CHEMLAB

• Chromatography

  • Components to be separated are distributed between two phases: stationary and mobile
  • Used for analyzing, identifying, and monitoring the separated components of a mixture
• Stationary phase

  • can be adsorbent powder or gel (solid)
  • loaded in the column as a slurry (liquid)
  • homogeneous, no air bubbles, no dry patches
  • silica gel and alumina (highly polar)
• Stationary phase is uniform in shape, no reaction with acids, bases, and any solvents during the experiment as it has a high mechanical stability and should be chemically inert
• Eluent

  • Fraction of the mobile phase that transports the sample components
  • Typically an organic solvent
• Column Chromatography

  • A purification method to separate compounds in a solution
  • A solution mixture is carried by a solvent through a column
  • Molecules in the column travel at different rates based on their chemical properties and affinity with SP
  • Compounds that interact weakly with SP are faster to exit the column
  • Compounds that interact strongly qith SP are slower to elute
• Mobile phase

  • can be liquid, gas, or supercritical fluid
  • solvent + sample mixture
• Nonpolar solvents elute nonpolar compounds

  Polar solvents elute both nonpolar and polar compounds
• Safety Data Sheet

  Hexane: harmful when inhaled, ingested, to the eyes, skin, and environment; flammable

  Acetone: harmful when inhaled, ingested, to the eyes, skin; flammable

  Magnesium sulfate (MgSO4), anhydrous: harmful when inhaled, ingested, to the eyes, skin

  Acid-washed sand: harmful to environment
• Adsorption, Partition, Ion Exchange, Size Exclusion, Affinity


  BAsed on the interaction of solute to the stationary phase
• TLC, Paper Chromatography

  Based on chromatographic bed shape that is 2D
• Column chromatography

  Based on chromatographic bed shape that is 3D
• Liquid, gas, supercritical fluid chromatography

  Based on physical state of mobile phase
• Adsorption Chromatography

  • a liquid or gaseous solution of the sample called the MP is passed through an adsorbent called SP
  • The components of the mixture travel through the adsorbent at different rates due to their IMF of attraction and interactions
  • process by which molecules or ions bind to the surface of a solid or liquid material due to attractive forces between the adsorbate (the substance being adsorbed) and the adsorbent (the surface to which the adsorbate binds).
  • Solvent molecules compete with solute molecules for sites
• Normal Phase Chromatography


  SP - Polar (silica gel, alumina)

  MP - Nonpolar
• Reversed Phase Chromatography

  SP - hydrophobic nonpolar (C-18 bonded silica or ODS)

  MP - Polar (water + organic solvent)
• Adsorbent

  • Most used in column chromatography are silica gel (SiO2) and alumina (Al2O3)
  • Sold in different mesh sizes indicated by number on bottle label (e.g., silica gel 60, silica gel 230-400)
  • Number = mesh of the sieve used to size the silica; number of holes in the mesh/sieve through which the crude silica particle is passed in manufacturing
  • Larger mesh size = smaller adsorbent particles

  columns

  Silica gel 70–230: gravity column chromatography

  Silica gel 230–400: flash chromatography
• Column Chromatography

  • Elution Chromatography
  • A compound that is very soluble with solvent but not strongly adsorbed, moves down the column by gravity relatively fast compared to the compound that is attracted to the adsorbent. The elution can be isocratic or gradient/
• Wet packing

  1. Vertical column is packed with a slurry (suspension of a polar adsorbent with a solvent)

  2. Slurry is poured into the column

  3. As the solvent drains, it leaves a packed bed of the adsorbent material on the column's walls

  4. Helps ensure the column is packed evenly and uniformly
• Dry packing

  1. Column is packed with the adsorbent material without a solvent

  2. Dry adsorbent material is poured into the column

  3. Column is then tapped or gently agitated to settle the material into a packed bed
• Dry packing


  Generally used for smaller columns, or when the solvent used in wet packing may interfere with the chromatographic separation
• Isocratic

  • a single constant mobile phase composition (solvent or solvent mixture) is used throughout the entire chromatographic run
  • This means that the mobile phase composition remains constant, which can simplify method development and is often used for separating compounds that have similar retention times.
  • typically faster than gradient elution but may not be as effective for separating complex mixtures or compounds with different polarities.
• Gradient

  • Uses changing mobile phase composition during chromatographic run to improve separation of compounds
  • The change in composition is typically a linear or nonlinear gradient, where the solvent composition is adjusted to elute different compounds at different times.
  • often for separating complex mixtures or compounds with different polarities, as it can improve resolution and reduce analysis time
  • requires more complex equipment and method development 
• Flash Column Chromatography

  • Classical Column is time-consuming and compound bands tend to tail when quantity of material becomes large.
  • The rate of elution is controlled by air or nitrogen pressure supplied on top of the column; Adsorbent used has a much smaller particle size than that of CC.
• High-Performance Liquid Chromatography (HPLC)

  • the solvent is pumped through the column at high pressure and the sample is injected into the solvent stream.
  • As components are eluted from the column, they pass through a detector and are converted into electrical potential.
  • number of components is estimated by the number of peaks and the amount is proportional to the area under each peak.
• High-Performance Liquid Chromatography

  • the measurement is used to identify components of small volume and analyzed sample is discarded as waste
  • highly versatile
  • separates mixtures based on their SP
  • it is like CC, but with a higher flow rate and higher pressure
  • uses smaller beads that increase interaction
• Retention Time


  The time required for a component to exit the column, or elute
• Peak size

  • Used to quantify the amount of compound in a solution
  • Represents the amount of compounds that passed the detector
• In an HPLC chromatogram, from left to right, highly polar to nonpolar.

  • Absorbance with broad bands contains mostly water
  • Absorbance with strong (thin) bonds is mostly organic solvent

  Gradient elution can prevent peak broadening of the less polar components, improving separation and shortening elution time.
• Partition Chromatography

  The partitioning of a solute between two immiscible liquids, as in solvent extraction, except that one of the liquids is held stationary on a solid support such as silica gel, diatomaceous earth, cellulose, polytetrafluoroethylene (PTFE) or polystyrene.
• Partition coefficient in Chromatography


  When the flow of the mobile phase has been put at a stop at any interval of time, the solute acquires an equilibrium distribution between the two phases. In each phase, the concentration is given by the partition coefficient in chromatography which is represented as K = CS/CM. When the solute is equally distributed amongst the two phases, then K = 1.
• Liquid-liquid Chromatography

  Gas-Liquid Chromatography


  Types of partition chromatography
• Liquid-liquid Chromatography

  • Instead of an adsorption column, a sheet of adsorbent paper is utilized.Based on their differential migratory velocities, the components are divided. To make the chromatograms visible, they are stained after separation.

  Paper Chromatography
• Gas-Liquid Chromatography

  • Vapor-phase chromatography
  • separation of the sample mixture is carried by an inert gas with a tube.
  • Stuffing of the tube is done with finely divided inert solids that are coated with non-volatile oil.
  • According to the rate determined by both its solubility in oil and its vapor pressure, the migration of every component takes place.
• Ion Exchange Chromatography

  • Involves the separation of ionizable molecules based on their total charge
  • SP is an ion exchange resin
• Size Exclusion Chromatography

  • Gel Filtration Chromatography
  • Separates molecules based on their sizes
  • SP is a gel made up of porous beads
  • Large molecules cannot enter the beads and are excluded. They have less volume to traverse and elute sooner.
  • Small molecules enter the aqueous spaces within the beads, have more volume to traverse, and elute later.
• Affinity Chromatography

  • based on a specific binding interaction between an immobilized ligand and its binding partner. 
  • Only molecules with specific affinity for the ligand can bind with the ligand. 

  Then, to remove ligands from column, one can:

  • Use a buffer with different pH/ionic strength to remove molecules of interest
  • Use a competitive inhibitor (further removed by dialysis/ change pH/ ionic strength)
  • Ex: antibody/antigen, enzyme & substrate, receptor & ligand, protein & nucleic acid, and enzyme/inhibitor interactions
• Thin-layer Chromatography

  • used for analysis; one of the easiest and most versatile methods because it is cheap, simple, fast, sensitive, and reproducible.
  • In synthetic chemistry: it is used for identifying compounds with a set of standards; used for monitoring the progress of the reaction, and determining the purity of the product
  • In organic chemistry: the goal is to obtain a pure compound hence it’s important to separate a mixture into its chemical components to isolate one compound or to assess purity of mixture
• Thin-layer chromatography

  It also permits the optimization of the solvent system for a given separation problem → used eventually to separate them by column chromatography
• Thin-layer chromatography


  It is a method for separating compounds by their rate of movement (Rate of Flow, Rf) through a thin layer of silica gel (the stationary phase) coated on a plate as support
• Rate of Flow (Rf)

  • It is computed as distance traveled by the component divided by distance travelled by the solvent