Guideline for Preparation of Mobile Phase Required for HPLC : Pharmaguideline

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Guideline for Preparation of Mobile Phase Required for HPLC

Learn guidelines for preparation and validation of the mobile phase for HPLC analysis.

Mobile Phase Preparation

1. Use HPLC grade/ AR Grade / GR grade chemicals.
2. Weigh / Measure each component of the mobile phase separately.
3. Degas each solvent and buffer by sonication and then filter each component of the mobile phase separately through 0.45µ filter (Use the filter paper as per suitability with solvent).
4. Mix the individual solvent and buffer as per the specified proportion.
5. Adjust the pH of the mobile phase wherever specified (i.e. before or after mixing of individual components) between ± 0.05 unless otherwise specified.

6. Sonicate the mobile phase for 10 minutes or more as required.
7. Ensure that mobile phase is free of particles. Do not use the mobile phase if particles are observed.
8. Enter the details of mobile phase preparation in laboratory note book.
Related: Theoretical Plates and their Determination in HPLC Analysis

Procedure for mobile phase validation

1. Prepare the mobile phase as per the test procedure and keep on bench top in well closed condition.
2. Evaluate system suitability parameter(s) as per the test procedure at initial, after one day and after two days.
3. The mobile phase is considered stable if all the system suitability parameter are within the specified limits.


Mobile phase is to be prepared daily if mobile phase validation is not done.
In case if sequence is required to be run in continuation for more than 24 hours & sufficient mobile phase is available in the reservoir then sequence can be allowed to run by demonstrating system suitability of analysis by injecting bracketing standard.
For stability validated mobile phase – As per frequency indicated.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
.moc.enilediugamrahp@ofni :liamENeed Help: Ask Question

2 comments: Post Yours! Read Comment Policy ▼

  1. Useful information thank you.

  2. Thanks for the information. May i know the reference for this please, sir.


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