How to calculate void volume in HPLC?

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In the realm of High Performance Liquid Chromatography (HPLC), the precise measurement of T0, or the time at which solutes elute without interaction with the stationary phase, is fundamental.

Let’s discuss T0 and column volumes and explore measurement methods. Uracil, commonly used in the industry, serves as a practical example. By injecting uracil or simply the sample solvent, a disturbance or sine wave at the baseline, indicative of T0, can be observed. This provides a method to physically measure T0.

Estimating Void Volume

To estimate T0, consider this rule of thumb applicable to standard diameter HPLC columns. For columns with a 4.6 millimeter ID, the standard diameter, estimates suggest that every 10 cm of column length contains 1 mL of void volume. Despite potential calculations involving cylinder volume, remember the column includes both the mobile and stationary phases, with the latter making up roughly 70% of the column’s interior. This estimation suggests that every 10 centimeters of column contains approximately one milliliter of void.

Practical Application and Importance

Consider a practical scenario using a 5-centimeter column with a 4.6 millimeter ID. The void volume for this column is half a milliliter. Understanding the void volume is crucial for effectively re-equilibrating the column, necessitating five times the void volume for proper re-equilibration. For a column of this size, that equates to 2.5 milliliters. If running at a flow rate of 1 mL per minute, re-equilibration takes two and a half minutes.

Efficiency of Short Columns

Let’s expand on this concept. Doubling the flow rate to 2 mLs per minute for the same column halves the re-equilibration time to one and a quarter minutes. This efficiency underscores the advantage of shorter columns.

Calculating void volume For Larger Columns

Taking a larger column, say a 25 cm column with a 4.6 millimeter ID, the void volume increases to 2.5 milliliters. Re-equilibrating this column requires 12.5 milliliters of solvent, which translates to 12.5 minutes at a flow rate of 1 mL per minute. This example highlights the importance of understanding void volume. It also underscores the trend towards smaller particles and shorter columns for faster analysis and quicker re-equilibration.

Special Considerations for LC/MS Specifications

For columns with a 2.1 millimeter ID, often used in LC/MS specifications, the calculations adjust proportionally. A 10 cm column with this diameter has a void volume of 0.2 milliliters, necessitating a different approach to flow rate and re-equilibration calculations.


In conclusion, understanding the principles of T0 measurement and void volume estimation are foundational in HPLC. These concepts not only facilitate precise analyses but also contribute to the selection of column sizes and flow rates. This will optimize chromatographic performance and efficiency.

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