HPLC and LC-MS solvents: acetonitrile, methanol, formic acid and TFA

Peptide HPLC normally uses water and acetonitrile, each with 0.1% acid: TFA for UV detection, formic acid for LC-MS. An HPLC result depends on these liquids as much as on the column itself. This guide explains why those choices are made, how methanol compares, why mass spectrometry needs a different acid, and what the grades on solvent bottles mean.

For how the instrument separates compounds, see how HPLC works. Terms are defined in the glossary.

The mobile phase for peptide HPLC

Reversed-phase HPLC uses a non-polar column, usually C18, and a polar mobile phase that changes during the run. A typical peptide method uses two solvents:

• Mobile phase A: water with 0.1% acid.
• Mobile phase B: acetonitrile with 0.1% of the same acid.

The run starts with mostly A and the pump raises the share of B over time, in a gradient, so each peptide lets go of the column in turn. For the full mechanism, see how HPLC works.

The water needs to be ultrapure or HPLC-grade. Trace organic matter in it shows up as stray peaks. See lab water.

Dissolved air can form bubbles in the pump and detector, so many labs degas mobile phases first, for example in an ultrasonic bath; see vortex mixers, sonication and magnetic stirrers.

Acetonitrile

Acetonitrile is a small, colourless liquid that mixes with water in any proportion. It is the default organic solvent for peptide HPLC for several reasons:

• It is a strong solvent in reversed phase. Less of it is needed to move a peptide off the column than with methanol, so runs are shorter and peaks sharper.
• It is transparent to UV light down to about 190 nm. Peptides are detected at 210–220 nm, where the peptide bond absorbs. A solvent that absorbs there would raise the baseline and hide small peaks.
• It gives low pressure. Mixtures of acetonitrile and water are less viscous than mixtures of methanol and water, so the pump works at lower pressure.
• It doesn't donate hydrogen bonds, which gives a different selectivity from methanol and suits most peptides.

Mixing acetonitrile with water takes in heat, so the mixture cools below room temperature (Shimadzu). The combined volume is also not exactly the sum of the two. For accurate mixtures, measure each solvent separately before combining them, rather than filling one up to a mark with the other.

Acetonitrile is a by-product of making acrylonitrile, a raw material for plastics. When demand for those plastics fell in the 2008–2009 downturn, a worldwide acetonitrile shortage pushed many labs to try methanol instead (LCGC).

It is flammable and harmful, so it is handled in a fume cupboard according to its safety data sheet. See COSHH and safety data sheets.

Methanol

Methanol (CH₃OH) is the other common organic solvent for reversed-phase HPLC. It boils at about 65 °C.

Methanol's higher UV absorbance at 205–215 nm makes it less suited to detecting peptides at low wavelengths. It is still used where its different selectivity separates two peaks that acetonitrile can't, and in many small-molecule methods. Like acetonitrile, it is flammable and toxic, and is handled under its safety data sheet.

Acids in the mobile phase

Peptide methods add an acid, usually at 0.1%, to both solvents. It keeps the pH low, which:

• stops the carboxylic acid groups on the peptide from carrying a charge,
• suppresses unwanted interactions with the silica of the column, and
• gives every basic group a consistent positive charge.

The two common choices are trifluoroacetic acid and formic acid.

Trifluoroacetic acid (TFA)

TFA is a strong acid. At 0.1% it brings the pH to about 2. It also acts as an ion-pairing agent: its negative ion pairs with the positive charges on the peptide, which makes the peptide behave more consistently on the column and gives sharp, symmetrical peaks. For UV detection, TFA is usually the best choice for peptides.

The same TFA is why many purified peptides end up as TFA salts. See trifluoroacetic acid in peptides.

Formic acid

Formic acid is the simplest carboxylic acid. It is weaker than TFA, with a pKa of about 3.75, and at 0.1% gives a pH of about 2.7. It is a much weaker ion-pairing agent, so peptide peaks are often a little broader than with TFA.

Its advantage is in mass spectrometry. Formic acid supplies protons for electrospray ionisation without holding on to the peptide ions, so it gives a strong signal.

Why LC-MS needs different solvents

In LC-MS, the liquid leaving the column goes straight into a mass spectrometer. Electrospray ionisation turns the liquid into a fine spray of charged droplets that evaporate, leaving charged peptide ions. See LC-MS, electrospray and MALDI.

Two things follow:

• Everything in the mobile phase must evaporate. Non-volatile buffers, such as phosphate, don't evaporate. They deposit in the instrument's source, block it and suppress the signal. LC-MS methods use only volatile additives.
• The additive mustn't hold on to the peptide. TFA pairs so tightly with peptide ions that it suppresses the electrospray signal, sometimes by a large factor (Apffel et al., J. Chromatogr. A 1995). That is why LC-MS methods usually use 0.1% formic acid, or a much lower TFA concentration, accepting slightly broader peaks.

This is one reason a peptide's UV purity and its LC-MS trace can look a little different. The two runs may use different acids. Purity is taken from the UV trace; MS confirms identity. See mass spectrometry and identity confirmation.

Volatile buffers

When a method needs a pH other than strongly acidic, LC-MS uses buffers that evaporate:

For how a buffer holds its pH, see buffers and pH.

Solvent grades

The same solvent is sold in different grades, tested for different things.

Using a lower grade than the method needs causes ghost peaks, a drifting baseline or a noisy mass spectrum. See reading a chromatogram. Once opened, solvents pick up water and contaminants from the air, so bottles are kept capped and used within a reasonable time.

Related guides

For the instrument and what HPLC can and can't show, see how HPLC works and HPLC purity explained. For how peptides are purified with the same solvents on a larger scale, see how peptides are purified. An independent lab tests every lot we sell for purity by HPLC and identity by mass spectrometry. For polarity, boiling points and UV cut-offs across other common solvents, see the solvent miscibility and polarity chart. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.