Spectroscopy for Pharmaceutical Analysis and Instrumental Method of Analysis.pptx

Spectroscopy for Pharmaceutical Analysis and Instrumental Method of Analysis.
Atomic spectroscopy, Molecular Spectroscopy, Beer Lambert's Law, Fundamental Laws of Photometry, application of beer lambert law in equilibrium constant, Chromophore, Auxochrome, Bathochromic shift, Hypsochromic shift, Hypochromic and Hyperchromic effects, Effect of solvent on absorption spectra

1. DISCOVER . LEARN . EMPOWER Mr. Yunes Alsayadi Assistant Professor of Pharmaceutical Analysis E 10695 UNIVERSITY INSTITUTE OF PHARMA SCIENCES Pharm.D Spectroscopy PST-392

2. Contents Theory of electronic, atomic and molecular spectra. Fundamental laws of photometry Beer-lambert’s Law Determining An Equilibrium Constant Using Spectrophotometry and Beer’s Law Chromophore Auxochrome Bathochramic Shift or Effect Hypsochromic Shift or Effect Hyperchromic Effect Solvent effect on absorption spectra 2

3. ELECTRONIC SPECTROSCOPY All organic compounds are capable of absorbing electromagnetic radiation because all contain valency electrons that can be excited to higher energy levels. The excitation energies associated with electrons forming most single bonds are high. E lectronic Spectroscopy relies on the quantized nature of energy states. Given enough energy, an electron can be excited from its initial ground state or initial excited state (hot band) and briefly exist in a higher energy excited state. Electronic transitions involve exciting an electron from one principle quantum state to another. Without incentive, an electron will not transition to a higher level. Only by absorbing energy, can an electron be excited. Once it is in the excited state, it will relax back to it's original more energetically stable state, and in the process, release energy as photons. 3

4. The science of spectroscopy grew out of studies of the interaction of electromagnetic energy with matter. When light shines on an object, for example, we know that part of the light is scattered (reflected) and part is absorbed. Of the initial part that is absorbed, some is later emitted as light of a different color or wavelength. Spectroscopy is that science which attempts to determine what specific energies and amounts of incident light are absorbed by specific substances, and what specific energies and amounts are later re-emitted. 4

5. Regions of the electromagnetic spectrum

6. Electromagnetic radiation moves in waves

7. Optical instruments called spectrometers reveal in photographic or printed records—as a series of specific wavelengths or frequencies—the light energies absorbed and emitted. These records, in turn— referred to as spectra—provide us with important information pertaining to the atomic and molecular structure of the substances on which the electromagnetic energy is focused. 7

8. Often, during electronic spectroscopy, the electron is excited first from an initial low energy state to a higher state by absorbing photon energy from the spectrophotometer. If the wavelength of the incident beam has enough energy to promote an electron to a higher level, then we can detect this in the absorbance spectrum. Once in the excited state, the electron has higher potential energy and will relax back to a lower state by emitting photon energy. This is called fluorescence and can be detected in the spectrum as well. 8

9. 9

10. Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm This limits the transitions that can be observed: 10

11. 11

12. An electron in a bonding s orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergo    transitions) shows an absorbance maximum at 125 nm. Absorption maxima due to    transitions are not seen in typical UV-VIS spectra (200 - 700 nm) 12

13. Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n   transitions. These transitions usually need less energy than     transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups with n   peaks in the UV region is small. 13

14. ▣ Most absorption spectroscopy of organic compounds is based on transitions of n or  electrons to the  excited state. ▣ These transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the  electrons. 14

15. 15 Chromophore Excitation  max , nm Solvent C=C  →  * 171 hexane C=O n→  *  →  * 290 180 he x ane he x ane N=O n→  *  →  * 275 200 ethanol ethanol C-X X=Br, I n →  * n →  * 205 255 he x ane he x ane

16. ANALYTICAL TECHNIQUES –SPECTROSCOPY Spectroscopy is the branch of science dealing with the study of interaction of electromagnetic radiation with matter. It is a powerful tool available for the study of atomic and molecular structure and it is also used in the analysis of most of the samples. Types of Spectroscopy :- The study of spectroscopy is divided into two types. They are, 1.     Atomic spectroscopy  2.     Molecular spectroscopy. 16

17. Atomic spectroscopy is the determination of elemental composition by its electromagnetic or mass spectrum. The study of the electromagnetic spectrum of elements is called Optical Atomic Spectroscopy. Electrons exist in energy levels within an atom. These levels have well defined energies and electrons moving between them must absorb or emit energy equal to the difference between them. Since every element has a unique electronic structure, the wavelength of light emitted is a unique property of each individual element. 17 Atomic Spectroscopy

18. The science of atomic spectroscopy has yielded three techniques for analytical use: 1. Atomic Absorption 2. Atomic Emission 3. Atomic Fluorescence The process of excitation and decay to the ground state is involved in all three fields of atomic spectroscopy. Either the energy absorbed in the excitation process, or the energy emitted in the decay process is measured and used for analytical purposes.

19. If light of just the right wavelength impinges on a free, ground state atom, the atom may absorb the light as it enters an excited state in a process known as atomic absorption . By measuring the amount of light absorbed, a quantitative determination of the amount of analyte element present can be made.  In atomic emission , a sample is subjected to a high energy, thermal environment in order to produce excited state atoms, capable of emitting light. The energy source can be an electrical arc, a flame, or more recently, a plasma. Atomic emission using electrical arcs has been widely used in qualitative analysis. Emission techniques can also be used to determine how much of an element is present in a sample. For a "quantitative" analysis, the intensity of light emitted at the wavelength of the element to be determined is measured.  19

20. 20 The third field of atomic spectroscopy is atomic fluorescence . This technique incorporates aspects of both atomic absorption and atomic emission. Like atomic absorption, ground state atoms created in a flame are excited by focusing a beam of light into the atomic vapor. Instead of looking at the amount of light absorbed in the process, however, the emission resulting from the decay of the atoms excited by the source light is measured. The intensity of this "fluorescence" increases with increasing atom concentration, providing the basis for quantitative determination. Most scorpions glow a  blue-green color  when illuminated by ultraviolet light or natural moonlight.

21. Molecular spectroscopy A molecule is a collection of positively charged atomic nuclei  surrounded by a cloud of negatively charged electrons. Molecular spectra  result from either the absorption or the emission of electromagnetic radiation as molecules undergo changes from one quantized energy state to another. The mechanisms involved are similar to those observed for atoms but are more complicated. The additional complexities are due to interactions of the various nuclei with each other and with the electrons, phenomena which do not exist in single atoms.

22. There are two primary sets of interactions that contribute to observed molecular spectra. The first involves the internal motions of the nuclear framework of the molecule and the attractive and repulsive forces among the nuclei and electrons. The interaction of electromagnetic radiation with these molecular energy levels constitutes the basis for electron spectroscopy, visible, infrared (IR) and ultraviolet (UV) spectroscopies, Raman spectroscopy, and gas-phase microwave spectroscopy. The first set of interactions can be divided into the three categories given here in decreasing order of magnitude: Electronic, Vibrational, And rotational.

23. The other set encompasses the interactions of nuclear magnetic and electrostatic moments with the electrons and with each other. The second set of molecular interactions form the basis for  Nuclear magnetic resonance (NMR) spectroscopy,  Electron spin resonance (ESR) spectroscopy, And nuclear quadrupole resonance (NQR) spectroscopy. The first two arise, respectively, from the interaction of the magnetic moment of a nucleus or an electron with an external magnetic field. The nature of this interaction is highly dependent on the molecular environment in which the nucleus or electron is located.

24. Differences between atomic spectra and molecular spectra Atomic Spectroscopy It deals with the interactions of electromagnetic radiation with atoms. Molecular Spectroscopy It deals with the interaction of electromagnetic radiation with molecules.

25. Photometry  is the science of the  measurement  of  light , in terms of its perceived  brightness  to the  human eye It is distinct from  radiometry , which is the science of measurement of  radiant energy  (including light) in terms of absolute power. In modern photometry, the radiant power at each wavelength is weighted by a  luminosity function  that models human brightness sensitivity. Fundamental Laws of photometry

26. There are two principal laws utilized to produce photometric measurements. They are known as the inverse square law and the cosine law . The inverse square law determines the correlation between illumination from a constant-intensity source and its distance from the surface. It states that “ “The intensity of illumination of surface (E)  or illumination of surface (E)  is inversely proportional to the square of the distance between the surface and source”. In other words, the it states that the illuminance decreases with the square of the distance between the light source and the illuminated surface. This means: if the distance between the light source and illuminated surface is doubled, four times the luminous intensity is needed for the same illuminance. Fundamental Laws of photometry

27. Lambert’s Cosine Law Lambert’s cosine law states that the strength of light on a surface of a fixed area varies with incident angle. When light hits a surface obliquely, the illumination of the surface is directly proportional to the cosine of the angle θ between the direction of the incident light and the surface normal. It means that the radiant intensity from the ideal diffusely reflecting surface and cosine of the angle θ between the direction of incident light and surface normal are directly proportional. Mathematically, E (illumination from a surface) ∝ Cos Ɵ 27

28. 28

29. Lambert’s Law : It states the relationship between the radiant power of absorbed light with the thickness of the medium.

30. When a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of radiant power with the thickness of the medium is directly proportional to the radiant power of the incident light. dP is change in radiant power, db is small thickness of sample, k 1 is proportionality constant and minus sign indicate radiant power decrease - d P P db -dP = k 1 P db Lambert’s Law

31. Beer’s Law :  When monochromatic light passes through a ‘transparent medium’, the rate of decrease of transmitted radiation with the increase in the concentration of the medium is directly proportional to the intensity of the incident light. Beer's Law states that the concentration  of a chemical solution is directly proportional to its absorption of light . Combined formed is called Lambert-Beer’s law.

32. The law deals with the relationship between the radiant power of the incident light and transmitted light as a function of both the thickness of the medium and the concentration of the absorbing species. Lambert’s law Absorbance = Constant x Thickness of the medium x C o ncen t r a t i on of t he Beer’s Law A bs o r b a n c e = C o ns t a nt medium Lambert-Beer’s law

33. Lambert-Beer’s Law The combined law may be given by the relation – x Absorbance = Constant x [Thickness of the medium] [Concentration of the medium] For a given material, the sample path length and concentration of the sample are directly proportional to the absorbance of the light .

34. • the number of molecules capable of absorbing light of a given wavelength, the the extent of light absorption. The more effectively a molecule absorbs light of a given wavelength, the greater the extent of light absorption. Fro m t h ese guidi n g ideas , BE E R – LAMBE R T LAW may be formulated: ▣ Where, ▣ A = absorbance ▣ I° = intensity of light incident upon sample cell ▣ ▣ ▣ ▣ I = intensity of light leaving sample cell c = molar concentration of solute l = length of sample cell Ɛ = molar absorptivity

35. Deviations to the law The Beer-Lambert law maintains linearity under specific conditions only. The law will make inaccurate measurements at high concentrations because the molecules of the analyte exhibit stronger intermolecular and electrostatics interactions which is due to the lesser amount of space between molecules. This can change the molar absorptivity of the analyte. Not only does high concentrations change molar absorptivity, but it also changes the refractive index of the solution causing departures from the Beer-Lambert law. 35

36. Applications The important use of the Beer Lambert law is found in electromagnetic spectroscopy.  To analyze the drugs, for that, let’s take an example of a tablet: Let’s suppose we have a tablet and we don’t know which drug is present in it. Though we may know the drug, then the question arises about what its molar concentration is. In electromagnetic spectroscopy, we use electromagnetic radiation (we may take UV rays), which scans the tablet and determines the qualitative (drug present) and the quantitative (concentration) property of the tablet. The same method can be used in determining the molar absorbance of bilirubin in blood plasma samples. We use Beer Lambert Law to conduct a qualitative and quantitative analysis of biological and dosimetric materials that may contain organic or inorganic materials. We can determine the concentration of various substances in cell structures by measuring their absorbing spectra in the cell. 36

37. Limitations of the Beer-Lambert law The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include: Deviations in absorptivity coefficients at  high concentrations (>0.01M)  due to electrostatic interactions between molecules in close proximity. Scattering of light due to particulates  in the sample. Fluorescence or phosphorescence  of the sample Changes in refractive index at high analyte concentration Shifts in chemical equilibria as a function of concentration Non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band stray light. 37

38. Determining An Equilibrium Constant Using Spectrophotometry and Beer Lambert’s Law One of the fundamental problems in chemistry is how to determine the extent of a reaction. While not every chemical reaction goes to completion, they usually approach an equilibrium state. When the system reaches equilibrium, the concentrations of the reactants and products no longer change over time. This does not mean that the reaction has ceased. In fact, the reaction continues to progress forward, as well as backward. It is said to be in a dynamic state of equilibrium where the rate of the products being formed from the reactants is exactly the same as the rate of the products being decomposed to form the reactants. For the general equilibrium reaction aA + bB < = > cC + dD (1) 38

39. When studying the equilibrium of chemical systems, one of the most important quantities to determine is the equilibrium constant, Keq . At equilibrium at a given temperature, the mass action expression is a constant, known as the equilibrium constant, Keq . The equilibrium expression for the reaction in Equation 1 is given as: The value of the equilibrium constant may be determined from experimental data if the concentrations of both the reactants and the products are known. Additionally, all equilibrium concentrations can be calculated if a single equilibrium concentration is known along with all other “initial” concentrations. 39

40. It may be recalled that in spectrophotometric studies, the Beer-Lambert law, or Beer’s Law, can be used to determine the concentration of highly colored species. Mathematically, Beer’s Law can be stated as: A = abc where “a” is the molar absorptivity, “b” is the path length, “c” is the concentration, and “A” is absorbance. Molar absorptivity, a, is a proportionality constant that has a specific value for each absorbing species at a given wavelength. The path length, b, is the distance across the solution in centimeters and is dependent upon the size of the cuvette. In this case, the path length will be kept constant at 1.00 cm. The concentration, c, of the absorbing species is in moles of solute per liter of solution. For more details, refer this link: https://adamcap.com/schoolwork/1470/#:~:text=The%20reaction%20is%20represented%20by,and%20ultimately%20the%20equilibrium%20constant . 40

41. Chromophores It is Greek word Chroma means colour , phoros means bearer. The term chromophore was previously used to denote a functional group of some other structural feature of which gives a color to compound. For example- Nitro group is a chromophore because its presence in a compound gives yellow color to the compound.  But these days the term chromophore is used in a much broader sense which may be defined as “any group which exhibit absorption of electromagnetic radiation in a visible or ultra-visible region “It may or may not impart any color to the compound. Some of the important chromophores are: ethylene, acetylene, carbonyls, acids, esters and nitrile groups etc. 41

42. Auxochrome An auxochrome (from Ancient Greek α ὐξάνω auxanō "increase" and χρῶμ α chrōma "colour") is  a group of atoms attached to a chromophore  which modifies the ability of that chromophore to absorb light. It is a group which itself does not act as a chromophore but when attached to a chromophore, it shifts the absorption towards longer wavelength along with an increase in the intensity of absorption. Some commonly known auxochromic groups are: -OH, -NH2, -OR, -NHR, and –NR2. For example: When the auxochrome –NH2 group is attached to benzene ring. Its absorption change from λ max 225 to λmax 280. 42

43. Bathochromic Shift or Red Effect The shift of an absorption maximum to a Ionger wavelength due to the presence of an auxochrome , or solvent effect is called a bathochromic shift or red shift. For example, benzene shows λmax 256 nm and aniline shows λmax 280 nm. Thus, there is a bathochromic shift of 24 nm in the λmax of benzene due to the presence of the auxochrome NH2. 43

44. Hypsochromic Shift or Blue Effect The shift of an absorption maximum to a shorter wavelength is called hypsochromic or blue shift. This is caused by the removal of conjugation or change in the solvent polarity. Hyperchromic Effect. An effect which leads to an increase in absorption intensity εmax is called hyperchromic effect . The introduction of an auxochrome usually causes hyperchromic shift. For example, benzene shows B-band at 256 nm, εmax 200, whereas aniline shows B-band at 280 nm,due to the hyperchromic effect of the auxochrome NH2. 44

45. Solvent effect on absorption spectra Solvents play an important role in UV spectra. Compound peak could be obscured by the solvent peak. So, a most suitable solvent is one that does not itself get absorbed in the region under investigation. A solvent should be transparent in a particular region. A dilute solution of sample is always prepared for analysis. 45 Most commonly used solvents are as follows. in another solvent. Solvent Absorption Maxima (nm) Water 191 Ether 215 Methanol 203 Ethanol 204 Chloroform 237 Carbon Tetrachloride 265 Benzene 280

46. Two broad categories of shift phenomena have been defined: (1) Many, especially the larger, shifts are attributed to specific chemical effects of the solvent on one or both electronic states of the chromaphore . Some important specific effects are: hydrogen-bond formation; proton or charge transfer between solvent and solute; and solvent-dependent aggregation, ionization, and isomerization equilibria. Such specific effects are outside the scope of this discussion. (2) The second broad category of shifts are attributed to physical interactions between the solute and solvent molecules. These interactions must be considered even when the chromaphore is not involved in a solvent-dependent reaction. 46

47. The presence of specific and nonspecific interaction between the solvent and the solute molecules are responsible for the change in the molecular geometry, electronic structure and dipolar moment of the solute. These solute-solvent interactions affect the solute’s electronic absorption spectrum and this phenomenon is referred to as solvatochromism . Moreover, the behaviour of a solute in a neat solvent is very different from the behaviour in mixed binary solvent systems. In these kinds of systems, the solute may induce a change in the composition of the solvents in the cybotactic region compared to that in the bulk leading to preferential solvation. This situation commonly results from specific (hydrogen bonding) and non-specific (dielectric effects) interactions. 47

48. THANK YOU For queries Email: [email protected] 48