Tuesday 31 October 2017

Gradient HPLC: Is your mobile phase causing a ghost peak? Here's an easy way to find out...

Ghost peaks from mobile phase in gradient HPLC
Although not usually a problem in HPLC methods that use isocratic elution, ghost peaks resulting from the mobile phase can be a problem for gradient elution methods. Contaminants present in the mobile phase can behave like sample components and interact with the column during the gradient, leading to a ghost peak (or peaks) in the chromatogram.

However, it is easy to diagnose if your ghost peak is from the mobile phase. Simply run a blank gradient. It’s better if you can do this without performing an injection (to rule out the injection as the source of the ghost peak). In Empower, it is called ‘condition column’. If the peak is coming from the mobile phase then increasing the equilibration time at the beginning of the gradient will increase the size of the peak. For example, you could compare holding at the starting conditions for 5 minutes to holding at 10 minutes. The ghost peak will double in size if it is coming from the mobile phase.


Wednesday 11 October 2017

Validation Challenges for 'Area Percent' Methods

Analytical methods which use area normalisation, or ‘area percent’, as a calibration model are common in the analysis of biomolecules by chromatography and electrophoresis. They present particular challenges when it comes to validation.
The approach is to assign a value of 100% to the area of all the peaks in a chromatogram or electropherogram and then quantify individual peaks as a percentage of that total. Although a very simple idea, the reality is that these methods can be quite complex. In particular, the interpretation of what the percentage value assigned to each peak actually means. If a correlation between this value and the amount of the component present in the sample for analysis is desired then it has to be assumed that the size of the peaks relates to how much is actually there.

This may not actually be the case. For example, if using the popular detector of UV, then the size of the peaks will relate to not just how much is there but also the type of chromophores present in the molecules. Additionally, an area percent approach assumes that there is a linear correlation from zero to 100% which allows each peak to be expressed as a percentage, this may not be the case if the size of the peaks are not within the dynamic linear range of the detector.

When it comes to method validation, it is desirable to address these assumptions and demonstrate that they are justified and this may be achieved if there are reference materials for the components in the sample. However, often the reason for using this approach is that external reference materials for the sample components separated by the method are not available. In this case method validation is challenging.
If you want to learn more about validating methods then you may be interested in the following courses from Mourne Training Services Ltd: