![[Readers Insight] Choose Methanol or Acetonitrile? The Organic Mobile Phase in HPLC](http://www.welch-us.com/cdn/shop/articles/1080_7c636f0f-edc5-4f09-ae81-c57bf62ca14c_750x.jpg?v=1763358164)
[Readers Insight] Choose Methanol or Acetonitrile? The Organic Mobile Phase in HPLC
Author: Chromatography Mound
Introduction
When chromatographers talk about the organic solvent in the mobile phase, methanol and acetonitrile are the first two that come to mind. Some practitioners prefer methanol, while others prefer acetonitrile. But what exactly differentiates them?
In this article, I will share my experience and thoughts on this matter.
Conclusion First: No Universal "Better" Choice
In my opinion, there is no standard answer to whether methanol or acetonitrile is "better". The appropriate solvent is the one that more effectively addresses the specific separation problem at hand. For analytes of highly polarity, methanol is generally more suitable in retarding their elution; for analytes of lower polarity, acetonitrile generally improves their tailing.
In the following sections, I will further explain the logic behind choosing between methanol and acetonitrile.
Polarity and Retention Behavior
Water has a polarity of 10.2, methanol 6.6, and acetonitrile 6.2. Therefore, water is the most polar, and acetonitrile the least polar among them. When mixing methanol or acetonitrile with water, the polarity of methanol-water mixtures ranges from 6.6 to 10.2, while the polarity of acetonitrile-water mixtures ranges from 6.2 to 10.2. It is obvious that acetonitrile-water system offers a slightly larger adjustable polarity range.
Furthermore, methanol and acetonitrile show different performance in different separation tasks. In the separation of multi-component mixtures, acetonitrile performs better as the organic mobile phase in fine-tuning gradient programs.
For polar analytes (amines, carboxylic acids, alcohols, etc.), methanol is more recommended as it increases retention; but for less-polar analytes, acetonitrile is better as it shortens elution time while maintaining resolution. Acetonitrile is also preferred for analytes that tend to produce broad or tailing peaks, yielding more desirable peak shapes.
Viscosity and Column Pressure
Acetonitrile-water mixtures have lower viscosity than methanol-water mixtures under any given ratio across the range, especially between 20% to 60% organic proportion where the difference is most significant. Higher viscosity results in higher pressure to HPLC columns: excessive column backpressure during separation may cause peak distortion, leaks, or instrument errors. Acetonitrile has a clear advantage in lowering viscosity.
Heat Effects
When mixing methanol with water, the breaking and formation of hydrogen bond achieves a dynamic balance, showing an overall exothermic process; when mixing acetonitrile with water, the formation of hydrogen bond cannot compensate the lost energy from the breaking, resulting in an endothermic process.
If an analysis uses on-line multi-pump mixing to prepare the mobile phase, methanol can produce heat upon mixing and thereby induce baseline drift, while acetonitrile does not present this problem.
UV Absorption
Methanol absorbs in the 205–210 nm range, while acetonitrile absorbs around 190 nm. When separating analytes of low maximum absorbance wavelengths (aspartame, neotame, organic acids, etc.), acetonitrile improves baseline stability, thereby enhances overall method sensitivity.
Compatibility with Buffers
When the aqueous mobile phase contains buffer salts, methanol is preferred. This is due to high ionic strength affecting acetonitrile-water miscibility, which is less affecting on methanol-water. Even if buffer salts precipitate, methanol will not separate from water. Therefore, methanol has a high tolerance limit for buffer salt usage than acetonitrile.
Summary
In summary, the primary criterion in choosing between methanol and acetonitrile is whether the polarity is suitable for separating target components in your sample matrix.
Methanol can be used whenever the mobile phase is pre-mixed offline, the analyte's maximum absorbance wavelength is greater than 210 nm, and the viscosity is within the column's acceptable range. Acetonitrile can be used whenever the buffer salts in aqueous mobile phase (if present) do not affect the mixing of acetonitrile and water.
Rather than applying rigid "formulas", analysts should adjust solvent selection and gradient strategy in a way that meet the real analytical requirements.