Instrumental Methods of Analysis PDF

CLIN PHARM 169: PHARMACEUTICAL ANALYSIS AND QUALITY CONTROL INSTRUMENTAL METHOD OF ANALYSIS Professor, Ethel Andrea C. Ladignon, RPh, MS | Reviewed: Sep 27, 2024 | Last Edited: October 1, 2024 This font color is for extr...

CLIN PHARM 169: PHARMACEUTICAL ANALYSIS AND QUALITY CONTROL INSTRUMENTAL METHOD OF ANALYSIS Professor, Ethel Andrea C. Ladignon, RPh, MS | Reviewed: Sep 27, 2024 | Last Edited: October 1, 2024 This font color is for extra information from the OUTLINE professor. SPECTROSCOPY/SPECTROPHOTOMETRY SPECTROSCOPY/SPECTROPHOTOMETRY INTRODUCTION TO SPECTROMETRIC INTRODUCTION TO SPECTROMETRIC METHODS METHODS Spectroscopy ELECTROMAGNETIC RADIATION ○ Science that deals with the interactions of PROPERTIES OF ELECTROMAGNETIC various types of radiation with matter RADIATION Spectrometry ○ Measurement of intensity of radiation with a SPECTROSCOPY/SPECTROPHOTOMETRY: photoelectric transducer or other type of METHODS electronic device UV-VISIBLE SPECTROPHOTOMETRY ELECTROMAGNETIC RADIATION (MOLECULAR ABSORPTION) PROPERTIES OF ELECTROMAGNETIC ABSORPTION SPECTRUM RADIATION BEER-LAMBERT LAW EMR AS A WAVE ABSORPTION SHIFTS Superposition CHROMOPHORIC SHIFTS ○ When two or more waves overlap in space, the resulting disturbance is equal to the HYPSOCHROMIC SHIFT algebraic sum of the individual disturbances. BATHOCHROMIC SHIFT Diffraction ○ The bending and spreading of waves around IR SPECTROPHOTOMETRY an obstacle STRUCTURE ELUCIDATION Transmission ○ The passage of electromagnetic radiation MOLECULAR EMISSION through a medium. SPECTROPHOTOMETRY Refraction FLUORESCENCE SPECTROPHOTOMETRY ○ When light waves change direction as they pass from one medium to another (MOLECULAR EMISSION) Reflection VARIABLES FOR FLUORESCENCE AND ○ When incident light (incoming light) hits an PHOSPHORESCENCE object and bounces off. Scattering QUANTUM YIELD ○ When light bounces off an object in a variety TRANSITION TYPES of directions. The amount of scattering that takes place depends on the wavelength of MOLECULAR STRUCTURE the light and the size and structure of the STRUCTURAL RIGIDITY object Polarization TEMPERATURE ○ Describes the magnitude and the direction of SOLVENT the electric field of the wave. EMR AS A PARTICLE pH Emission CONCENTRATION ○ When a substance gives off electromagnetic DISSOLVED OXYGEN radiation Absorption ○ When electrons in a substance take up energy from electromagnetic radiation types of light Relaxation This font color is for PPT content and ○ When electrons fall from an excited state to NOTE a lower energy state (usuakky the ground figure/table descriptions. state). ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 1 bit.ly/TransDatabase  SPECTROSCOPY/SPECTROPHOTOMETRY: METHODS Type of Wavelength Quantum Spectroscopy Range Transition Gamma ray 0.005- 1.4 Â Nuclear emission Figure 2. Bonding and anti bonding orbitals of a chromophore X-ray absorption, 0.1-100 Â Inner electron emission, For a compound to be suitable for Uv/Vis spectra, if it fluorescence, and contains a chromophore (if there are double or triple diffraction bonds in the compound), it is capable of absorbing light, which can then elicit a value of absorbance in the Vacuum UV 10-180 nm Bonding electrons spectrophotometer. No chromophore, nothing to be absorption determined in the sample or standard. UV/Vis 180-780 nm Bonding electrons ABSORPTION SPECTRUM absorption, emission, and Applications: fluorescence Qualitative ○ Identification IR absorption 0.78-300 μm Rotation/vibration ○ Purity and Raman of molecules Quantitative scattering ○ Single component analysis ○ Mixture analysis ○ Preformulation and Formulation Microwave 0.75-3.75 mm Rotation of Partition coefficient absorption molecules Solubility Drug release Electron spin 3 cm Spin of electrons in resonance a magnetic field Why do we measure absorbance at 𝜆𝑚𝑚𝑚 ? ○ To be able to determine the maximum absorption NOTE: Those in bold text are the only types that are applicable ○ Where you have the most accurate results to the spectroscopy discussion of Ma’am Ethel. UV-VISIBLE SPECTROPHOTOMETRY (MOLECULAR ABSORPTION) Figure 1. Wavelength range Figure 3. UV-Visible spectrum of amoxicillin As you go left, you have higher energy electron volts. The highest energy elicited is the Gamma rays. Gamma rays have the shortest wavelength but the BEER-LAMBERT LAW highest energy, the relationship is inversely Transmittance formula: proportional. T = l t / lo Radiation in the UV/Visible region is absorbed through %T = 100 lt / lo excitation of the electrons involved in the bonds Beer-Lambert Law Formula: between the atoms making up the molecule so that the electron cloud holding the atoms together redistributes A = -logT itself and the orbitals occupied by the bonding A = 𝜺𝜺𝜺 electrons no longer overlap. where: A = absorbance ε = molar absorptivity (L·mol-1·cm-1) b = pathlength C = concentration (mol·L-1) Transmittance is inversely proportional with Absorbance. ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 2 https://docs.google.com/spreadsheets/d/1b22oYSfhn-46eo06DTYg9kFsmU4qyBgPR_bxS5ln0h8/edit?usp=sharing  Aromatic ring effects absorbs a higher wavelength at the visible region Presence of Auxochrome ○ Benzene is a less effective chromophore ○ Auxochrome: Functional group that does not itself absorb at UV region but has an effect of shifting Figure 4. How a Spectrophotometer works chromophore peaks to lower wavelength and increases their intensity ○ Two or more chromophores separated by more than 1 Light source travels at Collimator (lens). As it transmits single bond the total absorption is equal to the sum of light through the lens, the monochromator distributes absorption characteristics of each chromophore light through the slit or wavelength selector to be ○ There is an increase in maximum 𝜆𝑚𝑚𝑚 , by the emitted to the cuvette. substitution of the (auxochrome) polar group in the Higher absorbance = higher concentration visible region. ○ Functional group that is not absorbed in the visible ABSORPTION SHIFTS region but has an effect of shifting chromophores into longer wavelengths and increasing their intensities. The additive characteristics ○ When a molecule contains two or more chromophores separated with more than one single bond. ○ Total absorption = to the sum of absorption characteristics of each chromophore like ethylene and 1,5 - hexadiene. They absorbed the same wavelength but doubled for 1,5 - hexadiene than ethylene in terms of radiations absorbed for similar concentrations. CHROMOPHORIC SHIFTS Figure 5. Effect of Conjugation Figure 7. Solvents effects for Chromophoric Shift Figure 6. Absorption Shifts HYPSOCHROMIC SHIFT The “blue” shift Conjugation Transitions are generally shifted to shorter wavelengths ○ What happens in conjugation, as the number with increasing polarity of solvents. of pi electrons increases, the localization increases as well BATHOCHROMIC SHIFT ○ What happens in the spectrum, due to the The “red” shift increase of localization the molecules of the The reverse is observed sample gets stabilized and hence reach a Transitions were the peak shifts to longer wavelength with state of lower energy increasing solvent polarity. ○ This lowering of energy causes a change in The polarity of solvents causes a pronounced effect on the wavelength towards a higher wavelength position of and intensity of absorption bands. And it is due called the red shift. to the presence of solvents involved in the sample like the ○ Conjugation presence of polar hydrolytic solvents like water. ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 3 https://docs.google.com/spreadsheets/d/1b22oYSfhn-46eo06DTYg9kFsmU4qyBgPR_bxS5ln0h8/edit?usp=sharing  ○ The hydrogen bonds with the lone pair of electrons of ○ 1303-909 cm-1: fingerprint region auxochrome results in the energy of auxochrome ○ 4000-1300 cm-1: functional group region lowers to an equal amount of the bond formation The most important is the broad -OH at about 3350 cm-1. energy. Hence the energy gap between increases so *Students use this usually for their thesis but it is preferred hypsochromic will observe in the transition. for pure compounds not like for some compounds in a IR SPECTROPHOTOMETRY crude extract. The high frequency portion - Functional Group: 4000-1300 Electromagnetic radiation ranging between 2500 and cm-1 20000 nm is passed through a sample and is absorbed by ○ The characteristics are stretching frequency such as the bonds of the molecules in the sample causing them to hydroxyl group (OH group), amino group (NH group) stretch or bend. The wavelength of the radiation absorbed and the carbonyl group (CO group). is characteristic of the bond absorbing it. There is a lack of strong absorption bands in the region of Stretching Vibration 909 - 650 cm-1 that indicate a non aromatic structure. ○ Involves in the continuous change of interatomic For the Fingerprint region - 1303-909 cm-1; frequently distance along the axis of the bonds between two complex with the bands originating and interacting bonds. vibration. MOLECULAR EMISSION SPECTROPHOTOMETRY Figure 8. 2 Types of Stretching Bending ○ Change on the angle between two bond ○ Four types of Bending Scissoring Wagging Rocking Twisting Figure 9. Four Types of Bending Figure 11. Molecular emission spectra FLUORESCENCE SPECTROPHOTOMETRY (MOLECULAR EMISSION) Figure 10. Example of IR Spectrum Unique for a certain functional group. Figure 12. Fluorescence Spectrophotometry Used for identification test Excitation is needed in the panel so that it shows results in the display panel. STRUCTURE ELUCIDATION Interpretation by looking at the peaks and depends of the points stated below: ○ 909-650 cm-1: presence of aromatic structure ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 4 https://docs.google.com/spreadsheets/d/1b22oYSfhn-46eo06DTYg9kFsmU4qyBgPR_bxS5ln0h8/edit?usp=sharing  VARIABLES FOR FLUORESCENCE AND PHOSPHORESCENCE QUANTUM YIELD also called “Quantum efficiency” The ratio of the number of molecules that are luminescent Figure 15. Example of Fluorescence Molecule to the total number of excited molecules. For highly fluorescent molecules, the quantum efficiency Phosphorescence Molecule approaches one. Molecules that do not fluoresce have quantum efficiencies that approach zero. TRANSITION TYPES Figure 16. Example of Phosphorescence Molecule STRUCTURAL RIGIDITY The structural rigidity in a molecule favors fluorescence. The explanation for lower quantum efficiency or lack of rigidity is caused by the enhanced internal conversion rate (kic) which increases the probability that there will be radiationless deactivation. Nonrigid molecules can also undergo low-frequency vibration which accounts for small energy loss. TEMPERATURE Figure 13. Fluorescence Energy Transition As the temperature increases, the frequency of the collision increases which increases the probability of deactivation by Fluorescence energy transition external conversion. ○ As photons have spin-0, only transitions between SOLVENT states of the same spin are spin-allowed. Therefore, Solvents with lower viscosity have higher possibility of where S0 is the singlet ground state (or highest deactivation by external conversion. Fluorescence of a occupied molecular orbital), and S1 is the singlet molecule decreases when its solvent contains heavy atoms excited state (or lowest occupied molecular orbital), such as carbon tetrabromide and ethyl iodide, or when the absorption edge will tend to correspond to the S0 heavy atoms are substituted into the fluorescing → S1 transition. Similarly, the emission peak will also compound. tend to correspond to the S1 → S0 transition. pH Phosphorescence energy Transition ○ an electron is initially excited through absorbance of a The fluorescence of aromatic compounds with basic or acid photon. The electron can then non-radiatively relax substituent rings are usually pH dependent. into the triplet state through intersystem crossing CONCENTRATION (ISC). This transition is not spin-allowed but can occur The power of fluorescence emission F is proportional to due to spin-orbit coupling. the radiant power is proportional to the radiant power of the excitation beam that is absorbed by the system. Figure x. Equation that describes the relationship of concentration to fluorescence and phosphorescence DISSOLVED OXYGEN Dissolved oxygen reduces the intensity of fluorescence in solution, which results from a photochemically induced oxidation of fluorescing species. Figure 14. Phosphorescence Energy Transition MOLECULAR STRUCTURE Fluorescence Molecule ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 5 https://docs.google.com/spreadsheets/d/1b22oYSfhn-46eo06DTYg9kFsmU4qyBgPR_bxS5ln0h8/edit?usp=sharing  https://www.britannica.com/science/principle-of- superposition-wave-motion Wave Behaviors - NASA Science. (n.d.). Retrieved from https://science.nasa.gov/ems/03_behavior REFERENCES: Absorption and Emission — Definition & Overview - Expii. (n.d.). Retrieved from https://www.expii.com/t/absorption-and-emission- definition-overview-8302 D. A. Skoog, et al. "Principles of Instrumental Analysis" 6th Edition, Thomson Brooks/Cole. 2007 D. C. Harris and M.D. Bertolucci "Symmetry and Spectroscopy, An Introduction to Vibrational and Electronic Spectroscopy" Dover Publications, Inc., New York. 1989. Libretexts. (2023, January 30). Photons. Retrieved from https://chem.libretexts.org/Bookshelves/Physical_and _Theoretical_Chemistry_Textbook_Maps/Supplement al_Modules_(Physical_and_Theoretical_Chemistry)/ Quantum_Mechanics/02._Fundamental_Concepts_of _Quantum_Mechanics/Photons Libretexts. (2021, September 8). Polarization of waves. Retrieved from https://eng.libretexts.org/Bookshelves/Materials_Scie nce/Supplemental_Modules_(Materials_Science)/Opti cal_Properties/Polarization_of_Waves Reflection, Transmission and Absorption GigaHertz- Optik. (n.d.). Retrieved from https://www.gigahertz- optik.com/en-us/service-and-support/knowledge- base/basics-light-measurement/light-color/reflec- trans-abs/ T.-S. Ahn, et al. Rev. Sci. Instrum. 78, 086105 (2007). The Editors of Encyclopaedia Britannica. (2024, September 7). Principle of superposition | Definition, Examples, & Facts. Retrieved from ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 6 https://docs.google.com/spreadsheets/d/1b22oYSfhn-46eo06DTYg9kFsmU4qyBgPR_bxS5ln0h8/edit?usp=sharing ADALLA, MAGALUED, MATAAC, SOBERON, TEÑOSO 7 https://docs.google.com/spreadsheets/d/1b22oYSfhn-46eo06DTYg9kFsmU4qyBgPR_bxS5ln0h8/edit?usp=sharing