores: (Chrom = colour, phore = carrier). They are functional groups which confer colour on substances capable of absorbing UV and/or visible light (200 - 800 nm). They have unsaturated bonds (double or triple bonds) such as -C=C;-C=O,N=N , -C=N and etc... Example Excitation λmax, nm ε Solvent Chromophore C=C Ethene π __> π* 171 15,000 hexane C≡C 1-Hexyne π __> π* 180 10,000 hexane n __> π* 290 15 hexane C=O Ethanal π __> π* 180 10,000 hexane n __> π* 275 17 ethanol N=O Nitromethane π __> π* 200 5,000 ethanol C-X X=Br Methyl bromide n __> σ* 205 200 hexane X=I Methyl Iodide n __> σ* 255 360 hexane The molecule containing a chromophore is called chromogen 12-Oct-24 30  Made with Xodo PDF Reader and Editor A chromophore (literally color-bearing) group is a functional group, not conjugated with another group, which exhibits a characteristic absorption spectrum in the ultraviolet or visible region. Some of the more important chromophoric groups are: However, although the next molecule contains two double bonds, they aren't conjugated. They are separated by two single bonds. If any of the simple chromophores is 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 chromophores. 12-Oct-24 31  Made with Xodo PDF Reader and Editor Auxochromes:  An auxochrome doesn’t itself absorb radiation, but if present in a molecule , it can enhance the absorption by chromophore or shift the wavelength of absorption when attached directly to the chromophore. Examples:  hydroxyl groups [ OH ]  Amino groups [ NH2 ]  Halogens [ Cl, Br, I ] 12-Oct-24 32  Made with Xodo PDF Reader and Editor Factor Affecting on the absorption spectra 1- Effect of pH on absorption spectra 2- Effect of Solvents on absorption spectra 12-Oct-24 33  Made with Xodo PDF Reader and Editor 1- Effect of pH on absorption spectra: The spectra of compounds containing acidic or basic groups are dependent on the pH of the medium (e.g.) phenols and amines. The UV spectrum of phenol in acid medium (where the molecular form predominates) is completely different 'from its spectrum in alkaline medium (where the phenolate anion predominates). The spectrum in alkaline medium exhibits bathochromic shift with hyperchromic effect. The red shift is due to the participation of the pair of electrons in resonance with the π-electrons of the benzene ring, thus increasing the delocalization of the π-electrons. OH O O OH- H+ - in acid medium 12-Oct-24 34  Made with Xodo PDF Reader and Editor Another example about ph.ph. indicator Remember: Increasing the amount of delocalization shifts the absorption peak to a longer wavelength 12-Oct-24 35  Made with Xodo PDF Reader and Editor On the other hand, UV spectrum of aniline in acid medium shows hypsochromic (blue) shift with hypochromic effect (decrease in absorption intensity). 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).. NH 2 in acid medium + NH 3 Blue shift 12-Oct-24 36 Made with Xodo PDF Reader and Editor 12-Oct-24 37 Made with Xodo PDF Reader and Editor 12-Oct-24 38 Made with Xodo PDF Reader and Editor 12-Oct-24 39  Made with Xodo PDF Reader and Editor 2- Effect of Solvents on absorption spectra: Less polar solvents (e.g. hydrocarbons) interact less strongly with the solute than do polar solvents (e.g. water and alcohols) The polar solvents may have a strong effect on the position of λ max due to its effect on the energy of transition. 12-Oct-24 40  Made with Xodo PDF Reader and Editor 2- Effect of Solvents on absorption spectra: π-π*Transitions: Two cases arise: (a) π-π*bands of dienes: Not shifted by any change of solvent polarity due to absence of charge separation in either ground or excited states. (b) π-π‫ ٭‬bands of enones: are red shifted on increasing solvent polarity due to stabilization of excited state by dipole-dipole solvent interaction. This stabilization leads to lowering the energy of the excited state (i.e.) smaller transition energy and hence longer λ (↓E→↑λ ) 12-Oct-24 41  Made with Xodo PDF Reader and Editor n-π* Transition: Blue shift with increasing solvent polarity due to stabilization of the ground state by hydrogen bonding: ,R-C=O…….H-OR Hydrogen bonding lowers the energy of the ground state (i.e.)increase energy of transition and hence decrease λ (Fig. b). 12-Oct-24 42 Made with Xodo PDF Reader and Editor 12-Oct-24 43  Made with Xodo PDF Reader and Editor 1 10/19/2024  Made with Xodo PDF Reader and Editor Lecture 3 Pharmaceutical Analytical Chemistry iii  SPECTROSCOPY Dr. Bassam Shaaban, PhD 2 10/19/2024  Made with Xodo PDF Reader and Editor Contents  Principles of spectrophotometry  Light and radiation  Electromagnetic spectrum  Light as energy  Interaction of photons with matter  Absorption spectrum, characteristics and shifts  Types of electronic transitions  Chromophores, and auxochromes  Factors affecting absorption spectrum (pH, solvent)  Quantitative spectrophotometry (laws of light absorption)  Instrumentation (basic component of spectrophotometer)  Applications of spectrophotometry  Made with Xodo PDF Reader and Editor Factor Affecting on the absorption spectra Hyperchromic Hypsochromic Bathochromic Absorbance APEX Hyporchromic Wavelength, nm  Made with Xodo PDF Reader and Editor Factor Affecting on the absorption spectra 1- Effect of pH on absorption spectra 2- Effect of Solvents on absorption spectra 10/19/2024 5  Made with Xodo PDF Reader and Editor 1- Effect of pH on absorption spectra: The spectra of compounds containing acidic or basic groups are dependent on the pH of the medium (e.g.) phenols and amines. The UV spectrum of phenol in acid medium (where the molecular form predominates) is completely different 'from its spectrum in alkaline medium (where the phenolate anion predominates). The spectrum in alkaline medium exhibits bathochromic shift with hyperchromic effect. The red shift is due to the participation of the pair of electrons in resonance with the π-electrons of the benzene ring, thus increasing the delocalization of the π-electrons. OH O O OH- H+ - in acid medium 10/19/2024 6  Made with Xodo PDF Reader and Editor Another example about ph.ph. indicator Remember: Increasing the amount of delocalization shifts the absorption peak to a longer wavelength 10/19/2024 7  Made with Xodo PDF Reader and Editor On the other hand, UV spectrum of aniline in acid medium shows hypsochromic (blue) shift with hypochromic effect (decrease in absorption intensity). 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).. NH 2 in acid medium + NH 3 Blue shift 10/19/2024 8 Made with Xodo PDF Reader and Editor 10/19/2024 9 Made with Xodo PDF Reader and Editor 10/19/2024 10 Made with Xodo PDF Reader and Editor 10/19/2024 11  Made with Xodo PDF Reader and Editor 2- Effect of Solvents on absorption spectra: Less polar solvents (e.g. hydrocarbons) interact less strongly with the solute than do polar solvents (e.g. water and alcohols) The polar solvents may have a strong effect on the position of λ max due to its effect on the energy of transition. 10/19/2024 12  Made with Xodo PDF Reader and Editor 2- Effect of Solvents on absorption spectra: π-π*Transitions: Two cases arise: (a) π-π*bands of dienes: Not shifted by any change of solvent polarity due to absence of charge separation in either ground or excited states. (b) π-π‫ ٭‬bands of enones: are red shifted on increasing solvent polarity due to stabilization of excited state by dipole-dipole solvent interaction. This stabilization leads to lowering the energy of the excited state (i.e.) smaller transition energy and hence longer λ (↓E→↑λ ) 10/19/2024 13  Made with Xodo PDF Reader and Editor n-π* Transition: Blue shift with increasing solvent polarity due to stabilization of the ground state by hydrogen bonding: ,R-C=O…….H-OR Hydrogen bonding lowers the energy of the ground state (i.e.)increase energy of transition and hence decrease λ (Fig. b). 10/19/2024 14  Made with Xodo PDF Reader and Editor Interaction of Photons with Matter 10/19/2024 15  Made with Xodo PDF Reader and Editor Interaction of Photons with Matter When a monochromatic light passes through a cell containing an absorbing substance, then the effects occurring will include: Reflection, refraction, scattering, absorption, and transmission. Scattering from solution (Is) Reflection at interfaces (Ir) Transmitted intensity (It) Incident intensity (I0) Light source Refraction from solution (If) Io (incidence light) = Ia + Ir + It + If + Is  Made with Xodo PDF Reader and Editor When light interacts with matter it can do one of several things, depending on its wavelength and what kind of matter it encounters: it can be transmitted, reflected, refracted, diffracted, adsorbed or scattered. 10/19/2024 17  Made with Xodo PDF Reader and Editor Interaction of Photons with Matter For Clear Solution scattering (Is) = 0 light reflection (Ir) Cancelled by Blank refraction (If), absorption (Ia), and transmitting of light (It). Transmitted intensity (It) Incident intensity (I0) Light source Io (incidence light) = Ia + It + Is + If + Ir  Made with Xodo PDF Reader and Editor The amount of radiation absorbed may be measured in a number of ways: Transmittance Transmittance : T = It / I0 % Transmittance: %T = 100 T Absorbance: A = log10 (1 / T) = -log10 (T) A = log10 (100 / %T) A = 2 - log10 (%T) The absorption of a solution increases as the transmittance decreases 10/19/2024 19  Made with Xodo PDF Reader and Editor The relationship between absorbance and transmittance is illustrated in the following diagram: So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite. 10/19/2024 20  Made with Xodo PDF Reader and Editor Lambert's Law: When a beam of monochromatic radiation enters an absorbing medium; its absorption intensity increase exponentially with the increase of thickness [b] of the medium traversed mathematically log I0/It=Kb concentration C held constant K is the proportionality constant , I0 and It are intensity of incident and transmitted radiations. 10/19/2024 21  Made with Xodo PDF Reader and Editor Beer's Law: It relates absorption capacity to the concentration of an absorbing solute. It stated that absorption is proportional to the number of absorbing molecules in the light path log lo/l=K C where K is proportionality constant and C is the concentration while b is held constant. 10/19/2024 22  Made with Xodo PDF Reader and Editor Beer's-Lambert's law: This is a combination of both laws; Log lo/l= abc where (a) is a constant called absorptivity, (b) is the pathlength in cm and (c) is the concentration in grams/Liter. Log lo/l is usually substituted by A (Absorbance), then the equation become; A = abc The value of (a) will clearly depend upon the method of expression of the concentration. 10/19/2024 23  Made with Xodo PDF Reader and Editor Laws of Light Absorption Beer’s - Lambert’s Law: A =abC Expressions of a a absorptivity, A = ab c if concentration (c) expressed as gram / Liter. ε (Epsilon), Molar absorptivity, A =εbc if concentration (c) expressed as molar solution. A one percent one centimeter 1% A1% 1cm A = A1cm b c if c is expressed in g/100 mL Made with Xodo PDF Reader and Editor Laws of Light Absorption  Made with Xodo PDF Reader and Editor Deviations from Beer-Lambert Law: When the results obey Beer-Lambert law, the concentration versus absorbance gives a straight line passing through the origin (calibration curve) as indicated by the solid line in figure below. 10/19/2024 26  Made with Xodo PDF Reader and Editor In some cases, deviation from Beer-Lambert law occur which may be: (1) Real deviation: At high concentrations, due to crowding, molecular interaction and association as well as charge distribution. (2) Instrumental deviation (errors): a) Irregular deviation due to: Unmatched cells, unclean handling and unclean optics. b) Regular deviation: due to: - Errors in wavelength scale. - Slit width control. - Stray light is any radiation of wavelength other than those which are absorbed. Also includes any light reaches the detector without passing through the sample. 10/19/2024 27  Made with Xodo PDF Reader and Editor c) Other errors:  Non-linear response of photo cells.  Radio and TV interferences.  Unstabilized power supply. (3) Chemical deviations:  pH effects.  Solvents interaction due to high concentrations.  Temperature effects.  Dipole interactions  Time factor affect oxidation, reduction, or hydrolysis reactions which may be occur. 10/19/2024 28 Made with Xodo PDF Reader and Editor 19-Oct-24 29  Made with Xodo PDF Reader and Editor Methodology and Instrumentation UV-VIS Instrumentation 400-800 nm Colorimetry UV Spectrophotometry 200-400 nm Visual measurement Standard series Balancing (varying depth) SPECTROPHOTOMETER  Made with Xodo PDF Reader and Editor Photoelectric Colorimeters & Spectrophotometers - Used for electric measuring the light absorbed by the sample. - Monochromatic light is used instead of polychromatic light frequently used in visual methods. Components of Instruments: 1. Light source 2. Monochromator 3. Sample cell 4. Detector 5. Recorder (meter) Types of Instruments: 1. Single-beam spectrophotometers. 2. Double-beam spectrophotometers.  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Single-Beam Spectrophotometers: Detector Monochromator Light Sample Amplifier Meter cuvette source  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Single-Beam Spectrophotometers:  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Double-Beam Spectrophotometers: Blank cuvette Detector 1 Monochromator Amplifier Meter Beam Light splitter source Sample Detector 2 cuvette  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Double-Beam Spectrophotometers:  Made with Xodo PDF Reader and Editor 10/19/2024 Dr. Bassam 36 Made with Xodo PDF Reader and Editor 10/19/2024 37  Made with Xodo PDF Reader and Editor 1 27-Oct-24  Made with Xodo PDF Reader and Editor Lecture 4 ANALYTICAL III  SPECTROSCOPY Dr. Bassam Shaaban, PhD 2 27-Oct-24  Made with Xodo PDF Reader and Editor Contents  Principles of spectrophotometry  Light and radiation  Electromagnetic spectrum  Light as energy  Interaction of photons with matter  Absorption spectrum, characteristics and shifts  Types of electronic transitions  Chromophores, and auxochromes  Quantitative spectrophotometry (laws of light absorption)  Factors affecting absorption spectrum (pH, solvent)  Instrumentation (basic component of spectrophotometer)  Applications of spectrophotometry  Made with Xodo PDF Reader and Editor Methodology & Instrumentation 27-Oct-24 4  Made with Xodo PDF Reader and Editor Methodology and Instrumentation UV-VIS Instrumentation 400-800 nm Colorimetry UV Spectrophotometry 200-400 nm Visual measurement Standard series Balancing (varying depth)  Made with Xodo PDF Reader and Editor Photoelectric Colorimeters & Spectrophotometers - Used for electric measuring the light absorbed by the sample. - Monochromatic light is used instead of polychromatic light frequently used in visual methods. Components of Instruments: 1. Light source 2. Monochromator 3. Sample cell 4. Detector 5. Recorder (meter) Types of Instruments: 1. Single-beam spectrophotometers. 2. Double-beam spectrophotometers.  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Single-Beam Spectrophotometers: Detector Monochromator Light Sample Amplifier Meter cuvette source  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Single-Beam Spectrophotometers:  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Double-Beam Spectrophotometers: Blank cuvette Detector 1 Monochromator Amplifier Meter Beam Light splitter source Sample Detector 2 cuvette  Made with Xodo PDF Reader and Editor Types of Spectrophotometers Double-Beam Spectrophotometers:  Made with Xodo PDF Reader and Editor Components of Spectrophotometers Light source: - UV measurement: hydrogen or deuterium discharge lamp (190 – 375 nm) - Visible measurement: Tungsten lamp (350 – 1000 nm) Monochromator: Function: To select light beam of certain wavelength. - Filter - Prisms - Grating  Made with Xodo PDF Reader and Editor a- Filters Functioning via selective absorption of unwanted wavelengths and transmitting the complementary color which is needed to be absorbed by the sample to be analysed. Filters used to isolate certain wavelengths of light. Filter made of glass and contain chemicals (dyes) that absorb all radiation except that desired to be passed.  Made with Xodo PDF Reader and Editor b- Prisms Functioning via refraction of light. When electromagnetic radiation passes through a prism, it is refracted, because the index of refraction of the prism material is different form that in air. Shorter wavelengths are refracted more than longer wavelengths By rotation of prism, different wavelength of the spectrum can be made to pass through an exit slit and through the sample  Made with Xodo PDF Reader and Editor C- Gratings Consist of large number of parallel ruled very close to each other on a highly polished surface, e.g. aluminium, or aluminized glass (600 groove/mm). Each ruled groove functions as a scattering center for light falling on its edge and through diffraction and interference the grating disperses the light beam into almost single wavelength. Incident light Diffracted light  Made with Xodo PDF Reader and Editor Components of Spectrophotometers Sample Cell (cuvette) : – Transparent – Quartz for UV measurements – Glass or Quartz cell for VIS measurements – Pathlength: usually 0.5, 1 or  1 cm  Made with Xodo PDF Reader and Editor Components of Spectrophotometers Detectors : Receive light emerged from the sample, which excite electrons and generate an electric current that proportional to the received light intensity. Electrons Anode (iron) Cathode Light (selenium) beam 1- Photocell 2- Photomultiplier tube  Made with Xodo PDF Reader and Editor Components of Spectrophotometers Recorders : The function of the meter to translate the received current (current intensity proportional to light emerged from sample) to signals on paper or computer (integration of data).  Made with Xodo PDF Reader and Editor APPLICATIONS OF SPECTROPHOTOMETRY  Made with Xodo PDF Reader and Editor Application of spectrophotometry It includes application of spectroscopy in Qualitative & Quantitative analysis including both organic and inorganic pharmaceutical compounds and determination of some physical constants.  Made with Xodo PDF Reader and Editor Application of spectroscopy I- Qualitative Analysis: II-Quantitative Analysis: λmax ,Absorptivity, UV- The substance to be VIS absorption spectrum analyzed is dissolved in usually give indication of suitable solvent. The the sample to be absorbance readings are analyzed. taken in the expected range.  Made with Xodo PDF Reader and Editor Qualitative Analysis by Spectrophotometry 1. Identification of morphine: H since morphine is a phenolic compound, H3C N the observation of bathochromic shift with hyperchromic effect in KOH is consistent with, but not definite proof of, the presence of morphine in the sample. O HO OH Since other phenolic compounds show similar behavior, this test is a definite proof of the absence of morphine in the sample. For definite proof for the presence of morphine, better method (e.g. infrared spectroscopy) should be used.  Made with Xodo PDF Reader and Editor Qualitative Analysis by Spectrophotometry 2. Identification of phenolphthalein: HO OH O O- C O OH - C C O COO - H+ Benzenoid (colorless, pH 7) Quinonoid (pink, pH 9) max in UV region max of 550-555 nm  Made with Xodo PDF Reader and Editor Qualitative Analysis by Spectrophotometry 3. Identification of barbiturates (toxicological analysis): H O O- O- N R1 N R1 N R1 O OH - OH - O O N R2 N R2 N R2 H O H O O- Up to pH = 8 pH = 10 pH = 12 Lactam form Lactin forms  Made with Xodo PDF Reader and Editor Qualitative Analysis by Spectrophotometry Absorption spectra of barbiturates at different pH values H O N R1 O pH = 12 N R2 H O pH = 10 O- N R1 O N R2 H O pH = up to 8 O- N R1 210 220 230 240 250 260 270 O N R2 Wavelength, nm O-  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry General Procedure: 1. Determination of proper max : 350 450 550 650 Wavelength, nm  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry 2. Generating the calibration curve at max : Standard solutions Unknown Unknown conc. Is determined by: Absorbance Linear equation: A = a + b C Calibration curve: graphically Concentration  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry. Analysis of inorganic compounds: - Determination of copper via ammine complex. Cu2+ + 4 NH3 [ Cu(NH3)4 ]2+ ( Blue color) - Determination of ferric via thiocyanate complex. Fe3+ + SCN- [ Fe(SCN)]2+ ( Blood red color) - Determination of ferrous via 1,10-phenanthroline complex. 2+ N N 3 + Fe2+ Fe N N 3 1,10-phenanthroline Dense red color  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry. Analysis of organic compounds: - Analysis of aromatic amines by diazotization and coupling : + N N Cl- NH2 NaNO2 HCl NH-(CH 2)2-NH 2 Amine N=N NH-(CH 2)2-NH 2 Red color, max of 450-520 nm  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry. Analysis of organic compounds: - Analysis of carbonyl compounds by conversion into phenyl hydrazones via reaction with 2,4-dinitrphenylhydrazine: R C=O H 2 NHN NO 2 R O 2N Carbonyl compound 2,4-dinitrophenyl hydrazine R C=NHN NO 2 R O 2N phenyl hydrazone derivative Orange color, max of 430-480 nm  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry - Analysis of phenols via coupling with diazotized primary aromatic amines: + OH N N Cl - N=N OH R + R SO 3 H SO 3 H Phenolic Diazotized Orange-red color compound amine  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry Spectrophotometric Titration (Photometric End Point Detection) : Opening for the stirrer Opening for the burette Stirrer Quartz window Apparatus for spectrophotometric titration  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry Spectrophotometric Titration (Photometric End Point Detection) : Absorbance Volume of titrant S + + P + T + + S (Sample ), P (Product), T (Titrant)  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry Spectrophotometric titration of KMnO4 with Fe2+ at 540 nm Absorbance Volume of titrant 27-Oct-24 33  Made with Xodo PDF Reader and Editor Quantitative Analysis by Spectrophotometry Advantages of Photometric Titrations:  No interference from other substances because the end point depends on the change in the absorbance curve and not on the absorbance value.  Can be used for titrating very dilute solutions.  More accurate than direct photometry.  Can be applied to all types of reactions (redox, acid-base, complex,…….etc.  Made with Xodo PDF Reader and Editor Advantages of Spectrophotometry 1. Wide applicability: large number of organic and inorganic compounds absorb light in the UV-visible region. 2. High sensitivity: concentrations in the range of 10-4 to 10-6 M can be analyzed by spectrophotometry. 3. Moderate to high selectivity: due to selective reactions, selective measurements, and different mathematical treatment. 4. Good accuracy: relative errors in concentration measurement are in the range of 0.1 to 2%. 5. Ease and convenience: easily and rapidly performed with modern instruments.  Made with Xodo PDF Reader and Editor 10/27/2024 Dr. Bassam 36  Made with Xodo PDF Reader and Editor Dr. Bassam Shaaban 37 27-Oct-24  Made with Xodo PDF Reader and Editor Fluorimetry Lecture  Made with Xodo PDF Reader and Editor Molecular Photoluminescence Spectroscopy  Made with Xodo PDF Reader and Editor Molecular Fluorescence and Phosphorescence [Photoluminescence] General Interactions of Light and Matter When a beam of radiation strikes any object it can be absorbed, transmitted, scattered, reflected or it can excite fluorescence. With fluorescence ,a photon is first absorbed and excited the molecule to a higher energy state. The molecule then drops back to an intermediate energy level by re-emitting a photon. Since some of the energy of the incident photon is retained in the molecule or is lost by a non-radiative process such as collision with another molecule. The emitted photon has less energy and hence a longer wavelength than the absorbed photon. Like scatter, fluorescent radiation is also emitted uniformly in all directions.  Made with Xodo PDF Reader and Editor An analyte in an excited state possesses an energy,E2,that is greater than that when it is in a lowerenergy-state,E1, ΔE=E2-E1 ΔE is excess energy which must be released There are several ways an excited atom or molecule can give up its excess energy and relax to its ground state Relaxation occurs through : 1-Nonradiative a-release of heat A* → A + heat b-decomposition reaction excess energy is used A* → X+Y up in the chemical reaction c-reaction between A* and another species A* + Z → X+Y  Made with Xodo PDF Reader and Editor 2- Radiative: Emission of photons {with lower energy (longer wavelength) than was absorbed} A* → A+hν  Made with Xodo PDF Reader and Editor Lifetime : The length of time that an analyte stays in an excited state before returning to a lower-energy state.  Relaxation: Any process by which an analyte returns to a lower-energy state from a higher-energy state.  Made with Xodo PDF Reader and Editor A molecule at room temperature normally resides in ground state. The ground state is usually a singlet state S0 with all electrons paired. Electrons that occupy the same molecular orbital must be “paired" and have opposite spins. If electrons have the same spin they are “unpaired" and the molecule is in a triplet state T1. Singlet State Triplet State Electrons are paired and spin Electrons are unpaired by in opposite direction S0 flipping and spin in the same direction T1 Higher energy Lower energy  Made with Xodo PDF Reader and Editor Molecular fluorescence and phosphorescence To appreciate the origin of molecular fluorescence and phosphorescence, we must consider what happens to a molecule following the absorption of a photon. Let’s assume that the molecule initially occupies the lowest vibrational energy level of its electronic ground state. The ground state is a singlet state labeled S◦. Absorption of photon of correct energy excites the molecule to one of several vibrational energy levels in the first excited electronic state,S1, or the second excited electronic d state,S2, Both of which are singlet states. Relaxation to the ground state from these excited states occurs by a number of mechanisms that are either (a) Radiationless, in that no photons are emitted, or involve (b) The emission of a photon. (a) Radiationless Deactivation: It involves the following forms: 1- Vibrational relaxation 2- Internal conversion 3- External conversion 4- Intersystem crossing “ISC” Made with Xodo PDF Reader and Editor Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor  1-Vibrational relaxation: It is a form of radiationless relaxation in which an analyte moves from a higher vibrational energy level to a lower vibrational energy level in the same electronic level. It takes place during collision between excited molecules & molecules of solvent. Also collision with solvent molecules at this point rapidly removes the excess energy from the higher vibrational level of S1  2-Internal conversion: A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level. It occurs between lowest vibrational level of an excited electronic state & the upper vibrational level of another electronic state.  3-External conversion: A form of radiationless relaxation in which energy is transferred to the solvent or sample matrix  Made with Xodo PDF Reader and Editor 4-Intersystem crossing (ISC): A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level with a different spin state[ one electron to reverse its spin] and the molecule transfer to a lower- energy triplet state. From here the molecule can return to the ground state by emission of photon (phosphorescence).  Made with Xodo PDF Reader and Editor (b) Radiative Deactivation “the emission of a photon”: 1-Fluorescence: Fluorescence occurs when a molecule in the lowest vibrational energy level of an excited electronic state returns to a lower energy electronic state by emitting a photon. Since molecules return to their ground state by the fastest mechanism.  Made with Xodo PDF Reader and Editor  2-Phosphorescence:  A molecule in the lowest vibrational energy level of an excited triplet electronic state normally relaxes to the ground state by an intersystem crossing to a triplet state or by external conversion. Phosphorescence is observed when relaxation occurs by the emission of a photon.  Made with Xodo PDF Reader and Editor Main difference between fluorescence & phosphorescence Fluorescence Phosphorescence 1-The spin state of the excited 1-Change in spin state of electron electron doesn’t change during during transition from X‫ ٭‬to G transfer from excited state to the 2-Relaxation from lowest triplet ground state (G) state to G (electron unpaired) 2- Relaxation takes place from T1 → G lowest singlet state (electron paired) _↑↑__T1 unpaired to singlet ground state S1→ S0 _↑↑__G __↓__S1 3-Lifetime longer 10-4- 104 sec __↑↓__G paired __↑__S0 4-Measurement takes place even 3-it occurs very soon i.e lifetime after excitation ceases. 10-6 --10-9sec. It decays rapidly after the excitation source is removed. 4- Measurement take place during excitation  Made with Xodo PDF Reader and Editor Excitation and Emission Spectra If we plot the intensity of fluorescence obtained when the sample is irradiated with monochromatic radiation (λexcitation ) versus wavelength an emission spectrum is obtained. If we plot the excitation and emission spectra of a compound on the same chart the displacement of emission band to a longer wavelength (λ) (is known as stock’s shift).  Made with Xodo PDF Reader and Editor Instrumentation of Fluorescence Component of the instrument 1-Source 5-Read of output excitation 3-The detector 2-Sample cell 4-Monochromator  Made with Xodo PDF Reader and Editor 1-Source of excitation. Either xenon arc lamp ( ultra violet) or mercury arc lamp ( visible). 2-Sample cell Transparent in all sides either glass ( visible) or quartz ( ultra violet ) usually present at right angle with respect to the source and detector. 3-The detector Phototube or photomultiplier tube.  Made with Xodo PDF Reader and Editor 4-Monochromator Filters or grating. Two monochromators are used. One is used for wavelength selection of excitation and the second is used for emission. 5-Read output Recorder or meter scale.  Made with Xodo PDF Reader and Editor 1- Source of radiation Mercury-Xenon arc lamp 2- Primary monochromater 1 3 (λ excitation selector) 2 3- Sample cell transparent from all sides 4 4- Secondary monochromator (filter or grating) (λ emission 5 selector) 5- Detector (photomultiplier tube) 6 6- Read output recorder or meter scale Made with Xodo PDF Reader and Editor Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor  NB. Although the radiation emitted is observed at right angles to the exciting radiation, some of the exciting radiation can be detected by the emission detector because it is scattered by solvent molecules (Rayleigh scatter) or by colloidal particles in solution (Tyndall scatter).  The presence of this scatter makes the use of the second monochromator necessary and also means that the fluorescence band has to be shifted by at least 20 nm beyond the excitation band for fluorescence measurements to be made without interference.  Another weaker type of scatter which may be observed is Raman scatter, In Raman scatter, which is solvent dependent, the wavelength of the incident radiation is shifted to a longer wavelength by about 30 nm when methanol is used as a solvent and about 10 nm when chloroform is used as a solvent. Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor Fluorimetry Lecture  Made with Xodo PDF Reader and Editor Molecular Photoluminescence Spectroscopy  Made with Xodo PDF Reader and Editor Molecular Fluorescence and Phosphorescence [Photoluminescence] General Interactions of Light and Matter When a beam of radiation strikes any object it can be absorbed, transmitted, scattered, reflected or it can excite fluorescence. With fluorescence ,a photon is first absorbed and excited the molecule to a higher energy state. The molecule then drops back to an intermediate energy level by re-emitting a photon. Since some of the energy of the incident photon is retained in the molecule or is lost by a non-radiative process such as collision with another molecule. The emitted photon has less energy and hence a longer wavelength than the absorbed photon. Like scatter, fluorescent radiation is also emitted uniformly in all directions.  Made with Xodo PDF Reader and Editor An analyte in an excited state possesses an energy,E2,that is greater than that when it is in a lowerenergy-state,E1, ΔE=E2-E1 ΔE is excess energy which must be released There are several ways an excited atom or molecule can give up its excess energy and relax to its ground state Relaxation occurs through : 1-Nonradiative a-release of heat A* → A + heat b-decomposition reaction excess energy is used A* → X+Y up in the chemical reaction c-reaction between A* and another species A* + Z → X+Y  Made with Xodo PDF Reader and Editor 2- Radiative: Emission of photons {with lower energy (longer wavelength) than was absorbed} A* → A+hν  Made with Xodo PDF Reader and Editor Lifetime : The length of time that an analyte stays in an excited state before returning to a lower-energy state.  Relaxation: Any process by which an analyte returns to a lower-energy state from a higher-energy state.  Made with Xodo PDF Reader and Editor A molecule at room temperature normally resides in ground state. The ground state is usually a singlet state S0 with all electrons paired. Electrons that occupy the same molecular orbital must be “paired" and have opposite spins. If electrons have the same spin they are “unpaired" and the molecule is in a triplet state T1. Singlet State Triplet State Electrons are paired and spin Electrons are unpaired by in opposite direction S0 flipping and spin in the same direction T1 Higher energy Lower energy  Made with Xodo PDF Reader and Editor Molecular fluorescence and phosphorescence To appreciate the origin of molecular fluorescence and phosphorescence, we must consider what happens to a molecule following the absorption of a photon. Let’s assume that the molecule initially occupies the lowest vibrational energy level of its electronic ground state. The ground state is a singlet state labeled S◦. Absorption of photon of correct energy excites the molecule to one of several vibrational energy levels in the first excited electronic state,S1, or the second excited electronic d state,S2, Both of which are singlet states. Relaxation to the ground state from these excited states occurs by a number of mechanisms that are either (a) Radiationless, in that no photons are emitted, or involve (b) The emission of a photon. (a) Radiationless Deactivation: It involves the following forms: 1- Vibrational relaxation 2- Internal conversion 3- External conversion 4- Intersystem crossing “ISC” Made with Xodo PDF Reader and Editor Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor  1-Vibrational relaxation: It is a form of radiationless relaxation in which an analyte moves from a higher vibrational energy level to a lower vibrational energy level in the same electronic level. It takes place during collision between excited molecules & molecules of solvent. Also collision with solvent molecules at this point rapidly removes the excess energy from the higher vibrational level of S1  2-Internal conversion: A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level. It occurs between lowest vibrational level of an excited electronic state & the upper vibrational level of another electronic state.  3-External conversion: A form of radiationless relaxation in which energy is transferred to the solvent or sample matrix  Made with Xodo PDF Reader and Editor 4-Intersystem crossing (ISC): A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level with a different spin state[ one electron to reverse its spin] and the molecule transfer to a lower- energy triplet state. From here the molecule can return to the ground state by emission of photon (phosphorescence).  Made with Xodo PDF Reader and Editor (b) Radiative Deactivation “the emission of a photon”: 1-Fluorescence: Fluorescence occurs when a molecule in the lowest vibrational energy level of an excited electronic state returns to a lower energy electronic state by emitting a photon. Since molecules return to their ground state by the fastest mechanism.  Made with Xodo PDF Reader and Editor  2-Phosphorescence:  A molecule in the lowest vibrational energy level of an excited triplet electronic state normally relaxes to the ground state by an intersystem crossing to a triplet state or by external conversion. Phosphorescence is observed when relaxation occurs by the emission of a photon.  Made with Xodo PDF Reader and Editor Main difference between fluorescence & phosphorescence Fluorescence Phosphorescence 1-The spin state of the excited 1-Change in spin state of electron electron doesn’t change during during transition from X‫ ٭‬to G transfer from excited state to the 2-Relaxation from lowest triplet ground state (G) state to G (electron unpaired) 2- Relaxation takes place from T1 → G lowest singlet state (electron paired) _↑↑__T1 unpaired to singlet ground state S1→ S0 _↑↑__G __↓__S1 3-Lifetime longer 10-4- 104 sec __↑↓__G paired __↑__S0 4-Measurement takes place even 3-it occurs very soon i.e lifetime after excitation ceases. 10-6 --10-9sec. It decays rapidly after the excitation source is removed. 4- Measurement take place during excitation  Made with Xodo PDF Reader and Editor Excitation and Emission Spectra If we plot the intensity of fluorescence obtained when the sample is irradiated with monochromatic radiation (λexcitation ) versus wavelength an emission spectrum is obtained. If we plot the excitation and emission spectra of a compound on the same chart the displacement of emission band to a longer wavelength (λ) (is known as stock’s shift).  Made with Xodo PDF Reader and Editor Instrumentation of Fluorescence Component of the instrument 1-Source 5-Read of output excitation 3-The detector 2-Sample cell 4-Monochromator  Made with Xodo PDF Reader and Editor 1-Source of excitation. Either xenon arc lamp ( ultra violet) or mercury arc lamp ( visible). 2-Sample cell Transparent in all sides either glass ( visible) or quartz ( ultra violet ) usually present at right angle with respect to the source and detector. 3-The detector Phototube or photomultiplier tube.  Made with Xodo PDF Reader and Editor 4-Monochromator Filters or grating. Two monochromators are used. One is used for wavelength selection of excitation and the second is used for emission. 5-Read output Recorder or meter scale.  Made with Xodo PDF Reader and Editor 1- Source of radiation Mercury-Xenon arc lamp 2- Primary monochromater 1 3 (λ excitation selector) 2 3- Sample cell transparent from all sides 4 4- Secondary monochromator (filter or grating) (λ emission 5 selector) 5- Detector (photomultiplier tube) 6 6- Read output recorder or meter scale Made with Xodo PDF Reader and Editor Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor  NB. Although the radiation emitted is observed at right angles to the exciting radiation, some of the exciting radiation can be detected by the emission detector because it is scattered by solvent molecules (Rayleigh scatter) or by colloidal particles in solution (Tyndall scatter).  The presence of this scatter makes the use of the second monochromator necessary and also means that the fluorescence band has to be shifted by at least 20 nm beyond the excitation band for fluorescence measurements to be made without interference.  Another weaker type of scatter which may be observed is Raman scatter, In Raman scatter, which is solvent dependent, the wavelength of the incident radiation is shifted to a longer wavelength by about 30 nm when methanol is used as a solvent and about 10 nm when chloroform is used as a solvent. Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor Fluorimetry Lecture  Made with Xodo PDF Reader and Editor fluorescent quantum yield  A quantitative expression of the efficiency of fluorescence is the fluorescent quantum yield , Φ, which is the fraction of excited molecules returning to the ground state by fluorescence. Quantum yields range from , when every 1 molecule in an excited state undergoes fluorescence, to 0 when fluorescence does not occur. Φ Number of molecules that fluorescence = Total number of excited molecules The intensity if fluorescence,If, is proportional to the amount of radiation from the excitation source that is absorbed and the quantum yield for fluorescence where k is a constant accounting for the efficiency of collecting and detecting the fluorescent emission. From Beer’s law  Made with Xodo PDF Reader and Editor Relation between concentration and fluorescence intensity Florescence Intensity (If) is directly proportional to the concentration of the emitter at low concentration (< 10 -5 Molar solution). If = 2.3 K Φf P0 (ε b c ) K = photon measured / photon emitted P0 = Radiation power. Φf = quantum yield or quantum efficiency Φf = no. of molecule fluoresce / no. of molecule excited The intensity of fluorescence increase with an increase in quantum efficiency, incident power of the excitation source, and the molar absorpitivity and concentration of the fluorescing species.  Made with Xodo PDF Reader and Editor For quantitative analysis Plot of If versus C , constitutes fluorescence calibration curve If C  Made with Xodo PDF Reader and Editor Variables affecting fluorescence intensity 1- Quantum yield or quantum efficiency 2- Transition type in fluorescence 3- Fluorescence and Structure 4- Temperature and solvent effect 5- Effect of PH  Made with Xodo PDF Reader and Editor 1-Quantum yield or quantum efficiency No. of fluoresced molecules Qf = No. of excited molecules It reachesunity in highly fluorescent substances and reaches zero in non- fluorescent substances.  Made with Xodo PDF Reader and Editor 2- Transition type in fluorescence Fluorescence is seldom seen in σ* - σ transition using ultraviolet wavelength less than 250 nm ( highly energetic so cause rupture of the bonds.) Fluorescence emission due to less energetic transition П*- П and П*- n transition.π  Made with Xodo PDF Reader and Editor NB: pyridine, furan, thiophene and pyrrole do not exhibit molecular fluorescence. However, fused-ring structures exhibit fluorescence Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor 4- Temperature and solvent effect Φ decreases by increasing temperature due to collision: Temp.↑(collision ↑) or solvent viscosity ↓→ probability of collisional relaxation↑→ quantum efficiency of fluorescence ↓  Made with Xodo PDF Reader and Editor 5-Effect of pH Fluorescence of aromatic compounds with acidic or basic pH-dependent groups (phenol , aniline) Aniline PH-NH2 with lone pair of electrons in alkaline medium fluorescence at about 400 nm while in acid medium it does not fluoresce. Phenol in acid medium differ in its fluorescence from phenate form in alkaline medium [not fluoresce].  Made with Xodo PDF Reader and Editor 6. The Influence of high solute concentrations  As concentration increases, a peak emission is reached then emission decreases because absorption increases more rapidly than the emission.  We say the emission is quenched (decreased) by self- absorption, which is the absorption of excitation or emission energy by analyte molecules in the solution.  At high concentration, even the shape of the emission spectrum can change, because absorption and emission both depend on wavelength.  Made with Xodo PDF Reader and Editor 7. Effect of Dissolved Oxygen  Dissolved oxygen largely limits fluorescence since it promotes intersystem crossing because it is paramagnetic. However, dissolved oxygen affects phosphorescence more than it does to fluorescence. Although one would think that as far as intersystem crossing is increased in presence of oxygen, phosphorescence is expected to increase. On the contrary, phosphorescence is completely eliminated and quenched in presence of dissolved oxygen. This may be explained on the basis that the ground state of oxygen is the triplet state and it is easier for an electron in the triplet state to transfer its energy to triplet oxygen rather than performing a flip in spin and relax to singlet state. Therefore, oxygen will be excited and what we really observe is oxygen emission rather than phosphorescence. It is for this reason that oxygen should be totally excluded to be able to detect phosphorescence.  Made with Xodo PDF Reader and Editor 8. The heavy atom effect or heavy ion effect:  The presence of so - called heavy atoms such as bromine or iodine in either the parent molecule (internal heavy atom effect) or the solvent (external heavy atom effect) increases the probability of intersystem crossing. The heavy atom effect is illustrated in Tables below.  Made with Xodo PDF Reader and Editor 3-Fluorescence and Structure Fluorophore: Molecule that absorbs light but then returns to the ground state by emitting some of the light as a photon rather than losing all the energy as heat. Any molecule that absorbs ultraviolet radiation could fluorescence. The greater the absorption by a molecule, the greater its fluorescence intensity. Many aromatic and heterocyclic compounds fluorescence. Compounds with multiple conjugated double bonds are favorable to fluorescence. One or more electron –donating groups such as – OH,-NH2&-OCH3 enhances the fluorescence. Groups such as –NO2,-COOH, -CH2COOH, -Br, -I,& azo gps tend to inhibit fluorescence.  Made with Xodo PDF Reader and Editor Table: Effect of substitution on the fluorescence of Benzene derivatives * Relative Intensity of Fluorescence  Made with Xodo PDF Reader and Editor Effect of structure rigidity Quantum efficiency or quantum yield increase with increasing the number of fused rings. > > Anthracene naphthalene benzene  Made with Xodo PDF Reader and Editor Effect of structure rigidity Fluorescence is favored by rigidity of structure therefore: Eg. Fluorene (more rigid) where Qf = 1 , Biphenyl (less rigid) where Qf = 0.2 O Zn N C H2 2 Fluorene Biphenyl The Zinc complex Lack of rigidity enhance internal conversion and decrease Qf Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor Compounds which are intrinsically fluorescent (has native fluorescence) are easily determined at very low concentration by simple fluorimetric method for example , phenobarbitone, quinine , emetine , adrenaline, cinchonine, reserpine, vitamin A, riboflavine , thalium І(while thalium ІІІ does not fluoresce), cerium ІІІ (while cerium ІV does not fluoresce), UO2+ and many other pharmaceuticals and natural products.  Made with Xodo PDF Reader and Editor Also non-fluorescent substances can be determined after chemical reaction. Inorganic ions can be determined either by formation of fluorescent chelates upon reaction with fluorimetric reagents e.g. 8-hydroxyquinoline (for Al), benzoin(for Zn) or flavonol (for Zr) or by measuring the quenching of fluorescence of a fluorescent substance in presence of some ions  Made with Xodo PDF Reader and Editor Fluorescence Quenching  One difficulty frequently encountered in fluorescence is that of fluorescence quenching by many substances. These are substances that, in effect, compete for the electronic excitation energy and decrease the quantum yield (the efficiency of conversion of absorbed radiation to fluorescent radiation see below).  Iodide ion is an extremely effective quencher. Iodide and bromide substituent groups decrease the quantum yield.  Substances such as this maybe determined indirectly by measuring the extent of fluorescence quenching.  Some molecules do not fluoresce because they may have a bond whose dissociation energy is less than that of the radiation. In other words, a molecular bond maybe broken, preventing fluorescence.  Made with Xodo PDF Reader and Editor Advantages of fluorimetry 1- Highly sensitive to very low concentrations(10-5 M). 2- highly selective two λex and λem 3- Only aromatic compounds fluoresce while alicyclic or acyclic do not fluoresce therefore useful in their mixture analysis.  Made with Xodo PDF Reader and Editor Disadvantages of fluorimetry 1-Quenching by presence of non-fluorescent compounds. 2-Many organic compounds undergo photochemical reaction by U.V. radiation. 3-It does not exhibits high precision or accuracy +/- 2 to 10 ℅ however its sensitivity and selectivity makes it a method of choice Made with Xodo PDF Reader and Editor  Made with Xodo PDF Reader and Editor Multi Component Analysis Dr. Aya Mohamed Reda  Made with Xodo PDF Reader and Editor Introduction Nowadays multicomponent formulation is used in various combinations of drug forms for enhanced therapeutic index, faster relief, and lesser side effects. The introduction of impurities directly or indirectly or the formation of impurities during various stages of the drug discovery may have an adverse effect, that can cause serious disorders; thus, the development of drug molecules, followed by essential analysis, has radically shifted health sciences research to a new level. The subsequent analysis of developed drug formulation is highly recommended to assess drug safety and side effects and most importantly to ensure the therapeutic effect to the highest level before being placed for approval and commercialization.  Made with Xodo PDF Reader and Editor Samples possessing several components put challenges in analysis and therefore multicomponent analysis has become the topic of choice for analytical chemists for the last few years in the field of drug analysis in clinical chemistry. Different analytical techniques can be applied for multicomponent analysis including; ✓ spectrophotometry ✓ chromatography ✓ electrophoresis Out of several techniques, spectrophotometry is preferred in the estimation of multi-component formulation due to their advantages of cost-effectiveness, less time taking, high specificity and accuracy, etc. 3  Made with Xodo PDF Reader and Editor Spectrophotometric Assay of Binary Mixtures Having a binary mixture of two drugs X and Y; the following methods can be used. Y X Simultaneous equation Derivative spectroscopy #  Made with Xodo PDF Reader and Editor I. Simultaneous equation method  Made with Xodo PDF Reader and Editor Simultaneous Equation Method (Vierordt's method) Th