High performance liquid chromatography (HPLC) is an important means of modern drug analysis. In daily experiments, analytical workers usually focus on the choice of mobile phase, chromatographic column and instrument method. Therefore, when abnormal peak shape occurs, analysts tend to ignore the improper solvent used to dissolve samples.

The concept of solvent effect

Generally, the solvent effect is the phenomenon of chromatographic peak deformation caused by the sample solvent strength being greater than the mobile phase strength. However, we will find that sometimes the strength of our sample solvent is not greater than that of the mobile phase, but the peak deformation still occurs due to the addition of some diluents. Therefore, in a broad sense, the concept of “solvent effect” should be extended to refer to the abnormal chromatographic behavior of some components in the sample due to the large difference in the presence of diluent and mobile phase.

Cause of solvent effect

1. The intensity of the solvent

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

2. Injection volume

When the sample volume is small, the solute diffused into the mobile phase accounts for the majority, and the diffusion is completed in a very short time, so the peak shape is not much different from that of the sample directly dissolved by the mobile phase. With the increase of sample volume, the amount of solute left in the solvent increases gradually. When the sample volume increases to a certain amount, the amount of solute left in the solvent becomes not negligible, which is shown as the bifurcation and tailing of chromatographic peaks on the chromatogram.

3. Solvent compatibility

Sometimes, when we cannot directly dissolve the sample by using the mobile phase or the organic phase in the mobile phase, many small partners will choose to use soluble solvents such as chloroform and toluene, or even “universal solvents” such as DMSO to dissolve the sample. The result of such injection is that the solvent is incompatible with the mobile phase, and the solute is difficult to diffuse in the mobile phase. For another example, surfactant is often added to the solvent in the dissolution curve of reference preparation. After the solvent with surfactant is injected, the distribution coefficient of solute in the solvent containing surfactant is quite different from that in the mobile phase, which will lead to the problem of peak shape and even the drift of retention time.

4. Ionization state

The difference of ionization state is mainly caused by the pH difference of solvent and mobile phase. We all know that in the reversed-phase system, the retention behavior will be significantly different due to the different existing states of the target compounds, and the existing state depends on the ionization state of the target compounds in different pH systems. If the ionization state of the sample in the solvent and mobile phase is quite different, and the sample solution has no time to buffer to the ionization state in the mobile phase in the process of contacting the mobile phase, it is a time. If it is longer, it is easy to show that the retention time is misaligned or peak shape is deformed, as shown in the example below.

The mobile phase is pH 3.0 buffer salt. The green spectrum is the reference. The solvent of the other three spectrogram samples from bottom to top are 0.1% phosphoric acid, purified water and pH 3.0 mobile phase, respectively

Solution of solvent effect

1. Solvent strength
The solution to this most common solvent effect is to adjust the diluent or mobile phase so that the elution capacity of the diluent is similar to that of the mobile phase, or slightly lower than that of the mobile phase. In general, in order to avoid the solvent effect caused by the difference in solvent strength, it is safer to use the organic phase ratio in the diluent to be similar to the mobile phase, if necessary, slightly higher than the mobile phase, but the solvent effect risk should be assessed against it.

2. Injection volume

Under normal circumstances, in order to ensure the peak shape, we control the injection volume within 5-20μL, try not to exceed 25μL. If a suitable injection volume cannot be reduced, in order to obtain a better peak shape, solvents with the same or close proportion to the mobile phase should be selected as far as possible.

3. Solvent compatibility

There are two ways to make solvent and mobile phase compatible:

1. If the mobile phase or the organic phase in the mobile phase cannot directly dissolve the sample, dissolve the sample in a solvent that can dissolve the sample into a high concentration of reserve solution, and then dilute it with the mobile phase to the desired concentration;

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

4. Ionization state

Differences in ionization states can be manifested in many cases as drift of retention time or instability. This can be improved by adjusting the pH of the sample solution to match the flow, or by increasing the buffering capacity of the mobile phase.

The above is a summary of the high performance liquid chromatography “solvent effect” principle and solution. A suitable solvent system must be selected before HPLC injection. If the solvent is not properly selected, the abnormal peak shape will occur to different degrees, which will affect the analysis results.

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