HPLC Tips: How to calculate void volume

In this video we delve into the critical aspect of calculating void volume in High Performance Liquid Chromatography (HPLC). Understanding the void volume, or the space within the column not occupied by the stationary phase, is essential for precise chromatographic analysis.

We start by introducing T0 measurement—how to determine the time at which solutes pass through the column without interacting with the stationary phase. Using uracil as a common example, we demonstrate practical methods for visually identifying T0 through disturbances or sine waves on the baseline after injection.

The video further explains how to estimate void volume using a simple rule of thumb, applicable to standard HPLC columns. We cover the significance of the column's internal diameter, particularly the standard 4.6 millimeter ID, and how every 10 cm of column length equates to approximately 1 mL of void volume.

This knowledge is crucial for selecting the right column size and optimizing flow rates, enhancing chromatographic efficiency and performance.

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