What is Peak Splitting?

Peak splitting is when a Gaussian peak gets a shoulder or a twin. They have the same base, are unexpected and can be caused by a number of factors. The splitting can affect all peaks or just one, and different effects can be attributed to different causes.

It is not always an instrument problem that causes peak splitting. Samples and sample preparation are also contributory factors as determinants of the degree of peak splitting.

Single Peak Splitting

If only one peak in a chromatogram is splitting or has a shoulder, the problem is likely to be something related to the separation. One way of checking this is to inject a smaller sample volume. If this results in two discernible peaks, the problem could be two components eluting close together.

In this case the separation resolution needs to be improved. This can be achieved by revisiting the method parameters such as mobile phase, temperature, flow rate or even column type.

Frits and Voids

If all of the peaks are split in an HPLC run, it is an indication of a problem happening before separation has taken place. There are two common causes for splitting of all the peaks namely:

• A blocked frit
• A void in the packing at the head of the column

A blocked frit causes a spreading out of the sample as it enters the column. Normally the sample flows through the frit in a parallel band. But if there is a blockage in the frit, then part of the sample is delayed and enters the column later.

Since the delivery of the sample to the column is spread out, the separation of the sample is spread out. Hence the peaks for each component are split, and all peaks in the chromatogram are split.

If there is a void in the packing material, some of the sample travels faster to the column. The effect is the same as with a blocked frit. The sample is spread out before it enters the column and all of the peaks in the chromatogram are split.

Fixing Problems

As with all analytical instrumentation problem solving, it pays to approach the issues in a systematic way.

Check the easy fixes first.

• Is the method set up correctly with the right column? Is the mobile phase ok?
• Does a reduced injection volume help?
• Does flushing or back-flushing the column help?
• Replace the frit or column. In modern columns it can be simpler to replace the column unless it is a specialized column.

If there is contamination on the guard or analytical column inlet

• Remove the guard column and attempt analysis
• Replace the guard
• If the analytical column is obstructed, reverse and flush it
• If problem persists, the column may be fouled with strongly retained contaminants
• Use appropriate restoration procedures
• If the problem persists, the inlet is likely to be plugged
• Change the frit or replace the column

Another possibility is that the sample solvent may be incompatible with the mobile phase. If so, change the solvent and whenever possible, inject samples in the mobile phase.

Prevention is better than cure. Using in-line filters and column guards can reduce the incidence of blockages in a chromatography system, so preventing peak problems. The problem of blocked frits is highlighted in: ACE Ultra Low Dispersion, High Quality UHPLC and HPLC Pre-column Filters.

Sample filtration should also be considered when there is a risk of insoluble material entering the system. Simple techniques can save many hours of troubleshooting and downtime.

Image Source

• HPLC via Argonne National Laboratory