Preparation of Mobile Phase of HPLC Column

Preparing the mobile phase for High-Performance Liquid Chromatography (HPLC) is a critical step in chromatographic analysis. The mobile phase, which consists of one or more solvents, carries the sample through the chromatographic column and is essential for separation and elution of analytes. Here are the general steps for preparing the mobile phase for an HPLC column (such as C18 Alkaline HPLC Column, Silica HPLC Column):

Materials Needed:

  • HPLC-grade solvents (e.g., acetonitrile, methanol, water)
  • HPLC-grade chemicals and buffers (if required)
  • Analyte samples
  • HPLC system with appropriate column and detector


  1. Check Solvent Purity: Ensure that all solvents and reagents used in the mobile phase preparation are of HPLC-grade to avoid contamination and ensure accurate results.
  2. Select Solvents: Determine the appropriate solvents for your chromatographic separation based on the nature of your analytes and the chromatographic method (e.g., reversed-phase, normal-phase, ion-exchange). Common solvents include acetonitrile, methanol, and water.
  3. Buffer Addition (if needed): In some cases, buffers may be required to adjust the pH and ionic strength of the mobile phase for specific separations. If buffers are needed, prepare them separately, ensuring they are HPLC-grade.
  4. Mixing Ratios: Calculate the required mixing ratios of the chosen solvents and buffers to create the desired mobile phase composition. The specific ratios depend on your chromatographic method and the elution properties of your analytes. Consult your HPLC method’s protocol or method development guidelines for guidance.
  5. Degassing: To avoid bubble formation and ensure stable and accurate flow rates, it’s essential to degas the mobile phase. This can be done by sonication or by using a degassing apparatus.
  6. Filtration: Filter the mobile phase using a suitable filter (e.g., 0.2 μm or 0.45 μm) to remove particulates and ensure the mobile phase is free of impurities that could affect chromatographic results.
  7. Equilibration: Once prepared, the mobile phase should be allowed to equilibrate to the desired temperature and pressure of the HPLC system to ensure stable chromatography.
  8. Testing: Before sample injection, run a blank or system suitability test to ensure that the mobile phase is flowing properly and that the system is free from leaks or other issues.
  9. Sample Preparation: Depending on your analysis, prepare your sample for injection into the HPLC system. This may involve sample dilution or concentration steps as required.
  10. Column Conditioning: Before the actual sample analysis, it’s important to condition the HPLC column by running the mobile phase through it to ensure that it is stable and equilibrated.
  11. Sample Injection: Load your prepared sample onto the HPLC system and initiate the chromatographic run according to your method’s specifications.
  12. Data Collection: Monitor the chromatogram, record data, and analyze the results.

Regularly verify and maintain the quality of your mobile phase to ensure the reliability and reproducibility of your HPLC analyses. This includes monitoring the pH, buffer concentration (if used), and solvent purity over time.


HPLC is the separation of sample components between the column packing and the mobile phase for mass exchange. Therefore, the mobile phase is required to have the following characteristics:

a. the flow relative to the sample has a certain solubility, to ensure that the sample components will not precipitate in the HPLC column (or remain in the column for a long time).

b. The mobile phase is inert and does not react chemically with the sample (except in special cases).

c. The viscosity of the mobile phase should be as small as possible so that a good separation effect can be obtained when using a longer analytical column; at the same time, the HPLC column pressure drop is reduced and the life of the liquid pump is prolonged (the temperature can be lowered to reduce the viscosity of the mobile phase).

d. The physical and chemical properties of the mobile phase should be compatible with the detector used. If a UV detector is used, it is best to use a solvent with a lower UV absorption.

e. The boiling point of the mobile phase should not be too low, otherwise, bubbles will be easily generated, which may make the experiment impossible.

f. After the mobile phase is prepared, it must be degassed. Removal of traces of gases dissolved in the mobile phase facilitates both the detection and prevention of trace oxygen in the mobile phase from interacting with the sample.