[Readers Insight] Why Do M-Shaped Peaks Occur?

In chromatographic analyses, a vexing problem commonly encountered during chromatogram interpretation is the appearance of M-shaped peaks. Even standards may face this phenomenon, yet its cause is often unclear. Adjusting the mobile phase or changing the column may seem to help slightly, or not at all, leaving analysts frustrated.

In this article, we explore the causes of M-shaped peaks and corresponding remedial approaches, from the author’s personal experience and point of view.

Solvent effects arise when the polarity difference between the diluent (reconstitution solvent) and the mobile phase is too large.

In reversed-phase systems the mobile phase is typically relatively polar, while the diluent, being often pure methanol or pure acetonitrile, is less polar. During elution, the outer portion of the injected droplet is preferentially eluted, causing part of the analyte to “elute faster”, which ultimately produces an M-shaped peak.

In this case, one can add a certain proportion of water to the diluent to increase its polarity, or use the mobile phase as the diluent directly. If both operations are constrained, reducing the injection volume may also improve the situation.

This situation is more common for acid–base or salt compounds, such as benzoic acid, sorbic acid, melamine, and some synthetic dyes. Such compounds may exist simultaneously as ionized and neutral species under certain pH conditions, which leads to different interactions with the stationary phase and the mobile phase, and thus to differences in retention time, forming an M-shaped peak.

For these cases, adjusting the mobile phase pH or adding buffer salts to keep the analyte in a single form can resolve the issue.

Isomers are generally categorized as functional-group isomers, positional isomers, cis-trans isomers, and stereoisomers (chiral isomers), among other less common types. Isomeric situations are difficult to resolve because the structures are similar and are hard to distinguish by spectrum or molecular weight, thus chromatography is typically required for separation.

In cases of isomers, one may attempt separation by adjusting the gradient elution program; if that is still unsatisfactory and there is no strict requirement to distinguish the individual isomers, increasing the proportion of organic modifier in the mobile phase to speed analyte elution can compress the M-shaped peak into a single peak for quantification.

For example, in the national standard GB 5009.35-2016 "Determination of synthetic colorants in food stuffs", Brilliant Blue, although having two forms in its standard material, had only one peak in the standard method (only in a later revision, GB 5009.35-2023, are the two peaks separated). 

Under the method’s elution conditions they do not separate; but in practice, if the gradient change is slowed a little, they do begin to split. Furthermore, fully separating these peaks is difficult and of limited practical value (as it would require naming both forms). Therefore, they are treated as a single Brilliant Blue peak in the 2016 version.

If samples contain substantial impurities, repeated large-batch analyses may contaminate the column inlet, causing droplet splitting during injection and resulting in M-shaped peaks.

This situation is relatively straightforward to address: 

• The first solution is to enhance upstream sample cleanup to reduce impurity load.
• Another solution is to install a guard column before the analytical column and replace the guard cartridge regularly. 
• Backflushing the column can also help to some extent with this contamination, but it may also cause secondary contamination of the column, so it is not recommended by the author.