Systematize Columns Resolution For Free

The most important thing in HPLC is to obtain the optimum resolution in the minimum time. A resolution value of 1.5 or greater between two peaks will ensure that the sample components are well (baseline) separated to a degree at which the area or height of each peak may be accurately measured.

Resolution is calculated using the separation of two peaks in terms of their average peak width at the base (tR2 > tR1). In the case of two adjacent peaks, it may be assumed that the peak width at the base wb1 ? wb2, and thus, the width of the second peak may be substituted for the average value.

Generally, increasing the organic solvent content, decreasing the pH, and decreasing the flow rate improve peak resolution, but they also affect the retention time, peak shape, and sensitivity. Therefore, you need to optimize these parameters by performing a gradient elution, a buffer titration, or a flow optimization.

Consider using smaller particle sizes and solid-core particles, which can further increase resolution by increasing efficiency. Longer columns generally mean smaller particles and larger pores. This improves peak resolution but increases back pressure and analysis time.

Issues such as incorrect pH, buffer concentration, or solvent composition can impact separation and lead to resolution failure. Sample Preparation: Proper sample preparation is crucial to obtaining accurate HPLC results.

Another effective method is to change the column particle size. Columns with smaller particle sizes produce higher plate numbers for sharper peaks and are able to resolve closely eluting peaks.

The advantage of switching to longer columns is that the theoretical plates will increase and so will the resolution. The number of theoretical plates (N) is directly proportional to the length of the column and inversely proportional to the particle diameter2.