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Choosing the right eluent in HPLC

In order to successfully design a chromatographic method, the choice of the eluent (flux, solvent) is crucial. Since this choice depends on the column used, it must be selected first. The detector also adjusts the selection of the eluent: UV detectors require eluents which show no residual absorption at the wavelength used in order for them to not interfere with the detection of the analytes. Some stabilizers for example, which prevent peroxide formation of e.g. dioxane or THF, are UV-active. Particularly in the case of gradient elution this is an aspect that must be considered specifically. So-called quenching effects due to insufficient degassing of the eluents should also be avoided.

The solvent of the substance can generally be referred to as the optimum eluent, since it can prevent chromatography to be influenced. If this cannot be ensured, the eluent and solvent should be structurally similar or at least well 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 answer this question, several requirements and circumstances have to be examined and observed. Of course, it is first and foremost to decide which substances have to be separated and how their selectivity is. However, the following considerations should also be taken into account when selecting the eluent: If the UV detector measures in the range of 200 nm and less, acetonitrile is advantageous because it has lower self-absorption than methanol in small wavelength areas. Above 230 nm, on the other hand, methanol is preferred, since its baseline here is significantly steadier than the one of acetonitrile.

Viscosity of the eluent mixtures

In case of gradient separations, the viscosity of the eluent mixtures must be taken into account. If it is too high, it can lead to an increase in pressure in the system during the measurement. That can result in an increased material consumption or the termination of the measurement. Methanol/water mixtures generally have a higher viscosity at a water content of 40-60% than corresponding acetonitrile/water mixtures. Moreover, methanol/water mixtures form significantly more air bubbles, so that a removal of those, for example in an ultrasonic bath, is not sufficient and the usage of restriction capillaries or backpressure regulators becomes necessary.

Because of its consistency Acetonitrile, as the more toxic of the two eluents, initially implies greater risk. However, the significantly higher elution force also means that considerably less eluent is necessary compared to methanol. In terms of price, methanol costs less than acetonitrile, but this is not directly comparable due to the higher consumption.

Conclusion: acetonitrile has more advantages

In conclusion, depending on the separation problem, both methanol and acetonitrile can be the more suitable eluent. In any case, the consistency of the substances to be separated, the wavelength of the UV detector, and the viscosity of the fluid (especially in the case of gradient separations) must be observed. Personal preferences must be evaluated regarding the financial and toxic burden of the two eluents. In most cases, however, acetonitrile leads to faster success without substantial problems.