Lecture 01, 2

• What is the definition of spectroscopy?

  The study of the interaction of electromagnetic radiation in all its forms with matter
• What is the wavelength range of visible light?

  400-800 nm
• What is the wavelength range of UV light?

  150-400 nm
• Why is UV-Vis radiation used to excite electrons to a higher energy state?

  Because UV-Vis radiation has enough energy to excite electrons to a higher energy state
• What is the name given to the part of a molecule that is responsible for light absorption?

  Chromophore
• What is the formula for the Beer-Lambert law?

  A = -log10(I/Io) = log (Io/I)
• How would you calculate the molar attenuation coefficient of paracetamol?

  Make a standard solution of paracetamol with known concentration, acquire its UV spectrum, select a wavelength, note the absorbance and other conditions, then calculate the molar attenuation coefficient
• How can you determine the concentration of an unknown protein solution using UV-Vis spectroscopy?

  Measure the absorbance of the unknown protein solution, then use the known molar extinction coefficient and the Beer-Lambert law to calculate the concentration
• How can you use a Beer's law plot to determine the concentration of an unknown analyte?

  Measure the absorbance of the unknown analyte solution, then use the linear equation from the Beer's law plot (A = ℇbc + b) to calculate the concentration
• What is the limitation of using Beer's law at higher concentrations?

  At higher concentrations the individual particles of the analyte no longer behave independently of each other
• How can the solvent and pH affect the absorption spectrum of an analyte?

  Solvent polarity and pH can affect the absorption spectrum of an analyte
• How can the presence of other molecules with chromophores affect the UV-Vis analysis of a sample?

  The presence of other molecules with chromophores that absorb in the same region of the spectrum will lead to incorrect results
• Why is it important to confirm the identification of a substance using UV-Vis spectroscopy with other methods?

  Because there is a possibility that two substances can have similar UV-Vis spectra, so the identification needs to be confirmed using other techniques
• What are the common structural features of the molecules that show similar UV-Vis spectra?

  The molecules have common structural features like the presence of double bonds and lone pairs of electrons
• What are the key practical considerations when developing a UV-Vis spectroscopy method for analysing a sample?

  Key considerations are: selection of solvent, selection of wavelength, preparation of standards, and proper sample handling
• What is the recommended absorbance range for quantitative measurements when preparing a calibration curve?

  0.2 to 0.8
• Why is it important to prepare the standards in the same way as the sample when developing a UV-Vis method?

  To ensure the standards and sample have the same solvent, pH, and presence of other components
• What are some of the applications of UV-Vis spectroscopy in the pharmaceutical industry?

  Quality control of active ingredients, excipients, and simple pharmaceutical products
• What type of cuvettes should be used for acquiring UV spectra?

  Quartz cuvettes
• What are the key principles of UV-Vis spectroscopy?

  • Absorption of UV-Visible radiation by molecules, typically in solution
  • Electrons are excited to a higher energy state (absorption)
  • The amount of absorption is proportional to the concentration (Beer-Lambert law)
  • Mainly used for quantitative analysis
• How can you use UV-Vis spectroscopy for quantitative analysis?

  • Measure the absorbance of the sample at a specific wavelength
  • Use the Beer-Lambert law (A = ℇbc) to calculate the concentration
  • Determine the molar attenuation coefficient (ℇ) from a standard solution
  • Alternatively, use a calibration curve (Beer's law plot) to determine the concentration
• What are the limitations of using the Beer-Lambert law for quantitative UV-Vis analysis?

  • Beer's law only holds true over a limited concentration range
  • At higher concentrations, the individual particles of the analyte no longer behave independently
  • Presence of other absorbing species can interfere
  • Instrumental factors like non-monochromatic light and stray light can cause deviations
• How would you develop a UV-Vis spectroscopy method for analysing a sample?

  Key steps:

  1. Select an appropriate solvent to dissolve the sample
  2. Choose the wavelength for measurement (ideally at the absorption maximum)
  3. Prepare calibration standards in the same solvent
  4. Measure the absorbance of the standards and construct a calibration curve
  5. Measure the absorbance of the unknown sample and use the calibration to determine the concentration
• How can the UV-Vis spectrum of a compound be used for identification?

  • Each compound has a unique UV-Vis spectrum based on its molecular structure
  • The spectrum can act as a "fingerprint" to identify the compound
  • However, two different compounds can sometimes have similar spectra
  • Therefore, identification using UV-Vis should be confirmed with other techniques
• How do the structural features of a molecule affect its UV-Vis spectrum?

  • Molecules with conjugated double bonds and lone pairs of electrons tend to have strong UV-Vis absorption
  • The position and intensity of absorption peaks are related to the specific chromophores present in the molecule
  • Structural similarities between molecules can lead to similar UV-Vis spectra
• How can the effects of solvent and pH be accounted for in UV-Vis analysis?

  • Solvent polarity and pH can affect the absorption spectrum of an analyte
  • To minimise these effects:
  • Use the same solvent for samples and standards
  • Maintain the same pH conditions for all measurements
  • Perform stability tests to ensure the analyte is not interacting with the solvent
• How can the presence of other absorbing species affect UV-Vis analysis?

  • The presence of other molecules with chromophores that absorb in the same spectral region as the analyte can lead to incorrect results
  • This interference can be minimised by:
  • Selecting a wavelength where the analyte has maximum absorption and the interferents have minimal absorption
  • Using separation techniques like HPLC-UV to isolate the analyte
  • Confirming the identity of the analyte using other analytical methods
• How can UV-Vis spectroscopy be combined with other analytical techniques?

  • UV-Vis spectroscopy can be coupled with separation techniques like HPLC
  • The HPLC-UV method allows for the separation and quantification of analytes in complex mixtures
  • UV-Vis detection provides structural information and can be used for identification in addition to quantification
  • Other combinations include GC-UV, CE-UV, and LC-MS-UV
• What are the key advantages of using UV-Vis spectroscopy?

  • Rapid and easy to perform
  • Relatively inexpensive instrumentation
  • Suitable for quality control of pharmaceuticals and other applications
  • Can be combined with other analytical techniques for enhanced capabilities
• How does the choice of solvent affect the UV-Vis analysis of a sample?

  • The sample must be fully soluble in the chosen solvent
  • The solvent should not interfere with the analyte's absorption spectrum
  • Solvent polarity and pH can influence the shape and position of absorption peaks
  • Stability of the analyte in the solvent must be confirmed
• How would you select the optimal wavelength for quantitative UV-Vis analysis?

  • Choose the wavelength where the analyte has its maximum absorption (absorption maximum)
  • This will provide the highest sensitivity and the best linear calibration curve
  • The calibration curve will be steeper at the absorption maximum, allowing better differentiation between samples with small concentration differences
• How can you prepare standards for a UV-Vis calibration curve?

  • Use chemical reference standards to prepare the calibration standards
  • Ensure the standards are prepared in the same solvent, pH, and with the same matrix as the sample
  • The standards should cover a concentration range where the absorbance is between 0.2 and 0.8 for best precision
  • The sample concentration should ideally fall in the middle of the calibration curve
• What precautions should be taken when handling samples for UV-Vis analysis?

  • Samples in organic solvents should be kept closed to prevent evaporation
  • Quartz cuvettes must be used for UV spectra, glass cuvettes for visible spectra
  • The cuvette walls should not be touched to avoid contamination
  • Proper sample preparation and handling is crucial to obtain accurate results
• What are some common applications of UV-Vis spectroscopy in the pharmaceutical industry?

  • Quality control of active pharmaceutical ingredients and excipients
  • Quantification of simple pharmaceutical products
  • Combination with other techniques like HPLC for enhanced analysis capabilities
  • Rapid and cost-effective analytical method compared to other techniques