Lab Tools
HPLC Mobile Phase Calculator
Calculate mobile phase compositions, prepare HPLC-grade buffers, and build gradient elution programs for pharmaceutical analysis.
Mobile Phase Mixer — Volume Calculator
Quick-fill ratios (Buffer:Organic):
Buffer Volume
—
mL
Organic Volume
—
mL
Modifier
—
Buffer Preparation — Exact Recipe Calculator
Gradient Table — Linear Elution Program
| Step | Time (min) | % Organic | % Aqueous | Note |
|---|
Organic Modifier Reference
| Modifier | UV Cutoff | Polarity | Notes |
|---|---|---|---|
| Acetonitrile | 190 nm | Medium | Most common; low viscosity; preferred for LC-MS |
| Methanol | 205 nm | High | Protic; higher viscosity; better for certain selectivity |
| THF | 220 nm | Low | Use <30%; strong eluent; hazardous; peroxide risk |
| Isopropanol | 205 nm | High | For hydrophobic analytes; high viscosity at >20% |
| Acetone | 330 nm | Medium | High UV cutoff limits use; only refractive index detection |
Frequently Asked Questions
The mobile phase in HPLC is the solvent system that transports analytes through the stationary phase column. It is typically a mixture of an aqueous buffer and an organic modifier. The ratio controls analyte retention time — more organic increases eluting strength for reverse-phase HPLC. Precise preparation is critical because even small deviations in composition can shift retention times and affect peak shape.
Always adjust the pH of the aqueous buffer before mixing with organic modifier. Organic solvents shift electrode readings and can damage pH electrodes. Use dilute phosphoric acid or sodium hydroxide for fine adjustments. Check pH after the solution equilibrates to room temperature. For volatile buffers such as ammonium acetate, adjust to your target pH and use promptly as the pH can drift.
Acetonitrile (ACN) is preferred for its low UV cutoff (190 nm), low viscosity, and strong eluting power. It generates lower back pressure than methanol when mixed with water. Methanol is protic and can provide different selectivity through hydrogen bonding interactions. Methanol mixtures with water have higher viscosity — especially in the 30–50% range — and may require lower flow rates on older systems. For LC-MS, ACN is strongly preferred as it produces better ionization.
Gradient elution is a technique where the mobile phase composition changes continuously during the HPLC run, typically increasing the organic modifier percentage. This allows separation of compounds with a wide range of polarities in a single run. After the gradient, the column must be re-equilibrated with the starting mobile phase for at least 5 column volumes before the next injection. The gradient table calculator provides the time vs. % organic program for a linear gradient.
For HPLC phosphate buffer: weigh the calculated amounts of NaH2PO4 and Na2HPO4 (from Henderson-Hasselbalch at your target pH), dissolve in HPLC-grade water, verify pH with a calibrated electrode, and adjust if needed. Filter through a 0.22 μm membrane filter before use. Keep concentration below 100 mM to avoid salt precipitation when mixed with organic modifier. Discard and prepare fresh every 3–5 days to prevent microbial growth.