HPLC Products

Problem

By Ghost Peaks, we refer to peaks which appear in the chromatogram, but do not originate from the sample.

When running a gradient, ghost peaks are almost always present. So it is really important to run a blank over and over until a constant result is obtained. When running a gradient for the first time, it is likely that contamination will wash off the column. So the column must either be flushed in a strong eluent prior to running the gradient, or gradient blanks must be run until reproducible results are obtained.

Ghost peaks eluted during a reversed phase gradient run are almost always due to impurities in the water. These collect on the column during the early part of the gradient. Then as the eluent becomes strong enough to elute them, they come off and peaks appear. So before running any samples, we need to know the retention time and size of each and every ghost peak.

The size of ghost peaks will be dependent upon the time the system is left to re-equilibrate between runs. So to be sure that there is no component of the sample co-eluting, we need to run the system with consisten re-equilibration times, and if it is left running over lunch, a further blank gradient is needed to clear the column.

If a ghost peak co-elutes with a peak of interest, we have a problem. It is not safe to simply subtract the expected peak area of the ghost peak. So we must either get rid of the ghost peak, or change the separation to resolve the components of interest. To learn how to optimise a gradient separation, book on the HPLC Method Development Course.

If the aim is to get rid of the ghost peak, consider where it comes from. It will be an organic component of the water used by the HPLC water supplier which has passed through the clean up process. Some suppliers have a better water source than others, and some HPLC Solvent manufacturers are better at cleaning up the water than others. So it may be enough to buy the water from a different supplier. However, no supplier can guarantee the impurity content of their water, so the situation may change from batch to batch!

If a lab water purifier is available, this may offer an acceptable purity. It will need a carbon scrubbing system, and since the cartridge collects the impurities, it will need to be changed regularly. Also beware of how long the water stays in the tank, and ensure that the air feed to the tank has an antimicrobial filter fitted to prevent bacteria from entering the tank.

If you really struggle to get rid of the ghost peaks, and have an old C18 column available which is not blocked, you could pump a litre of HPLC water through it. The ghost peaks should bind to the column, leaving the water clean for use, although do test it first. From time to time the old column will need to be washed with methanol to remove the organic impurities. But its a good use for an old column!