Eight major reasons for chromatographic peak branching

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1. Column contamination: There was no problem at first, but this issue arose after some time of use; it may be caused by contamination. At this point, it is necessary to intensify the flushing of the chromatography column, that is, to flush it with a solvent that is more potent than the method’s mobile phase. Rinsing the column with pure acetonitrile in Figure 1 eliminated the branching issue of peak 3. 2. Guard column failure: If the sample matrix is quite dirty, after prolonged use, issues such as peak splitting or tailing may occur. These problems are very likely caused by a failed guard column. As a general rough guideline, if the number of plates, pressure, or separation efficiency changes by more than 10%, it is necessary to replace the guard column. The protection column is a consumable; it is recommended to replace it directly rather than attempting to regenerate it. 3. Incorrect connector connection: If, during use, it is found that the shape of each peak is problematic, first check whether there are any connection issues. The pipeline may be too long or too short—this can lead to leaks or peak splitting/tailing, as shown in Figure 2. If the pipeline is too long or the seal ring is in the wrong position, leakage will occur. If the distance the tubing protrudes is insufficient, a dead space will be created, forming a mixing chamber that results in extra-column volume and distorts the peak shape. If you are using columns from different manufacturers, be sure to use the correct connectors and ensure that the connector at the end of the column is in the proper position. 4. Solvent effect: In reverse-phase LC, when using 100% organic solvents or 100% strong solvents (acetonitrile in Figure 4), large-volume injections cause the chromatographic peaks to elute from the column prematurely, resulting in peak distortion. To resolve this issue, the analyte can be evaporated and concentrated to reduce the injection volume. Alternatively, the injection solvent can be changed to water for dilution, which is more compatible with the mobile phase; even with a larger injection volume, peak distortion will not occur. Peaks that emerge earlier or those near the solvent front are more prone to tailing. In liquid chromatography, it is most ideal to use a small volume of sample dissolved in the mobile phase. 5. Sample overload: When the amount of sample injected exceeds the capacity of the column, the sample peak may show branching or tailing; the solution is to reduce the amount of sample injected. Such problems are generally more pronounced when using small-volume chromatography columns. Therefore, when switching from conventional 250 mm or 150 mm long columns to smaller-volume fast analysis columns in a chromatographic method, the injection volume must be reduced proportionally to the decrease in column volume. 6. Column collapse: Peak splitting caused by column collapse is difficult to repair. If the pH of the mobile phase is >7 or if it is used for an extended period of time near the critical point of the column’s pH tolerance, it is more likely to cause the silica filler to dissolve and collapse. If column collapse occurs, you will see that the shape of each peak in the chromatogram changes (peaks become tailing, wider, or split), as shown in Figure 5. It is possible to run the system in the opposite direction for a short time, but it is recommended to replace the column directly. During regular use, make sure to lower the temperature to below 40 ℃, especially when using a high-pH mobile phase ； Use high-temperature compatible chromatography columns, such as stereoprotected silica columns, mixed-type columns, polymer columns, zirconia columns, etc. Use a high-capacity or double-crosslinked stationary phase that is particularly suitable for operation at higher pH values (such as ZORBAX Extend-C18), or use polymer, hybrid, or zirconia reverse-phase columns. 7. Partial blockage of the pre-column frit: When analyzing dirty samples over a long period of time, in the absence of a guard column or an online pre-column filter, particles and strongly adsorbing substances are likely to cause blockage at the inlet frit of the column. This is accompanied by an increase in pressure. The usual solution is to backflush the chromatographic column. Chromatography columns with a particle size of 1.8um should try to avoid backflushing. Since a high-efficiency column packing process is being used currently, it is not recommended to replace the column inlet sieve plate, and it is also impossible to do so on many columns. Replacing the screen plate may affect column efficiency. Therefore, it is best to install an online filter (part number 5067-4638) and a guard column in front of the chromatography column. 8. Decline in column performance: When using a chromatography column, it is employed to analyze various target substances, with different mobile phases being used to separate different sample matrices. Over time, the column is affected by these substances and undergoes subtle changes, which can lead to peak splitting, tailing, or changes in retention times. It is very important to retain the chromatogram of test samples when using a new chromatography column; by making comparisons after it has been in use for a period of time, changes in performance can be detected. If the resolution decreases to the point that proper quantification is no longer possible, this chromatography column should be discarded and replaced.