What is Void Volume in Chromatography?

1. What is Void Volume?

The void volume (column void volume or dead volume) is the volume that the mobile phase occupies in a column. For instance, if 80% of the entire column’s capacity is filled up with stationary phase material, the void volume would be 20% of the total column volume.

2. How to Calculate Void Volume?

For columns with fully porous packings, use the formula  pi * (r)² * L * 0.66.

For columns with superficially porous packings, use the formula  pi * (r)² * L * 0.49.

Where:

L – column length in mm

L- Column length in mm

r – column internal radius in mm

0.66 – Pore volume (for fully porous packing)

0.49 – Pore volume (for superficially porous packing)

3. How to Expermentally Determine Void Volume?

The formulas shown above merely reflect an estimate of the void volume of a packed column (interstitial volume plus the pore volume).

To understand what the actual column void volume is for a particular column installed on a particular system, you must make an injection with a compound that does not retain on the packing material. An example of this is Uracil for reversed phase HPLC columns. Once a void marker peak is injected, you can use the following formula to determine column void volume:

Retention time of void peak (V_o) * Flow Rate

Example: If an unretained peak elutes at 2.32 min, and the flow rate is 0.85 mL/min, then:

V_o = 2.32 min  * 0.85 mL/min

V_o = 1.972 mL

4. Importance

Void volume is encountered in different types of chromatography techniques such as High Performance Liquid Chromatography (HPLC), gel filtration chromatography (SFC), or size exclusion chromatography (SEC) etc. Incorrectly estimated void volume can lead to several issues within the chromatographic process. This could result in suboptimal separations which can impact the accuracy of peak identification and quantification. Moreover, overlooking void volume can skew retention times, leading to misinterpretation of sample components and potentially causing erroneous conclusions in the analysis. Accurate determination of void volume is essential for various reasons:

1. Calculating retention factors: Void volume is used to calculate retention factors (k’), which are crucial for characterizing analyte retention and optimizing separations. 
2. Optimizing flow rates: Knowing the void volume helps in selecting appropriate flow rates for efficient separations. 
3. Understanding peak broadening: Void volume can contribute to peak broadening if not accounted for, impacting resolution. 

5. References

1. C. A Rimmer. Journal of Chromatography A, Volume 965, Issues 1–2, Pages 219-232