Professional Documents
Culture Documents
7 Key Differences in The Use of Methanol and Acetonitrile
7 Key Differences in The Use of Methanol and Acetonitrile
Acetonitrile
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 we outline 7 key differences to keep in mind when using methanol or
acetonitrile for analysis.
1.Column Pressure
Fig. 1 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 oC 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.
2.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. 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 reconsidering 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.
3.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. The nomogram in Fig. 5 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
been using acetonitrile as the mobile phase with a ratio to water of 50/50 (v/v),
when changing to methanol the equivalent ratio of methanol/water would be
60/40 (v/v).
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 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
Above we have introduced 7 key points in choosing between methanol and
acetonitrile as the organic solvent in HPLC analysis. 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 of 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.