- Acetonitrile and methanol are both used as solvents in liquid chromatography (LC) or high-performance LC (HPLC) systems. Even though they share many characteristics, acetonitrile is generally preferred as a solvent over methanol because it tends to give a better signal from an evaporative light scattering detector (ELSD).
- If ultra-high pressure liquid chromatography is used with tandem mass spectrometry (UHPLC/MS), acetonitrile is preferred; this is due to the fact that it forms less stable adducts compared to methanol. In addition, acetonitrile has a higher dielectric constant than methanol. This can increase the rate of migration of solutes in an electric field.
Both methanol and acetonitrile are common solvents used in HPLC
- Both methanol and acetonitrile can be used as mobile phase solvents in a one-component separation mode, or they can be co-solvent with water or other compatible solvents. They are both very polar, miscible with water, and relatively volatile.
- These qualities make them similar to each other in many aspects. However, there are also some key differences between the two compounds that should be considered when determining which reagent will be most useful for your particular needs.
Acetonitrile is preferred over methanol because it tends to give a better signal from ELSD
ELSD
- Acetonitrile is preferred over methanol for ELSD because it's more volatile and therefore a better solvent. Methanol is better suited for CID because it's more polar and less volatile than acetonitrile.
Dielectric Constant (∆e)
- One of the main purposes for using a mobile phase solvent with a high dielectric constant is to increase the ability of the analytes to dissolve in that solvent. The higher the dielectric constant, the more polar the solvent and the more likely an analyte can dissolve in that solvent. This is because water forms hydrogen bonds with itself and other molecules (such as acetonitrile), which helps it solubilize molecules that otherwise would not be able to dissolve well in water.
- The dielectric constant (∆e) is a measure of the ability of a solvent to dissolve salts. This can be useful for selecting an appropriate solvent for your experiment, especially if you are working with polar analytes. The higher the dielectric constant, the more polar the solvent and the more likely an analyte can dissolve in that solvent. Acetonitrile has a higher dielectric constant than methanol.
- Acetonitrile has a higher di-electron constant than methanol. Another purpose of using a high dielectric constant solvent is to increase the rate of migration of a solute in an electric field.
Dissolution of Solute
- The next reason for using methanol and acetonitrile is to increase the ability of the analytes to dissolve in that solvent. Methanol and acetonitrile are both high dielectric solvents and this property allow them to be used as mobile phases in reversed-phase liquid chromatography.
- The advantage of using these solvents is that they allow you to separate compounds that would not normally be able to be separated by other methods because they have similar boiling points, etc.
- The higher the dielectric constant, the more polar the solvent and the more likely an analyte can dissolve in that solvent.
Use in LC-MS
- In LC-MS, acetonitrile is used because it forms less stable adducts compared to methanol for volatility concerns. Methanol is preferred in LC-MS because it forms more stable adducts compared to acetonitrile.
Acetonitrile vs. Methanol for Reverse Phase Chromatography
Acetonitrile (ACN) and methanol (MeOH) are the most common organic modifiers in reversed-phase chromatography. Both solvents offer certain advantages and disadvantages. This guide takes a deeper look at acetonitrile vs. methanol for reverse-phase chromatography.
Absorbance
- LC grade acetonitrile is well suited for HPLC-UV assays, as it has the lowest absorbance of UV wavelengths. It also has low viscosity, high elution strength, and miscibility in water.
- LC Grade solvents for both acetonitrile and methanol are manufactured with the removal of UV-absorbing impurities. Mass Spectrometry Grade solvents such as acetonitrile and methanol meet all the requirements of modern LC-MS ionization methods (ESI/APCI – positive and negative mode). Due to their low level of ionic background and low ion suppression, these solvents ensure high reproducibility and high 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.
What is the difference between methanol and acetonitrile?
- Acetonitrile (MeCN) and methanol (MeOH) are the most commonly used organic modifiers in reversed-phase chromatography. Although both solvents offer certain advantages and disadvantages, one of their key strengths, from a chromatographic perspective, is that they offer substantially different selectivity, and as such, are valuable for method development purposes.
Methanol and acetonitrile are organic acids commonly used as the mobile phase in reverse-phase chromatography. The properties of these two organic acids differ, and below are key differences to keep in mind when using methanol or acetonitrile for analysis.
Column Pressure
Fig. shows an example of the solvent ratio and solvent delivery pressure for mixtures of water/acetonitrile and water/methanol.
- The pressure also tends to become lower as the viscosity of the solvent decreases due to the higher column temperature. Setting the column temperature between 25-40°C and comparing the column pressures for water/acetonitrile and water/methanol, we can see that the pressure is higher for methanol. When switching the mobile phase from acetonitrile to methanol, the pressure resistance of the equipment and column should be rechecked.
Absorption Spectrum
- Figs. 2 and 3 show the absorption spectra of acetonitrile and methanol, including both commercial-grade solvent for HPLC use and high-grade solvent. Commercial organic solvents for HPLC have been processed to remove virtually all impurities and to display absorbance within set limits between specified wavelengths.
Fig.2 Absorbance spectrum of acetonitrile
- It can be seen from Fig. 2 that the absorbance of HPLC-grade acetonitrile is particularly low at short wavelengths. This HPLC-grade acetonitrile is therefore suited to high-sensitivity analysis with UV detection in the short-wavelength region. Moreover, organic solvents which have been processed for LCMS analysis have both UV-absorbent impurities and residual metals removed.
- This acts to prevent background noise specific to LCMS analysis. When changing the organic solvent from acetonitrile to methanol, ghost peaks may be detected in gradient analysis due to the analytical conditions in the UV short wavelength range. In this case, we recommend considering the solvent grade. If the cause of the ghost peaks is unclear and causes problems in the analytical results, try the Ghost Trap DS, which removes impurities from organic solvents.
Fig.3 Absorbance spectrum of methanol
Elution Strength
- Fig.4 shows an example separation of parabens, which is p-hydroxybenzoic acid, with ODS column. It can be seen that when acetonitrile and methanol are mixed with water in the same ratio, an acetonitrile mobile phase displays greater elution strength.
Fig.4 Comparison of the elution strength of methanol and acetonitrile (p-hydroxybenzoic acid; parabens)
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- The nomogram in Fig. 5 below shows the ratios of methanol and acetonitrile to water with equivalent solvent strength, useful for approximate calculation of elution strength when changing between these solvents. If we have previously used acetonitrile as the mobile phase with a ratio to the water of 50/50 (v/v), when changing to methanol the equivalent ratio of methanol/water would be 60/40 (v/v).
Fig.5 Nomogram of solvent strength for reverse-phase chromatography (taken from "Practical HPLC" Method Development”)
Separation Selectivity
- The separation selectivity of acetonitrile and methanol differ, but since selectivity depends on the properties of the dissolved compound, it is not the case that selectivity is always higher for one or the other. In the separation of positional isomers, phenyl columns may be the most appropriate columns for reverse-phase chromatography.
- In addition, to hydrophobic interactions, π-π interactions of the phenyl stationary phase contribute to separation. Fig. 6 shows an example of the separation of positional isomers of cresol.
- Acetonitrile (CH3-C≡N) has a triple C-N bond and therefore π electrons, whereas methanol(CH3-OH) has no π electrons. For a phenyl column, using methanol as the mobile phase allows π-π interactions, which improves the separation.
Fig.6 Difference in selectivity between methanol and acetonitrile (for positional isomers of cresol)
Retention Behavior
- Methanol and acetonitrile have different chemical properties. Methanol is a protic solvent, whereas acetonitrile is a non-protic solvent, so we know that their elution behavior will differ. If adequate separation can not be obtained with an acetonitrile-based mobile phase, switching to a methanol-based mobile phase to change the elution order is one useful possibility for method development.
Fig.7 Differences in elution selectivity between methanol and acetonitrile
- Fig. 7 (above) shows an example of separation by methanol or acetonitrile of compounds in which one the hydrogen atom of a benzene ring is substituted with a carboxyl group or a hydroxyl group. When the three compounds are equally retained, it can be seen that the elution order of phenol and benzoic acid changes depending on the solvent used.
- Depending on the column type, there may be side effects from polar functional groups from the packing material in addition to the chemically-modified functional groups such as ODS groups and C8 (octyl)groups. There are also cases where the organic solvents and side effects from the functional groups together have a positive effect.
Fig.8 Separation of cephem antibiotics using an ODS column
- Figs. 8 (above) and 9 (below) show the separation of 13 cephem antibiotics using a reverse-phase column and acetonitrile or methanol respectively, with the same analytical conditions in both cases. The retention and elution order differ depending on whether acetonitrile or methanol is used.
- We also know that the elution order differs depending on the stationary phase. For example, when comparing ODS and C8 columns, the C8 column will generally have smaller retention values. However, due to the polar functional groups from the packing material, it is not simply the case that the retention is always smaller; the retention behavior also differs. Since the retention behavior is affected by these various factors, it is necessary to try different combinations of mobile and stationary phases to optimize analysis conditions.
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Fig.9 Separation of cephem antibiotics using a C8 column
Precipitation from mixing with a buffer
- In reverse-phase chromatography, buffers are used with water-based mobile phases. They are mixed with organic solvents for use, but depending on the type of buffer and organic solvents, too high a quantity of organic solvents may cause the buffer salt to be precipitated.
- Tables 1 and 2 show whether precipitation occurs for mixtures of commonly-used buffers with acetonitrile or methanol respectively. The values in the table show the ratio (v/v) at which precipitation starts to occur. We can see that for some buffers there is no precipitation for either organic solvent, but in general, methanol causes less precipitation.
* Values shown in the table may vary depending on laboratory conditions.
** ”〇” means no precipitation.
Heat of reaction from mixing with water
- For isocratic elution, water and organic solvent pre-mixed in the reserve bottle are used as the mobile phase. When mixed with water, methanol reacts exothermically. By contrast, acetonitrile reacts endothermically and therefore the temperature of the liquid will go below room temperature.
- As the acetonitrile mixture gradually returns to room temperature, bubbles tend to form in the liquid. Also, if the mixture is used as a mobile phase before it has returned to room temperature, retention times will be faster and only stabilize as the liquid approaches room temperature.
- Meanwhile, methanol produces heat when mixed with water, which has a degassing effect. This means that preparing a water and methanol mixture as the mobile phase requires less care than in the case of acetonitrile.
Summary
From the perspective of analytical workflow, there are differences in column pressure, UV absorption, and buffer compatibility, and when it comes to analytical separation, attention should be paid to elution power, separation selectivity and retention behavior. Understanding these differences in chemical properties of methanol and acetonitrile, together with appropriate column combinations, reduces the risk of problems in HPLC analysis and improves the efficiency of method development.
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