Acetonitrile vs. Methanol for Reverse Phase Chromatography

Acetonitrile (ACN) and methanol (MeOH) are the most widely used organic modifiers in reversed-phase chromatography. Each solvent has its own set of advantages and disadvantages. This guide provides a detailed comparison of acetonitrile and methanol for reversed-phase chromatography.

Absorbance  

LC-grade acetonitrile is ideal for HPLC-UV assays due to its low absorbance of UV wavelengths. It also offers low viscosity, high elution strength, and excellent miscibility with water.

Both LC-grade acetonitrile and methanol are produced with the removal of UV-absorbing impurities. Mass Spectrometry (MS) Grade solvents, such as acetonitrile and methanol, are specifically designed to meet the requirements of modern LC-MS ionization techniques (ESI/APCI in both positive and negative modes). With their low ionic background and minimal ion suppression, these solvents ensure high reproducibility and optimal ionization efficiency.  

Pressure  

Chromatography supplies, such as columns, experience pressure that varies depending on the type and mixture ratio of organic solvents. The pressure for methanol increases when mixed with water, but not so much for acetonitrile. Consequently, given the same flow rate, acetonitrile-based solutions apply less pressure to the column.  

Elution Strength  

Acetonitrile has a higher elution strength than methanol for reversed-phase chromatography. Therefore, one can expect shorter analyte retention for equal proportions of organic to water.  

Selectivity  

Selectivity tends to differ based on which solvent an individual uses. Methanol is a polar-protic solvent, whereas acetonitrile is a polar aprotic solvent and possesses a stronger dipole moment. Since methanol and acetonitrile are fully miscible with one another, an individual can blend them to fine-tune separation.  

Peak Shape  

For compounds such as salicylic acid (phenol with carboxyl or methoxy group in the ortho position), acetonitrile can cause significant tailing, which could be suppressed by using methanol. This is caused by differences in the way the mobile phases relate to the mutual absorption between the silica surfaces and target components, due to the chemical properties of the organic solvent molecules.   

Polymer-based reversed-phase columns generally result in broader peaks than silica-based columns. This is particularly common for aromatic compounds in polystyrene columns. This is especially noticeable when using methanol-based mobile phases, whereas it is not very noticeable for acetonitrile-based mobile phases.  

Mobile Phase Degassing  

When methanol mixes with water, the solution releases heat, which facilitates releasing any dissolved air bubbles, as opposed to acetonitrile, which cools the temperature by absorbing heat. With acetonitrile, air bubbles generate as the solution slowly returns to room temperature, meaning an individual must take more care when using acetonitrile.   

Availability and Price  

Acetonitrile is produced as a byproduct of acrylonitrile, which is a common component in plastics. Since 2008, the chromatography industry has dealt with a worldwide shortage of acetonitrile in which the availability has diminished, and prices have climbed. To combat this, many labs have switched to using methanol. 

When comparing acetonitrile to methanol for reverse phase chromatography, each solvent has its own advantages, such as methanol’s availability and lower cost, or acetonitrile’s elution strength.