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๐Ÿงซ Mobile Phase Preparation

Calculate exact volumes of each component for HPLC mobile phase preparation. Add up to 4 components, set the ratio and total volume.

Quick Presets
Components & Ratio
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Preparation Instructions
๐Ÿ’ก Preparation tip: Always prepare mobile phase in a clean volumetric flask. Mix thoroughly before use. Filter through 0.45 ฮผm membrane and degas by sonication or vacuum. For buffer-containing mobile phases, filter the buffer before adding the organic modifier.
โš ๏ธ Verify before use: Volumes assume simple volume-ratio mixing (v/v). Double-check critical mobile phase preparations independently, especially for validated methods.
๐Ÿ“– Example: Mobile phase 65% acetonitrile / 35% water, total 500 mL โ†’ measure 325 mL acetonitrile + 175 mL water. Mix and degas. Report: "Mobile phase: 65:35 (v/v) acetonitrile:water, prepared and degassed prior to use."
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Up to 4 components

Add up to 4 mobile phase components โ€” organic, aqueous, buffer and modifier.

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Ready-to-use instructions

Copy the preparation instructions directly into your lab notebook or report.

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Always free

No sign up, no limits. Use for every HPLC mobile phase preparation.

โœ” Formulas last verified against primary references: March 2025  ยท  Report an error
โš ๏ธ Report an incorrect calculation
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Frequently asked questions

How is this different from a gradient elution method?

This calculator prepares a fixed, isocratic mobile phase mixture at a single solvent ratio for the entire run. Gradient methods change the solvent ratio over time during the run, which requires programming the instrument's pump rather than premixing a single bottle โ€” this tool is for the premixed isocratic case.

Can I use this for more than 2 solvents?

Yes โ€” this calculator supports up to 4 mobile phase components, scaling each proportionally to your specified ratio and total volume needed.

Does the order I mix solvents in matter?

For most common HPLC solvent combinations, mixing order has minimal practical effect on the final ratio, but some solvent pairs generate heat or contract in volume when mixed (e.g. water and acetonitrile). For best accuracy, prepare each solvent volume individually before combining, rather than assuming volumes are perfectly additive.

Accuracy & how this is derived

Derivation: Each solvent's required volume is calculated as the total mobile phase volume multiplied by that solvent's fractional share of the specified ratio, following standard proportional volume/volume (v/v) mixing convention used in HPLC method preparation.

Validated against: Standard isocratic mobile phase preparation practice described in HPLC method development references.

โš ๏ธ For educational and research support only โ€” verify critical results independently before use in regulated, clinical, or publication-bound work.

โœ” Last updated: July 2026  ยท  Report an error

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Preparing and optimising HPLC mobile phases

Mobile phase composition controls HPLC retention, selectivity, and peak shape. Add organic solvent first to prevent salt precipitation, then add aqueous buffer. Degas by sonication or helium sparging. Higher organic content reduces retention in reversed-phase HPLC. Buffer pH controls ionisation of acidic and basic analytes. For LC-MS, use volatile buffers only: ammonium formate or ammonium acetate (0.1–10 mM). Avoid phosphate and borate buffers which damage mass spectrometer sources.