Separation chromotography

• What are pure substances and what are there properties?


  Pure substances contains only one type of atom or molecule.

  They have a fixed melting and boiling point
• What are mixtures?


  Mixtures contain more than one thing and do not have exact properties
• How are mixtures separated?

  Purification is the physical separation of a chemical substance of interest from foreign or contaminating substances
• Why do drugs need to be pure?

  –    Impurities could be harmful and/or cause additional effects

  –    Can also alter the ability to formulate a drug correctly

  -Can affect the stability and shelf-life: Blister packs are commonly used to protect medications from water, air, and microbes, which could degrade the drug and shorten shelf-life.
• What are the different ways drugs are purified?

  • Distillation: Separates liquids based on boiling points.
  • Filtration: Removes solids from liquids.
  • Centrifugation: Uses centrifugal force to separate solids from liquids.
  • Recrystallization: Purifies solids by dissolving and recrystallizing.
  • Liquid-Liquid Extraction: Separates compounds based on solubility in different solvents.
  • Chromatography: Separates components of a mixture using a stationary and mobile phase.
  • Solid Phase Extraction: Isolates compounds by passing a liquid through a solid phase.
• How would you know which method you should use to purify the drug?

  Separation of compounds is dependent on the properties of the molecules being different.

  This is dependent on physical properties, how many components there are and how much you have.
• What are the factors that affect solubility?

  • Type of solvent
  • Temperature
  • Nature of forces between solvent and solute (e.g. Ionic compounds: more soluble in polar solvents than nonpolar solvents.)
  • Functional groups
  • Ionisation
  • Molecular size
• Are carboxylic acids and aliphatic amines always charged?


  Yes at physiological pH they are always charged no matter what functional groups are attached in the molecule
• List these functional groups from most polar to least polar: Ketone, flurocarbons, ethers, amides, alkanes, ester, aldehyde, amines, alcohols?


        

  Amide

  Alcohol

  Amine

  Ketone

  Aldehyde

  Ester

  Ether

  Alkane

  Fluorocarbons           
• What are partially miscible solvents?

  Partially miscible solvents can dissolve in each other only up to a certain threshold.

  For example, isopropyl and a buffer they can mix without forming 2 phases unless you add too much of isopropyl and therefore will form 2 phases.
• Give examples of polar, less polar & non-polar solvents?

  Polar: water

  Less polar: ethanol, acetone

  Non-polar: chloroform, hexane, octanol
• Describe the liquid-liquid separation technique using beta-carotene?

  Hexane, an organic solvent, dissolves beta-carotene (a non-polar compound), while water serves as the second phase.

  After mixing, the immiscible solvents form two layers: the organic phase (top) and aqueous phase (bottom, containing glucose).

  The top layer contains the liquid with a lower density, whereas bottom layer will contain a liquid with a higher density

  The layers are separated using a separatory funnel, and the organic layer is purified by evaporation to remove the solvent, leaving purified beta-carotene.

  
• What requirements does the organic solvent need to meet for the liquid-liquid separation?

  –        Should readily dissolve substance to be extracted.

  –        Should not react with the substance to be extracted.

  –        Should not react with or be miscible with water (the usual second solvent).

  –        Should ideally have a low boiling point so it can be easily removed from the product
• Describe how you can separate aspirin and codeine?

  Dissolve aspirin and codeine in the organic solvent (toluene) and add H2O.

  If you add HCL, it will protonate the amine in codeine and form a salt and will not be soluble in toulene because its polar.

  The compound will move to the aqueous phase, which can then be separated by running it off in the tap.

  Collect in a conical flask and neutralise the compound by adding a base, so it wont be soluble anymore, thus become a solid
• What happens if the compound is soluble in both layers?

  Its a reversible process so the amount of compound that are in both layers varies depending on its properties.

  If the compound is more polar then more of it will be in the aqueous phase.
• How does the solute distribute itself in 2 liquids?

  It distributes itself in accordance with its partition coefficient.

  A dynamic equilibrium is established between the 2 liquids
• What is the partition coefficient?

  The ratio of the conc of the solute in one liquid over the conc in the other.
• What does the partition coefficient depend on?

  Temperature as solubility changes with temperature
• How do you calculate the partition coefficient?

  Po/a = Conc in organic/ conc in aqueous
• Whats log p?


  Used as a measure of the hydrophobicity of a drug

  Measures only the unionised form of the drug between octanol and water.

  
  

  If log P is less than 0 =hydrophilic

  log p above 0 = hydrophobic

  log P = 0 equal affinity for water and lipids
• Why doesnt log P measure the ionised form of the drug?

  Because the drug is only in water and therefore wont be ionised as theres no acid/base being added to it
• Whats lipinski's rule of 5?

  Drug-like molecules have a log P of less than +5:

  mw less than 500 Da

  Fewer than 5 HBD

  Fewer than 10 HBA

  Log P less than 5
• What does druglikeness mean?

  Properties of a molecule that make it likely to become a drug.

  Properties include:

  • Absorption: How well the drug is absorbed into the bloodstream
  • Distribution: How the drug spreads through the body to reach its target
  • Metabolism: How the drug is chemically broken down in the body, often in the liver.
  • Elimination: How the drug or its metabolites are excreted, typically through urine or feces.
• From looking at the structure of metformin would you say its polar or non-polar?

  Polar:

  It has a log p of -0.9

  A lot of electronegative atoms

  The molecule is small

  
• Whats chromatography?


  The physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary and other is mobile
• What determines whether a component prefers the mobile or stationary phase?

  Whether its ionic, dipolar or non-polar
• Whats the distribution coefficient?

  Partitioning of a solute between two immiscible phases.

  Kx = C (Stat)/ C (mob)

  C(stat) = concentration of compound X in the stationary phase

  C(mob) = the concentration of compound X in the mobile phase
• Describe the chromatographic process?


  The stationary phase consists of particles that provide a surface for the analytes to interact with.

  These interactions (e.g., adsorption, partitioning) determine how much time an analyte spends in the stationary phase.

  As the mobile phase flows over the stationary phase particles, analytes partition between the two phases based on their Kx.

  This differential movement leads to the separation of analytes based on their Kx values.

  
• What do the Kx values mean?

  • Weakly retained analytes (small Kx) elute first, moving quickly with the mobile phase, as they have weaker interaction
  • Strongly retained analytes (large Kx) elute later, as they spend more time bound to the stationary phase due to the strong affinity
• Whats the eluent?


  Fluid/solvent entering the column
• Whats the eluate?


  Fluid/solvent leaving the column
• Whats elution?


  Process of passing liquid through a chromatography column
• Describe solid phase extraction?


  • Conditioning: Prepare the stationary phase with a buffer to activate it, this ensures the stationary phase is well-prepared to retain the analyte and facilitates optimal interaction
  • Loading: Add the sample, allowing the analyte to bind to the solid phase, while other compounds flow through.
  • Washing: Use a solvent to remove unwanted compounds that don’t bind to the stationary phase.
  • Elution: Use a stronger solvent to release the analyte, which is then collected for further use, as you need to break the interaction between the analyte and stationary phase
• Whats adsorption chromatography?

  Separation based on differences between the adsorption affinities of the sample analytes for the surface of a solid stationary phase
• Whats commonly used for the stationary phase in adsorption chromatogrphy?

  Silica
• What is adsorption chromatography used for?

  Organic molecules based on non-covalent interactions between the stationary (solid) and mobile phase (liquid)

  H-bond

  electrostatic

  dipole-dipole
• Whats partition chromatograph?

  Separation based on differences between the solubility of the sample analytes in mobile (liquid) and stationary phase (liquid - solid but behaves as a liquid).
• Whats parition chromatography used for?

  Polar organic molecules based on non-covalent interactions:

  electrostatic

  dipole - dipole

  H-bond
• What are the two types of stationary phase?


  Normal phase: using silica/alumina. Polar compounds are going to be strongly retained as they can interact via H-b etc- increase elution

  Reverse phase: modified silica (OH is replaced by long alkyl chain). Less polar compounds are strongly retained due to hydrophobic interactions - increase elution
• What are the two types of stationary phase?

  Normal phase: using silica/alumina. Polar compounds are going to be strongly retained as they can interact via H-b etc- increase elution

  Reverse phase: modified silica (OH is replaced by long alkyl chain). Less polar compounds are strongly retained due to hydrophobic interactions - increase elution