11. Issues with peaks

As explained in previous sections, it is critical to choose the right column and chromatographic conditions to afford a proper separation of compounds, a good peak resolution, and also peak shape, which has an impact on how the peak is analyzed.

Chromatographic peaks are measured by asymmetry and tailing factor.

When the asymmetry factor is equal to 1.0, the peak is perfectly symmetrical. Peak tailing occurs when the asymmetry factor is greater than 1.2. Above 2.0, the tailing is very pronounced and the chromatographic condition is considered undesirable.

There are several factors influencing peak shape, but they mostly fall into the following 3 categories:

1. column mismatch – when the chemistry of the column and analyzed compound are mismatched, the interactions do not allow for proper separation.

2. pH has a profound impact on peak tailing

3. tubing design – improperly cut tubing, wrong length and ID of the tubing cause dead volumes, which manifest in peak tailing. Other factors, such as column overload (too much sample volume injected) can cause peak tailing.

 

The most common peak shape problems:

1. Peak tailing

The most common causes are: blocked column frit, void space in the column, unresolved interfering peak eluting at similar retention time, wrong mobile phase pH, and wrong column chemistry.

 

2. Peak fronting

The most common causes are: low column temperature, wrong sample solvent, sample injection overload, and bad column.

 

3. Split peaks

The most common causes are: contamination of the guard column or main column, and wrong chromatographic conditions resulting in poor peak separation.