HPLC Column Void Volume : Pharmaceutical Guidelines

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HPLC Column Void Volume

Void volume in High Performance Liquid Chromatography system analysis plays an important role in accuracy of the analysis. void volume affects the resolution of the HPLC system
High-Performance Liquid Chromatography system is a test used mainly in analytical chemistry for identification, quantitative and control of related substances in chemical mixtures. The technique depends mainly on the uniqueness of the molecular weights and the polarities of elements. Compounded mixtures are passed through the HPLC column and depending on the unique features stated above; they are separated before they can leave the HPLC column.

The HPLC set up is composed of two main parts, a mobile phase and a stationary phase. The stationary phase is a solid packed in a column, usually consisting of a silica gel while the mobile phase is a liquid solvent mixture. The mobile phase is pumped up the column where separation takes place followed by detection.

HPLC Void VolumeThere are several volumes in the column such as the column volume, bed volume, elution volume, retention volume and void volume to name a few.

The HPLC column void volume denoted Vm oV0 is in simple terms the volume of the mobile phase in the column. It is the part of a fraction that when added to the volume of the stationary phase makes up a whole fraction or 100% volume.

The HPLC column void volume is considered in the presence of the stationary phase particles, meaning that if there is no stationary phase, then the void volume cannot be calculated. The HPLC column void volume is also roughly the minimum amount of the solvent that has to be pumped through the column before any sample that is introduced by injection can be detected.
Related: Steps for HPLC Method Development

Determination of the HPLC column void volume is mainly approximate. If unknown, the volume is determined by the injection of an unretained standard that usually has minimal retention in any given phase. This means that its role is almost inert relative to the components of the mobile phase. A product of this unretained standard and the flow rate of the system gives the column void volume of that particular system. It should be noted that the packed volume of the column and the flow rate can also be utilized to the same effect in the calculation of the volume.

To determine the various volumes in High-Performance Liquid Chromatography system, the dimensions of the capillary column that holds the mobile phase are mostly used, i.e., its length and radius.
The general formula for determination of the HPLC column void volume is given by:
V= F x to
Where;
Vm is the void volume
F is the flow rate in Millilitres per minute
to is the column dead time
Alternatively, the dimensions of the column can be used as well as the percentage of the void volume as a factor of the total volume. Using this method, the formula becomes;
V= y x π x (i.d/2)2 x L 
where;
Vm is the void volume
y is the fraction of the void volume as a function of the total volume usually about 70%
i.d is the internal diameter of the column in centimeters
L is the length of the column in centimeters.

HPLC column void volume and the flow rate are useful in the calculation of a value known as the column void time, otherwise known as to. Knowing this value is helpful in the determination of other critical chromatographic values such as the resolution and the separation factor of a compound.





Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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