HPLC (High Performance Liquid Chromatography), GC (Gas Chromatography), and TLC (Thin Layer Chromatography) are a tandem of powerful analytical tool used to solve almost all separation activities in the laboratory.
In our first blog article on increasing the sensitivity in case of limit of detection (LOD) / limit of quantitation (LOQ) problems in HPLC methods, we addressed the increase in sensitivity primarily by reducing baseline noise. Now we will look on the same topic from another perspective – with the eyes of “method optimization”. What can be optimized to increase the peak size and thus the overall signal to noise ratio (S/R)?
During validation of purity testing methods of drugs or their active ingredients, sooner or later a point comes when the detection and quantification limits are needed to be determined. The limit of detection or LOD is defined as the lowest amount of an analyte in a sample that can still be detected. This is different from quantification - since the limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can still be reliably quantified.
HPLC methods are often part of routine analysis of drugs or drug substances in pharmaceutical QC laboratories under GMP conditions.
The shape of the peak of a HPLC chromatogram is extremely critical for its evaluation. Subtle changes in the shape during a quantitative measurement can lead to misleading results and in turn can ruin the entire measurements obtained throughout the experiment. Therefore, it is important to keep a watchful eye on the “peak shape”.
In another blog post, we have already discussed the problem of peak broadening under the topic "HPLC: What to do in case of peaks being too broad?” while focussing on symmetrical broad peaks. In this blog post, we will emphasize more on the asymmetric peaks.
Not only in university laboratories, but especially in the laboratories of the pharmaceutical industry or of contract research laboratories, HPLC methods play an important role e.g. as purity tests of medicines and their active pharmaceutical ingredient during quality control. Therefore, let's take a closer look at the HPLC system.
High Pressure Liquid Chromatography (HPLC) is a technique widely used to separate various substances in solution. A HPLC system could be thought as an example of a steady flow:
In HPLC systems everything is associated with each other. There is not one common solution for one specific problem. Of course, pressure is completely normal for high pressure liquid chromatography (HPLC). This is the backpressure of the eluents, which are pumped through very narrow capillaries onto a tightly packed column. So, what can go wrong? Quite a lot!