HPLC Mobile phase Preparation

How to Preparation HPLC Mobile phase?

HPLC Mobile Phase preparation procedure:

  • All mobile phase components (reagents and chemicals) shall be of equal or superior grade to those specified in the procedure.
  • Unless otherwise mentioned in the test procedure, solvent and buffer volumes are to be dispensed using appropriate graduated measuring cylinders. The measuring cylinder should be large enough to accommodate the total volume required.
  • A graduated pipette may be used to dispense volumes of 25 mL or less.
  • Follow ‘one solvent/buffer at a time’ rule. Never measure two components of mobile phase together in a measuring cylinder.
  • Solid components are to be weighed by using a balance with the appropriate level of accuracy. HPLC water must be used to prepare buffer solutions.
  • The pH of buffers is to be adjusted as mentioned in the test procedure.
  • The aqueous portion of the mobile phase (e.g. buffers) must be filtered through a membrane filter with a pore size of 0.45 µm or less. Water that has been dispensed through an in-line filter (pore size 0.45 µm or less) needs no re-filtration before adding it to the mobile phase.
  • Organic solvents may be added directly to the filtered aqueous portion without filtering, if the solvents are HPLC grade or better.
  • The mobile phase should be mixed thoroughly by stirring. The mobile phase must be degassed suitably prior to use.
  • While mixing buffers with solvents, care should be taken to avoid possible precipitation of buffers, if the composition has not been previously defined.
  • Care should be taken while degassing highly volatile mobile phases to prevent evaporation of a significant amount of the organic component and alter the composition of the mobile phase.

Documentation and Labelling of HPLC Mobile Phase

  • A label should be placed on the mobile phase bottle.
  • The label should include the following details (not to be limited).
    • Product Name.
    • Test performed.
    • Composition (by listing the component of the mobile phase along with their proportions).
    • Date of preparation.
    • Use before date and prepared by.
  • Record the preparation of the mobile phase in an appropriate Laboratory Note book.

Related: Principle of HPLC (Liquid Chromatography), Difference Between C18 and C8 HPLC Column

Assigning ‘Use Before’ Date for HPLC Mobile Phase

  • The expiration period for all mobile phases shall not exceed seven days from the date of preparation or based on visual observation, unless otherwise specified in the analytical procedure.
  • If the mobile phase is a mixture of 100% organic solvent then expiration date will be 30 days from the date of preparation.
  • The mobile phase should be stored in a sealed container.
  • Mobile phase shall be discarded after the ‘Use before’ date that is written on the label.
HPLC Mobile phase Labelling

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