Introduction to Chromatography

Introduction to Chromatography

• Mikhail Tswett (1872-1919) was a Russian botanist who invented chromatography in 1906 to separate plant pigments
• Tswett named the technique chromatography because the analysis resulted in a "written in color" separation along an adsorbent column
• "Chroma" means color and "graphein" translates to write
• Chromatography is useful in physical and biological sciences
• Between 1937 and 1972, 12 Nobel Prizes were awarded for work involving chromatography

Basics of Chromatography

• Chromatography is a physical separation method where components distribute between a stationary and a mobile phase moving in a specific direction
• Separation occurs due to differences in the distribution constant of sample components
• Chromatography separates and identifies components in a mixture
• Molecules distribute themselves between stationary and mobile mediums
• Molecules that spend more time in the mobile phase are carried along faster
• One phase is typically hydrophilic, while the other is lipophilic
• Analyte components interact differently with each phase
• Components spend varying times interacting with the stationary phase based on polarity, leading to separation
• Each component elutes from the stationary phase at a specific retention time (tR)
• As components pass the detector, the signal is recorded and plotted as a chromatogram

Classification by Mobile Phase

• Liquid Chromatography (LC): The mobile phase is a liquid (LLC, LSC)
  • The sample is dissolved and pumped through a column containing the stationary phase
  • LC is more versatile than gas chromatography as it processes heat-stable samples and volatile ones
  • Requires the sample to dissolve completely in the mobile phase
  • Common detection methods include UV spectroscopy, refractive index measurement, fluorescence, electrical conductivity, and mass spectrometry
  • Operation modes are classified as normal and reversed-phase chromatography
  • Normal Phase Chromatography: Stationary phase is hydrophilic (ex: silica particles) and mobile phase is a hydrophobic organic solvent (ex: hexane)
  • Reversed-Phase Chromatography: Stationary phase is hydrophobic, and mobile phase is a mixture of polar solvents (ex: water and acetonitrile)
  • Reversed-phase chromatography works for amino acids, peptides, proteins, nucleic acids, and carbohydrates because biomolecules are soluble in polar solvents
• Gas Chromatography (GC): A gas is a mobile phase, applicable only to heat-stable and volatile substances (GSC,GLC)
  • GC is not common for biomolecule analysis because large molecules like peptides and proteins are thermally destroyed before evaporation
  • Smaller molecules (amino acids, fatty acids, peptides, carbohydrates) are analyzed if chemically modified to increase volatility
  • Volatile metabolites (aldehydes, alcohols, ketones) produced by some cell cultures can be analyzed by GC

Classification by Stationary Phase Packing

• Thin Layer Chromatography (TLC): The stationary phase is a thin layer on glass, plastic, or aluminum plates
• Paper Chromatography (PC): The stationary phase is a thin liquid film on an inert support
• Column Chromatography (CC): The stationary phase is packed in a glass column

Classification by Separation Force

• Adsorption
• Partition
• Ion Exchange
• Gel Filtration
• Affinity

Liquid Chromatography for Bioanalysis

• Chromatography separates, isolates, and purifies proteins from complex sample matrices
• Proteins in cells must be separated from lipids and nucleic acids for analysis
• The target protein must be isolated from other proteins and purified
• Chromatography is essential for protein purification, using different techniques for analysis of proteins
• Techniques are classified according to the physical principle of separation
• Examples: reversed-phase, ion exchange, affinity, and size exclusion chromatography (SEC)

Protein Separation Methods

• Hydrophobicity uses reversed-phase chromatography
• Charge uses ion exchange chromatography
• Biospecificity uses affinity chromatography
• Size/form uses size exclusion chromatography

Reversed Phase Liquid Chromatography

• Normal phase chromatography was created long before reversed-phase chromatography
• Stationary phases were made of polar materials (paper, cellulose, silica gel), and mobile phases used non-polar solvents (ex: hexane, chloroform)
• Later, phase polarities were reversed using polar solvents (water, acetonitrile) with non-polar stationary phases
• Non-polar stationary phases were obtained by etherifying silica gel's polar hydroxyl groups with long alkyl chains
• Reversed-phase chromatography is ideal for separating smaller biomolecules (peptides, amino acids, carbohydrates, steroids) soluble in water/acetonitrile mixtures
• Protein separation can be problematic if organic solvents decrease the protein's solubility and cause denaturation
• The stationary phase consists of porous silica particles with non-polar surface groups from etherification of the silica particle's hydroxyl groups with silanes containing non-polar hydrocarbon chains
• Chain lengths from ethyl silane (C2) to n-octadecyl silane (ODS/C18) are used with octyl silane (C8) and ODS being common choices

Optimization of Reversed Phase Chromatography

• Analytical separations typically use 5 μm or smaller particles
• Preparative liquid chromatography uses larger particles to isolate compounds of interest
• Silica particles have a pore size of around 10 nm, providing a large surface area (100-400 m²/g) for interaction with the stationary phase
• A polar solvent system with an aqueous buffer and acetonitrile/methanol forms the mobile phase
• Gradient elution increases resolution and shortens separation times by increasing the organic solvent, decreasing mobile phase polarity and retention of less polar analytes
• Solvents are classified by elution strength and polarity

Solvents and Polarity

• Solvent order, from low to high polarity: Water < Methanol < Acetonitrile < N-propanol < Tetrahydrofuran
• Elution speed also increases along this order
• Buffer systems (ammonium acetate, phosphate, hydrogen carbonate) are added at ~20mM to adjust the mobile phase pH between 2 and 8
• Ion pairing reagents (typically 0.1%) increase hydrophobicity of charged analytes and form ion-pair complexes
• Anionic ion pairing reagents (TFA) bind to positively charged analytes
• Cationic ion pairing reagents (tetraalkyl ammonium salts) bind to negatively charged analytes
• Complexes are retarded by the stationary phase and easier to separate than unretained charged analytes

Modern Chromatography and HPLC

• Modern columns are packed with 1-5 μm particles
• High pressures (300-400 bar) are needed for ambient flow rates in these columns
• High-performance liquid chromatography (HPLC) is a typical setup for these conditions

HPLC Components

• Computer-controlled pumps move the mobile phase, and aqueous/organic solvents are mixed to the composition of choice
• Gradient elution gradually alters the composition during separation
• Sample volumes are injected using a manual loop/valve system or autosampler
• Sample volumes depend on column dimensions (nL to mL)
• The column is kept in an oven at a constant temperature
• Analytes pass through a detector after eluting
• UV detection is performed at 210 nm for peptides or 254/280 nm for proteins
• Diode array detectors (DADs) take several spectra per second for less ambiguous identification
• Fluorescence detection of derivatized amino acids and peptides gives high sensitivity
• Liquid chromatography systems are coupled with electrospray ionization mass spectrometers (ESI-MS)
• Mass spectrometry allows universal detection at high sensitivity and gives structural information, but not all buffers are compatible with mass spectrometers

Trends in Liquid Chromatography

• Development trends are leading to smaller LC systems
• Micro and nanoscale LC systems have been proven
• Shorter columns with smaller particles allow faster analysis, less solvent consumption, and require less sample

Chromatography Types by Scale

• Preparative uses mg-g sample amounts, >4mm column diameter, >1 mL/min flow rate
• Analytical uses μg-mg sample amounts, 2-4mm column diameter, 0.2-1 mL/min flow rate
• Micro uses μg sample amounts, 1mm column diameter, 0.05-0.1 mL/min flow rate
• Nano uses ng-μg sample amounts, <1mm column diameter, <0.05 mL/min flow rate