Why is methanol used in HPLC
Choosing the right eluent
In order to design a chromatographic method successfully, the choice of the eluent (solvent) is crucial. Since this depends on the column used, it must be selected first. The detector also limits the selection of the eluent: UV detectors require flow agents that have no residual absorption at the wavelength used in order not to interfere with the detection of the analytes. For example, some stabilizers that prevent the formation of peroxides, for example dioxane or THF, are UV-active. Particularly with gradient elution, this is an aspect that must be given special attention. So-called quenching effects due to insufficient degassing of the eluents should also be avoided.
The solvent of the substance can generally be described as the optimal eluent, since this means that any influence on the chromatography can be ruled out. If this cannot be guaranteed, the eluent and solvent should be structurally similar or at least easily miscible in order to avoid double peaks or the formation of shoulders in the peaks.
Methanol or acetonitrile as eluent in RP-HPLC?
In order to be able to answer this question, several requirements and conditions must be checked and observed. Of course, the primary decision is which substances have to be separated and what their selectivity is. However, the following considerations should also be made when choosing the eluent: If the UV detector measures in ranges of 200 nm and less, acetonitrile is advantageous because it has a lower self-absorption than methanol in the smaller wavelength ranges. On the other hand, above 230 nm, methanol is to be preferred, since its baseline here is much quieter than that of acetonitrile.
Viscosity of the eluent mixture
In the case of gradient separations, the viscosity of the eluent mixture must be taken into account. If this is too high, the pressure in the system can rise during the measurement, which can result in higher material or the measurement being aborted. With a water content of 40-60%, methanol / water mixtures generally have a higher viscosity than corresponding acetonitrile / water mixtures. In addition, methanol / water mixtures form significantly more air bubbles, so that removal of these in an ultrasonic bath, for example, is not sufficient and the use of restriction capillaries or back pressure regulators is necessary.
Acetonitrile, as the more toxic of the two eluents, initially entails a greater risk due to its nature. But due to the much higher elution power, significantly less is necessary than with a comparative use of methanol. In terms of price, methanol is lower than that of acetonitrile, but due to the greater consumption, this cannot be directly compared.
Conclusion: acetonitrile has more advantages
In conclusion, it can be said that, depending on the separation problem, both methanol and acetonitrile can be the more suitable superplasticizer. In any case, the nature of the substances to be separated, the wavelength of the UV detector and the viscosity of the flow agent must be taken into account (especially in the case of gradient separations). The financial and toxic burden of the two eluents must be weighed up personally. In most cases, however, acetonitrile leads to faster success without significant problems.
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