Chromatography Forum

I am wondering if anyone has any idea how we can calculate the volume of stationary phase in a C18 column with a particle size of 5um. Considering the point that carbon loading in a C18 column is from 10-20% on the silica particle. Please don't mix it up with the volume of particles in the column.

Volume balance on the column is
Vc=Vm + Vs + Vsi
Vc is the total column volume
Vm is the total volume of mobile phase
Vs is the total volume of stationary phase in the column
Vsi is the total volume of support (in this case silica in the column.

Vc = Pie r2l

But how can that help to get a baseline separation?

First of all, the particle size is essentially irrelevant; most of the surface area on silica is *inside* the pores.

What you really need to know is the surface area (which is typically given in m^2/gram), which is typically inversely related to the pore size, and the surface coverage (micromoles/m^2).

I'm with Tom and Gerhard here--that is, I agree with them. Pretty much you can use this equation to estimate the empty volume in a column which holds porous silica particles. The size of the particles and the volume of the ligands, if present, may be ignored for the sake of ease. Empty Volume of Column filled with Silica particles = (Column radius, mm)^2 x pi x column length, mm x 0.68. The numerical value, 0.68, represents the empty space within the porous silica. Please note that this is An Estimate Only.

I think that pycnometry using mercury metal is occasionally used to obtain a more accurate estimate of the empty volume within a HPLC column filled with silica-based particles.

Here is an idea how to calculate stationary phase volume:

k = K*β

k is the retention factor, K is the equilibration constant, and β is the phase ratio. An experiment can be designed (e.g. isocratic RP HPLC) so that k can be determined. K can be calculated by using Gibbs free energy delta Go = - R T ln K or estimated using logP. Once you have the phase ratio, everything else would be easy.