Have you ever encountered issues such as fronting, peak broadening, splitting, etc. during liquid phase system analysis? Why does reducing the injection amount of the same sample sometimes result in a better-looking chromatographic peak? In daily analysis work, we usually focus more on the selection of mobile phase and instrument methods, and neglect the importance of sample injection solvents. If the solvent selection is improper, it will lead to “solvent effects”, causing errors in qualitative and quantitative analysis and affecting the analysis results.
What is solvent effect?
Solvent effect refers to the phenomenon of chromatographic peak deformation caused by the solvent strength of the diluent being greater than that of the mobile phase.
For example, when a sample is dissolved in pure acetonitrile and injected into a reverse phase system with a mobile phase of acetonitrile-water (18:82), it will result in peak splitting or tailing, whereas if the sample is dissolved in the mobile phase, the peak shape will return to normal.
In simple terms, the occurrence of solvent effects is due to the mismatch between the solvent used to dissolve the sample and the mobile phase.
The “mismatch” between the sample solvent and the mobile phase includes:
● Differences in elution ability between the two.
● Differences in mutual solubility between the two.
● Differences in the solubility of sample molecules in the two.
● Differences in the distribution of sample molecules between the molecular and ionic states in the two.
The mechanism behind solvent effect.
Chromatographic analysis, the sampling process can be divided into three stages:
● The first stage is when the sample reaches the column.
● The second stage is when the sample part enters the column.
● The third stage is that all the samples enter the column.
Solvent effects, on the other hand, mainly occur during the second stage of the injection process, when the sample part enters the column.
During this stage, the mechanism of solvent effect can be explained in three parts:
● When the sample is injected into the system and enters the chromatographic column, the target compound in the mobile phase environment is eluted at a normal speed. Due to the strong solvent elution, the movement speed is fast, and the sample gradually splits in space.
● Within a certain time, the target compound in the solvent environment always moves faster, and the distance between the two parts expands. However, the moving fast target compound is gradually diluted by the mobile phase (note: because neither the solvent nor the mobile phase is retained), and its movement speed gradually decreases to the normal flow rate. Until the environment of the target compound at the front is completely replaced by the mobile phase, the distance between the two parts of the target compound stabilizes. Therefore, the greater the difference in elution intensity between the sample solvent and the mobile phase, the greater the final distance between the two parts of the target compound.
● All target compounds move normally in the chromatographic column, but the split is irreversible.
After passing through the detector, the same target compound gives two chromatographic peaks. The part of the target compound that diffused into the mobile phase before entering the column formed the later chromatographic peak, and its retention time is basically consistent with the true retention time of the target compound.
How to solve solvent effects
For this most common solvent effect, the solution is to adjust the diluent or mobile phase to make their elution strengths closer, or to have the diluent elution strength slightly lower than the mobile phase. In general, to avoid solvent effects caused by differences in solvent strength, the more reliable approach in reverse-phase chromatography is to maintain consistency between the diluent and the mobile phase. If the mobile phase or organic phase in the mobile phase cannot directly dissolve the sample, then the sample can be dissolved into a high-concentration stock solution using a solvent that can dissolve the sample, and then diluted to the desired concentration with the mobile phase.
2.Reducing the injection volume
When the sample solvent has a stronger elution power than the mobile phase, fronting peaks are easily formed due to the instantaneous effect of the sample solvent on the mobile phase. The degree of fronting is related to the injection volume, the larger the injection volume, the more pronounced the fronting and the more obvious the solvent effect. Conversely, the smaller the injection volume, the smaller the solvent effect. Therefore, reducing the injection volume can effectively weaken or eliminate the solvent effect.
Differences in ionization state often result in drift or instability in retention time. This can be improved by adjusting the pH of the sample solution to be consistent with the mobile phase or by increasing the buffering capacity of the mobile phase.
For gradient elution, a larger inner diameter pre-column tubing can be used without affecting the separation, as long as the separation efficiency is not compromised. (Note: If using isocratic elution, this method may decrease column efficiency; for gradient elution, it has little effect on column efficiency).