In general, we regard “solvent effect” as the phenomenon of chromatographic peak deformation when the strength of the sample solvent is greater than that of the mobile phase. Sometimes the strength of our sample solvent is not greater than that of the mobile phase due to the addition of some diluents, but the peak deformation still appears. Therefore, the concept of “solvent effect” in a broad sense should be extended to the great difference of some sample components between in the diluent and in mobile phase.


01 Solvent strength

This is the cause of the “solvent effect” in the general sense. For example, when the mobile phase is a low proportion of acetonitrile solution and the sample solvent is pure acetonitrile, the peak bifurcation or peak tailing will occur due to the strength of the solvent being greater than that of the mobile phase.

02 Injection volume

When the injection volume is small, most of the solute diffused into the mobile phase, and the diffusion is completed in a very short time. Therefore, the peak shape is not significantly different from that of the sample directly dissolved in the mobile phase. With the increase of the injection volume, the amount of solute remaining in the solvent gradually increases. When the injection volume increases to a certain amount, the amount of solute remaining in the solvent becomes non-negligible, which is shown as the bifurcation and trailing of peaks.

03 Solvent compatibility

Sometimes, when the mobile phase or the organic phase in it can not directly dissolve the sample, analysts may  use chloroform, toluene and other soluble solvents, or even DMSO such as “universal solvent” to dissolve the sample. The result of this injection is that the solvent is incompatible with the mobile phase and the solute is difficult to diffuse in the mobile phase. For intance, in the dissolution curves of reference preparations, surfactants are often added to the solvent. After the injection of the solvent with surfactant, the distribution coefficient of solute in the solvent with surfactant is greatly different from that in the mobile phase, which may lead to the problem of peak shape and the drift of retention time.

04 Ionization state

The difference of ionization state is mainly caused by the difference of pH in the solvent or mobile phase. It is known that the retention behavior of the target compound in the reversed phase system varies with the state of the target compound, and state depends on the ionization state of the target compound in the different pH system. If the ionization state of the sample in the solvent and the mobile phase is entirely different, and the sample solution can not be buffered to the ionization state in the mobile phase during the process of contacting the mobile phase, it is easy to show the retention time dislocation or peak deformation, as shown in the figure below.

(Mobile phase is pH 3.0 buffer salt. The green chromatogram shows the reference substance, and the other three sample solution is 0.1% phosphoric acid, purified water and pH 3.0 mobile phase from bottom to top.


01 Solvent strength

For this most common solvent effect, the solution is to adjust either the diluent or the mobile phase so that the elution capacity is similar or the diluent elution capacity is slightly lower than that of the mobile phase. In general, in order to avoid the solvent effect caused by the difference in the strength of the solvent, a more prudent approach in reverse phase chromatography is to take the same proportion of the organic phase in the diluent as the mobile phase, or the proportion can be slightly higher than the mobile phase if necessary, but the risk of the solvent effect should be assessed according to it.

02 Injection volume

In general, in order to ensure the peak shape, injection volume should be controlled between 5 and 20 μl, but do not to exceed 25 μl. If it is not possible to reduce the injection volume to a suitable size, a solvent with the same or close proportion to the mobile phase should be selected in order to obtain a better peak shape.

03 Solvent compatibility

At present, there are two methods to make the solvent and mobile phase compatible:

1. If the mobile phase or the organic phase in the mobile phase cannot directly dissolve the sample, use the solvent that can dissolve the sample to make a high concentration reserve solution, then dilute the mobile phase to the desired concentration.

2. Add the same cosolvent or surfactant In the mobile phase, or increase the proportion of organic phase.

04 Ionization state

In many cases, the difference in ionization state may appear as drift or instability of retention time. This can be improved by adjusting the pH of the sample solution to be the same with that of mobile phase or by increasing the buffering capacity of the mobile phase.

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