Choosing an HPLC mobile phase

1. Match the mode

In reversed-phase (the default for most small molecules), the column is non-polar (C18) and the mobile phase is polar: water/buffer plus an organic modifier — usually acetonitrile or methanol. In normal-phase, the column is polar (silica) and the mobile phase is non-polar: hexane or heptane plus a polar modifier such as 2-propanol or ethyl acetate.

2. Set strength with polarity

Use the polarity index to control retention: in reversed-phase, more organic (lower-P′) = stronger = faster elution; in normal-phase, more polar modifier = stronger. Get peaks eluting in a sensible window first.

3. Change selectivity by solvent type

If peaks co-elute, don't just change the ratio — swap the organic for one in a different selectivity group at the same strength. Methanol, acetonitrile, and THF are the classic trio to try.

4. Respect the UV cutoff

Your detection wavelength must sit above the UV cutoff of every component, including buffer and additives. For 210 nm work, acetonitrile (~190 nm) beats methanol (~205 nm); avoid acetone and aromatics for UV.

5. Check miscibility and viscosity

All components must be miscible — a common trap is that methanol and acetonitrile are only partially miscible with alkanes, so you can't jump straight from a reversed-phase to a normal-phase solvent without an intermediate. Watch viscosity too: methanol/water blends peak in viscosity around 40–50% and can spike backpressure; 2-propanol is very viscous. Acetonitrile keeps pressure low.

6. Buffers and practical notes

Keep buffers within their solubility limits in the organic (phosphate can precipitate at high organic %), filter and degas, and note that THF and ethers form peroxides. Always confirm solvent grade (HPLC/gradient grade) and consult each SDS.