CC LEC M3 U4

• Chromatography refers to techniques used to separate complex mixtures based on physical and chemical interactions between compounds and the stationary phase

  
• During chromatography, the mobile phase carries the sample through a bed, layer, or column containing the stationary phase
• In chromatography, solutes may reside only on the stationary phase, only in the mobile phase, or distribute between the two phases
• The four basic components in any chromatographic technique are the mobile phase, the stationary phase, the column, and the eluate
• Chromatography separates components based on their differential migration between the mobile and stationary phases
• There are two basic forms of chromatography: Planar chromatography and column chromatography
• Planar chromatography involves a stationary phase coated on paper or a solid surface, while column chromatography uses a pure silica or polymer stationary phase
• Column chromatography includes gas chromatography (GC) or liquid chromatography (LC), depending on the mobile phase being a gas or liquid
• Chromatographic separations are classified into ion-exchange, partition, adsorption, size-exclusion, and affinity mechanisms
• Most clinical applications use chromatographic separations based on ion-exchange and partition mechanisms
• Ion-exchange chromatography:

  • Separates solutes based on differences in their ionic charge or the magnitude of their ionic charges
  • Stationary phase: particle surfaces of a plastic resin or silica coated or bound with functional groups with fixed cationic or anionic charges
  • Counterion found in close proximity to the fixed charge and solute ions in the mobile phase exchange to maintain electrochemical neutrality
  • Solute ions eluted selectively by changing the mobile phase pH, ionic strength, or both
  • Cation-exchange particles contain negatively charged functional groups to separate cationic solutes
  • Anion-exchange packings separate anionic solutes using strongly basic quaternary amines with positive charges
  • Clinical applications include separation of amino acids, peptides, proteins, nucleotides, oligonucleotides, and nucleic acids
• Partition chromatography:

  • Separates solutes based on their differential distribution between two immiscible liquids
  • Classified as gas-liquid chromatography (GLC) or liquid-liquid chromatography (LLC)
  • LLC further categorized as normal phase or reversed phase
  • Reversed phase chromatography: stationary phase is nonpolar, mobile phase is relatively polar
  • Hydrophilic interaction chromatography (HILIC) is a version where analytes elute in order of increasing polarity
• Adsorption chromatography:

  • Separation based on differences between the adsorption and desorption of solutes at the surface of a solid
  • Sample and mobile phase compete for adsorptive sites on the solid stationary phase like silica or alumina
  • Used to separate low molecular weight compounds and gases at room temperature
• Size-exclusion chromatography:

  • Separates solutes based on molecular sizes and shape
  • Stationary phases include cross-linked dextran, polyacrylamide, agarose, polystyrene-divinylbenzene, porous glass
  • Used for separation of enzymes, antibodies, proteins (gel filtration) and triglycerides, fatty acids (gel permeation)
• Affinity chromatography:

  • Separation based on unique and specific biological interactions of the analyte and ligand
  • Selectivity achieved through biological interactions like enzyme-substrate, hormone-receptor, or antigen-antibody interactions
  • Used in clinical labs to separate analytes like glycated hemoglobin and lipoproteins
• Planar chromatography:

  • Stationary phase coated on a sheet of paper (paper chromatography) or bound to a solid surface (thin-layer chromatography)
  • In paper chromatography, the stationary phase consists of a layer of water or a polar solvent coated onto the fibers of a sheet of paper
• Paper chromatography uses a layer of water or a polar solvent coated onto paper fibers as the stationary phase for normal-phase partition chromatography
• Paper chromatography is utilized for the fractionation of sugar and amino acids
• Affinity chromatography uses the "lock-and-key binding" principle and is used for the exclusive use of SLU
• Thin-Layer Chromatography (TLC) involves applying the sample as a small spot or band near the bottom edge of the plate, then placing it in a closed tank containing the mobile phase that migrates up the plate by capillary action
• Migration of solutes in Paper Chromatography (PC) and TLC is expressed as a Retention factor (Rf) value, which is the ratio of the solute migration to solvent from migration
• Both PC and TLC can be used in the identification of drugs in urine, analysis of amino acids or glycosaminoglycans in urine, and analysis of lipids in amniotic fluids
• Column Chromatography includes Gas Chromatography (GC) and Liquid Chromatography (LC), with GC providing higher resolution for volatile components and LC for larger molecules like peptides, proteins, nucleic acids, and large polymers
• In Gas Chromatography (GC), a gas mobile phase carries volatile solutes through a column containing the stationary phase, with separation based on differential partitioning into the stationary phase
• Basic components of a gas chromatograph include a supply or carrier gas and flow controller, an injector or injection port, a chromatographic column, a column oven, a detector, and a computer to control the system and process data
• In Liquid Chromatography (LC), solutes distribute between a liquid mobile phase and a stationary phase, with High-Performance Liquid Chromatography (HPLC) being the most widely used form of LC
• Chromatography components:

  1. Solvent reservoir to hold the mobile phase

  2. Pump(s) to force the liquid mobile phase through the system

  3. Injector to introduce sample into the column

  4. Chromatographic column to separate solutes

  5. Column oven to maintain constant column temperature

  6. Online detector(s) to detect analytes as they elute from the column

  7. Computer to control the system and process data

  
• Column technology advancements have improved selectivity, stability, and reproducibility of LC analytical columns
• Liquid Chromatography is used for monitoring drugs and their metabolites, analyzing metal ions and organic compounds in solutions
• Gas Chromatography is used to identify and quantify drugs like alcohol, analyze volatile gases, and is considered the gold standard for drug testing
• Thin-Layer Chromatography detects pesticide residues in food and analyzes fiber dye composition in forensics
• Paper Chromatography is used for separating amino acids, RNA fingerprinting, and testing histamines and antibiotics
• Mass Spectrometry (MS) coupled with GC or HPLC allows definitive identification of eluted samples
• Tandem Mass Spectrometry (GC/MS/MS and LC/MS/MS) offers greater selectivity and lower detection limits
• MS coupled to GC or LC provides identification, quantitation, structural information, and molecular weight determination of compounds
• LC/MS has potential for measuring low-level and mixed-polarity analytes like vitamin D, testosterone, and immunosuppressant drugs
• MS is used in proteomics, pathogen identification, and toxicology
• Migration of solutes in PC and TLC is expressed as a Retention factor (Rf) value, or the

  relative distance of migration from the point of application. Rf value is the ratio of the solute

  migration to solvent from migration
• Basic components of a gas chromatograph:

  • Supply or carrier gas and flow controller
  • Injector or injection port to introduce samples
  • Chromatographic column to separate analytes
  • Column oven to heat the column
  • Detector that responds to solute/analyte concentration
  • Computer to control the system and process data
• A constant flow of carrier gas is required for column efficiency and reproducibility