If the dead volume is larger, the space leading to sample diffusion will also expand, which makes it easier to cause widened peak and worse peak shape. So how to define and calculate the dead volume?
Definition of Dead Volume
In a narrow sense, the dead volume refers to the void volume that has not occupied by the stationary phase in the column, that is, the volume of the mobile phase. Dead volume in a broad sense refers to the volume of the section from the injection position (injection inlet) to the detection position (detector flow pool)(PS：excluding the volume of packing materials in the column). Once the column is connected, the volume (shown in the orange line) is constant (dead).
How to Calculate the Dead Volume of HPLC Column?
For HPLC, how to calculate the dead volume?
The dead volume (V0)—— the free space excludes the stationary phase from the injection inlet to the detector flow pool, which consists of four parts: the volume from the injector to the pipe line of column, the particle gap of the stationary phase in the column (occupied by the mobile phase, Vm), the volume of the pipe line at the outlet of the column, and the volume of the flow pool of the detector. Among them, only Vm participate in the equilibrium process, and the other three parts only play a role in peak expansion. In order to prevent peak expansion, the volume of the other three parts should be minimized.
The dead time (t0)—— the retention time of the components failed to retain. That is, the time when the mobile phase (solvent) enters the column.
The dead volume of the measurement system needs to be measured by experiments regularly. The common method is injecting a special solution without interaction of the stationary phase (for example, uracil, a small amount of NaNO3 or methanol dissolved in the mobile phase, and 100% pure methanol) to get a peak. The dead time (t0) can be calculated from this peak , so do the dead time (t0) of the system.
The dead volume (V0)= dead time (t0)× flow rate of mobile phase (F。)
Precautions to Reduce Dead Volume
In the process of use or maintenance, if the replacement of fittings (such as pipes) is involved, the fittings consistent with the original specifications should be selected to ensure that the dead volume matches the original, which reducing the possibility of the increase of the dead volume and the broadening of the peak.
Check whether the pipeline joints match well (pay special attention to the use of peek pipe joints and check the connection between the pipeline and the column) to avoid the formation of joint cavities (as the figure shows).
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