MODULE 4 | Chromatography

• It is the technique for separating the components, or solute, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream (i.e., mobile phase) and a contiguous stationary phase.

  Chromatography
• Phases of Separation

  1. Stationary phase - remains fixed in place and acts as a constraint on many of the components in a mixture
  2. Mobile phase - carries the components of the mixture through the medium being used
• It is the output of the chromatography run. It is a visible record that shows the result, either in graph or layers, of the separation.


  Chromatogram
• It is typically a porous solid that is packed on a glass or metal tube or that constitutes the walls of an open-tube capillary.

  Stationary Phase
• The mobile phase may either be a liquid, a supercritical liquid, or a gas, while the stationary phase is either a solid or a liquid.
• Mikhail S. Tswett (or Tsvet)

  • Russian botanist who started/developed chromatography in the early 1900s
  • plant pigment is his basis
  • he coined the term "chromatography"
• It is what makes plants green.

  Chlorophyll
• It is what makes plant yellow, red, orange, and purple.

  Carotenoid
• specific technique Tswett used in separating plant pigments

  Column Chromatography
• Chromatography is from the Greek word "chroma" which means color and "graphein" meaning writing.
• This is how the colors were separated during the study of Tswett on plant pigments.

  
  

  Differential Distribution
• Column Chromatography

  more soluble : moves faster :: less soluble : moves slower
• Rafael E. Liesegang

  • 1927
  • developed paper chromatography
• Erika Cremer

  • 1944
  • developed gas chromatography
• Gas chromatography is valuable in petroleum products.
• Archer John Porter Martin & Richard Laurence Millington Synge

  • 1952
  • developed partition chromatography
  • novel prize awardee
• Jack Kirkland, Joseph Huban, & Csaba Horvath

  • 1960s
  • pioneer for liquid chromatography
  • Kirkland developed High Performance Liquid Chromatography (HPLC)
• substances that remains in place in the column

  Stationary Phase
• fluid that percolates through or along the stationary bed in a definite direction

  Mobile Phase
• Eluent - substance that separates and moves the mixture through the column
• Analyte - substance to be separated; also known as the sample/solute
• Eluite - samples that leaves the column; the sample being eluted
• Eluate - solvent (i.e., mobile phase) that leaves the column
• Column - physical device/instrument for the separation of chemical components
• Zone/Band - region where one/more components are located
• Alpha Synuclein (aSN) - a protein that is involved in the development of Parkinson's disease
• Sonification - method to extract aSN protein.
• Parkinson's Diseases

  • neurodivergent disease
  • disorder on the central nervous system mainly affecting the motor system
  • names after Dr. James Parkinson
• In Parkinson's Disease, neural degeneration occurs mainly in a small area of the midbrain called substantia nigra.
• Liquid Chromatography - type of chromatography where the mobile phase is a liquid, while the stationary phase can be solid or liquid
• Types of Liquid Chromatography

  1. Ion exchange chromatography
  2. Exclusion chromatography
  3. Partition chromatography
  4. Liquid-solid adsorption chromatography
• Size Exclusion Chromatography

  • molecules in the sample are separated according to their size
  • mobile phase is a liquid
  • stationary phase is a porous solid (normally a gel)
  • smaller molecules will elute later than the bigger ones

  
• Ion exchange chromatography

  • molecules in the sample are separated according to their charge
  • commonly used to separate charged biological molecules e.g., protein, peptides, amino acids, or nucleotides
  • mobile phase is a liquid
  • stationary phase is a solid

  
• Partition Chromatography

  • separation of components between two liquid phases viz original solvent and the film of solvent used in the column
  • also known as liquid-liquid chromatography
  • mobile phase and stationary phase are in liquid form
  • e.g., paper chromatography
• Paper chromatography

  • method of separating dissolved chemical substances by their different migration rates across the sheets of paper
• When a significantly higher operational pressure (50-350 bar) is used to move the mobile phase through the system, the technique is called High-Performance Liquid Chromatography (HPLC).
• Isoelectric point - pH where the protein has no charge
• Ion exchange chromatography

  buffer pH > isoelectric point : protein carries net negative charge :: buffer pH < isoelectric point : protein carries net positive charge
• Liquid chromatography typically uses the gravity force to pass the mobile phase through stationary phase.
• Typical Liquid Chromatography Equipment

  1. Mobile phase reservoir - where mobile phase is contained; normally in glass bottle
  2. Pumps - creates high pressure and create a flow of mobile phase in the system
  3. Sample Injector - where the sample is introduced in the system
  4. Stationary phase reservoir - where stationary phase is contained normally inside a column filled with beads or a porous material
  5. Detector - responsible for identifying and quantifying the separated analytes
  6. Fraction collector - collects the sample in different fractions according to the parameters selected