lecture 16-19

• Chromophore


  Colour bearer
• Chromophores


  • Absorb radiation
  • Emit light as a result
• The more conjugated (sharing of electrons (double and triple bonds))


  The longer and more intense the wavelength of absorption
• Auxochrome


  An OH group attached to a chromophore

  
• Auxochromes


  • Have no absorption above 200nm on their own
  • When attached to a chromophore, changes its absorption
• Auxochromes


  Attach to chromophores and increase conjugation (sharing of electrons)

  
• UV-Vis spectroscopy


  Generally considered a quantitative rather than qualitative technique
• IR spectroscopy


  Good for determining the functional groups within the molecule (i.e the structure of the unknown drug)
• UV spectroscopy


  Good for quantifying the compound (seeing how much there is)
• UV spectra cannot be used to give structure because of the peaks - it only gives parts where chromophores are absorbed (i.e. Where light is emitted)
• Beer-Lambert Law


  Used to determine the concentration of a drug
• A = ecl
• C = A/el
• HPLC is an analytical or other quality control test for identity, homogeneity or purity of a drug in a pharmaceutical product
• High Performance Liquid Chromatography (HPLC)


  Separates drugs based on their partitioning between a stationary phase and a mobile phase based on their relative affinity for the two phases

  
• Components of an HPLC system


  • Computer with chromatographic software
  • HPLC pump
  • Autosampler (injector)
  • UV monitor/diode array detector
  • Solvent reservoirs
  • Column - packed with different types of resin that allow for the chromatographic separation

  
• Normal phase HPLC


  • Polar or hydrophilic
  • Silica as the stationary phase
  • Contains side chains that are hydroxyl groups
  • Forms H bonds with functional groups of drug molecules (OH and NH)
  • High affinity for polar analytes (they stick to solid phase)
  • Increased non-polar solvent (hexane in this case) to improve separation (resolution) of analytes

  
• Reverse phase HPLC


  • Non polar or hydrophobic - forms hydrophobic interactions with non-polar functional groups
  • The more lipophilic the drug the better the drug distribution
  • Increasing polar solvent improves separation of analytes --> lipophilic/hydrophobic compounds are retained for longer as they are less soluble in solvent system

  
• Sample preparation for HPLC

  • Protein precipitation and centrifugation
  • Liquid-liquid extraction (LLE)
  • Solid phase extraction (SPE)
  • To deliver reproducible and specific results
• External calibration method


  • Area of sample compared to standard can determine concentration
  • Drug being analysed can be identified by its retention time
• Internal calibration method


  • Used to correct variations in the sample and analysis
  • Used to calibrate the instrument
  • Solutions of known concentration are prepared that contain standards of the same drug to be analysed
  • Compound of interest is different to the analyte
• Oxidation


  • Involves loss of electrons (LEO, OIL)
  • Loss of hydrogen or addition of oxygen
  • Oxygen radicals form from O2 - light can accelerate the generation of radical - as why storage of medications is important
  • Once one free radical starts --> has a snowball effect = quick degradation - propagation event
  • Antioxidant stops this - terminates free radicals
  • Oxygen radicals are oxidised during phase 1 metabolic reactions - do this by generating reactive oxygen species (free radicals)
• Reduction


  • Gain of electrons
  • Adding on hydrogen and loosing bonds to oxygen
• Functional groups prone to oxidation

  • Alkenes
  • Thiols and thio ethers
  • Aromatic rings
• Functional groups prone to reduction

  • Carboxylic acid or ketone
  • Azo and nitro groups
• Hydrolysis is not a redox reaction
• Photolysis


  • When something is radiated with light it goes to an antibinding orbital
  • Photolysis can lead to different products forming
  • Longer wavelengths penetrate deeper in the skin --> UNA is longer wavelength - drugs can degrade by photolysis
• Electrophilic aromatic substitution reaction

  • One of the H atoms on a ring is substituted with another functional group
  • Electrophile vs nucleophile - determines flow of electrons
  • When nucleophile attacks --> loses aromaticity
  • Substituted --> get aromaticity back
  • Activating and deactivating groups --> pushing more electrons into the ring = activating groups --> increasing electron density --> makes more reactive nucleophile
  • Electrophile will add into the least sterically hindered position (either para or ortho but not meta) - oxidised here
• Dealkylation


  • Replacement of an alkyl group (often a methyl group) with a hydrogen
  • Not reversible
  • Part of a phase 1 metabolic process
  • Catalysed by demethylase, peroxidase or a cytochrome P450 enzyme
  • Nor etc - means loss of methyl group from drug molecule
• Covalent attachment of drugs to their target

  • Nucleophile donates a pair of electrons
  • Electrophile accepts a pair of electrons
  • Reactions occur when nucleophile donates and electrophile accepts them
  • Single headed arrow = free radical reaction (1 electron)
  • Double headed arrow --> movement of two electrons
  • The more electron rich the nucleophile --> the more reactive
  • The more electron poor the electrophile is --> the more reactive
• Covalent drug


  • Drugs react with their target and form a covalent bond (irreversible reaction)
  • Examples: Alkylation and acylation
  • Suicide inhibitors
• Alkylation


  • Transfer of an alkyl group to a C, N, O or S
  • A new covalent bond is formed
  • Alkyl bond can form between the drug and the target - the drug acts as the electrophile - biological partner acts as a nucleophile
  • Amino acids can have nucleophiles of their side chains e.g. Alcohol on serine
  • All these functional groups have lone electrons and a proton that can be removed under certain pH conditions
• Acylation


  • Transfer of an acyl group to a molecule
  • A new covalent bond is formed
  • An acyl bond can form between a drug and the target - drug is the electrophile and partner is the nucleophile
  • The drug's electrophile is the carbonyl carbon of the acyl group
• Stationary Phase


  The solid component of an HPLC system that interacts with the sample. Can be a column packing or membrane.
• Mobile Phase


  The liquid component of an HPLC system that carries the sample through the stationary phase. Can be water, organic solvent, or a mixture of both.
• HPLC


  A laboratory technique used to separate, identify, and quantify the components of a mixture by interacting with a stationary phase and a mobile phase.
• Chromatography


  The combination of stationary and mobile phases that separates the components of a sample based on their interactions.
• Applications of HPLC


  Pharmaceutical analysis, food analysis, environmental monitoring, biological research (protein purification, gene expression)
• Pumps


  The HPLC pump(s) provide a precise and controlled flow rate of the mobile phase to the column.
• Column


  The column is the heart of the HPLC system, where the sample is injected and separated, and is typically a length of thin-walled glass or fused silica tubing coated with the stationary phase.