Choosing the Right Eluent in HPLC

The choice of eluent (flux, solvent) is crucial to the successful design of a chromatographic method. Since this choice depends on the column used, it must be selected first. The detector also affects the choice of eluent: UV detectors require eluents that have no residual absorption at the wavelength used so as not to interfere with the detection of the analytes. For example, some stabilizers that prevent the formation of peroxides, such as dioxane or THF, are UV active. Especially in gradient elution this is an aspect that has to be considered. So-called quenching effects due to insufficient degassing of the eluents should also be avoided.

In general, the solvent of the substance can be regarded as the optimal eluent, since it can prevent the chromatography from being influenced. If this is not possible, the eluent and solvent should be structurally similar or at least well miscible to avoid double peaks or peak shoulders.

Methanol or acetonitrile as eluent in RP HPLC?

In order to answer this question, several requirements and circumstances have to be examined and considered. First and foremost, the substances to be separated and their selectivity must be determined. 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 a lower self-absorption than methanol in small wavelength ranges. Above 230 nm, methanol is preferred because its baseline is much more stable than that of acetonitrile.

Viscosity of eluent mixtures

For gradient separations, the viscosity of the eluent mixtures must be taken into account. If the viscosity is too high, the pressure in the system may increase during the measurement. This can result in increased material consumption or the termination of the measurement. Methanol/water mixtures generally have a higher viscosity than acetonitrile/water mixtures when the water content is 40-60%. In addition, methanol/water mixtures form significantly more air bubbles, so that removal of these bubbles, e.g. in an ultrasonic bath, is not sufficient and the use of throttling capillaries or back pressure regulators becomes necessary.

Due to its consistency, acetonitrile, as the more toxic of the two eluents, initially poses a greater risk. However, its much higher elution force means that much less eluent is required than with methanol. In terms of price, methanol is less expensive than acetonitrile, but this is not directly comparable due to the higher consumption.

Conclusion: acetonitrile and its advantages

In summary, both methanol and acetonitrile may be the more suitable eluent for a given separation problem. In any case, the consistency of the substances to be separated, the wavelength of the UV detector, and the viscosity of the liquid (especially for gradient separations) must be considered. 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 significant problems.