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In the previous module you were introduced to the stationary phases which serve to retain selectively the sample components. After separation these components need to be transported to the detector for detection and quantification. Mobile phase is the life line of HPLC system as it transports the sample from the injector to the detector and its characteristics such as composition, pH etc, have a profound effect on separation of sample components. The mobile phase should have the following desirable characteristics to carry out this important function.
- Affordable cost
- Non hazardous
- Inertness towards sample constituents and stationary phase
- Sample components should be miscible fully with the mobile phase
- Detector should not respond to mobile phase or to changes in the mobile phase composition as in gradient elution
Mobile phases generally consist of water – organic solvent, aqueous buffer or mixtures of organic solvents with or without modifiers. In isocratic mode the composition of the mobile phase remains unchanged throughout the analytical run whereas in the gradient elution mode mobile phase composition changes through programming of the pump.
Choice of mobile phase depends on the mode of HPLC operation.
Mobile Phases in Reverse Phase chromatography
In reverse phase applications water is usually the base solvent. Other polar solvents such as Methanol, Acetonitrile or Tetrahydrofuran are added in fixed or varying proportions. pH is adjusted by buffers to modify separations of ionizable solutes. Ion-pairing reagents also enhance separation selectivity of charged analytes by increasing retention on hydrophobic bonding phases.
Mobile Phases in Normal Phase Chromatography
Typically non polar solvents such as hexane, heptane, iso-octane are used in combination with slightly more polar solvents such as isopropanol, ethyl-acetate or chloroform. Retention increases as the amount of non polar solvent increases in the mobile phase.
Mobile Phases in Ion Exchange Chromatography
Aqueous salt solutions are generally used as mobile phases. Moderate amounts of water miscible polar organic solvents such as methanol can be added to buffered mobile phases. Solvent strength and selectivity can be adjusted by control of pH, buffer and salt concentrations.
Mobile Phases for Size Exclusion Chromatography
The mobile phase composition is not varied as the detector is sensitive to such changes.
Choice of mobile phase is dependent on its ability to dissolve sample and maintain consistent viscosity at operating temperature. High polarity solvents such as acetone, alcohols, DMSO and water are not used with polystyrene packings. Ionic strength is maintained by addition of salts.
Precautions in usage of Mobile Phases
Observation of following precautions will save your valuable time and enhance laboratory throughput
- Use Milli Q grade water and HPLC grade solvents
- Degas solvents to prevent bubble formation
- Use same method of mixing solvents every time as mixing is not always additives
- Mobile phase changeover should be gradual and wash with solvent of intermediate polarity
- After use of buffer solutions always wash with water to prevent damage to pump parts by crystalline deposits on drying of buffers
- Do not store solvents in plastic container due to possible leaching of plasticizers
- Glass containers are not suitable for aqueous mobile phases with pH > 8.0 as metal ions will leach from glass. Stainless steel containers are suitable in such cases
- Solvent reservoir should have small opening and should be covered
- Oxidizable solvents such as chloroform or THF should be protected with the nitrogen gas cover
- Use solvent inlet filters in reservoirs. Periodically clean solvent lines by back flushing or sonication
The next module will introduce you to the commonly used detectors in HPLC
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