Switching between Normal Phase to Reversed Phase  Protocols

Normal phase solvents order of polarity:
hexane > dichloromethane > chloroform > isopropanol>ethanol, etc.

Reversed phase solvents order of hydrophobicity:
buffers > water > methanol >  acetonitril > ethanol > isopropanol


REVERSED PHASE to  NORMAL PHASE

waters ->  methanol  -> isopropanol -> hexane

  • 1. Remeber: H2O, ACN, MeOH do not mix with hexane! 
  • Do not keep any normal phase solvent in the reservoirs when working with reversed phase solvents!
  • 2. Isopropanol (IPA) mixes with hexane or with H2O, ACN, MeOH
  • When finishing with reversed phase solvents always wash the entire system with H2O: pump-autosampler-detector-sealwash and needlewash with H2O to remove residues of buffers.  Use a zero-dead-volume-union instead of the column.
  • Wash with MeOH: minimum of 10 ml should pass through each line A, B, C, D, and through the injector (Purge injector) and through the detector (just use the flow).
  • Wash with IPA: minimum of 10 ml should pass through each line A, B, C, D, and through the injector (Purge injector) and through the detector (just use the flow).
  • Only after the entire system is flushed with IPA you can introduce normal phase solvents such as hexane and chloroform.


NORMAL PHASE to  REVERSED PHASE

hexane -> isopropanol -> methanol -> waters

1. Remeber: H2O, ACN, MeOH do not mix with hexane! 

  • Do not keep any of those polar solvents in the reservoirs when working with normal phase solvents!

2. Isopropanol (IPA) mixes with hexane AND with H2O, ACN, MeOH

  • Always keep isopropanol as a reservoir when working with normal phase solvents, it will be used as the final wash solvents to the entire HPLC system.
  • When finishing with normal phase solvents always wash the entire system: pump-autosampler-detector-sealwash and needlewash with Isopropanol. Use a zero-dead-volume-union instead of the column if needed.
  • Wash with IPA: minimum of 10 ml should pass through each line A, B, C, D, and through the injector (Purge injector) and through the detector (just use the flow).
  • Wash with MeOH: minimum of 10 ml should pass through each line A, B, C, D, and through the injector (Purge injector) and through the detector (just use the flow). 
  • Wash with H2O: minimum of 10 ml should pass through each line A, B, C, D, and through the injector (Purge injector) and through the detector (just use the flow).
  • Only after the system was washed with H2O it is ready for reversed phase.