In the realm of high-performance liquid chromatography (HPLC), the term 'void volume' is notably significant. Understanding and managing void volume is essential for achieving effective chromatographic separations. It directly impacts the retention time and elution behavior of sample components.
This article delves into void volume, paving the way for an enhanced understanding of the role of this fundamental element in achieving precise and dependable analytical outcomes.
Definition Of Void Volume
The term ‘void volume’ is often used interchangeably with 'dead volume,' and denotes the volume of liquid (mobile phase) encased within the HPLC column. This volume is approximately "the minimum amount of solvent which must be pumped through the system before any injected sample can get to the detector," explains Tom Jupille, founder and moderator of Chromatography Forum. Jupille notes that despite its pivotal role, void volume does not directly impinge on the resolution in chromatographic procedures, meaning it does not factor into the calculation of resolution.
He adds that sometimes, 'void volume' can also represent a cavity located at the head of the column or the unswept volume in tubing or fittings. “Used in this context, void volume will decrease resolution (by providing a place for the peaks to broaden),” says Jupille. “It is not, however, incorporated directly into the calculation.”
Calculating Void Volume
While exact computation of void volume is elusive, approximation is attainable, especially if the percentage of pore volume in your packing is known. According to Jupille, with many common 'reversed phase' packings, void volume is about 35%. “It is more commonly measured, either approximately, by noting the time delay for the injection disturbance to perturb the baseline, or exactly, by injecting a non-retained but detectable solute,” he adds.
Risks Of Overlooking Void Volume
When void volume is disregarded or inaccurately estimated, it can lead to several issues within the chromatographic process. Not accounting for this critical factor may result in compromised separations, impacting the accuracy of peak identification and quantification. Furthermore, overlooking void volume can skew retention times, leading to misinterpretation of sample components and potentially causing erroneous conclusions in the analysis.