Separation through HPLC:

In sequence to achieve the desired separation through HPLC, various operating conditions involving retention time, pressure and number of plates require to be optimized. A main interest is short analysis time, or the plate count required to accomplish a difficult separation. First of all, a proper HPLC system such as adsorption, bonded-phase, reverse phase, ion-exchange, exclusion, affinity or any other form of chromatography must be selected. Then, all the parameters in the equations as mentioned in Unit 4 that depend on the properties of the mobile and stationary phases are determined. As already described in Unit 4, these are relative retention (α), capacity factor (k') and the plate count (N). It is desired that the compounds of interest should need at least ten times longer time to travel the column length than the unretained peak. Further, the viscosity of mobile phase and the diffusion coefficients of the solutes in the mobile phase are also of concern besides other characteristics of column packing.

The plate height (H = L/N where, L is the column length and N the number of plates) is reduced by the particle diameter (d_p) and may be represented as

h = H/d_p = L/N.d_p

It actually states the number of particle diameter (d_p) that constitutes one plate height.

Therefore, reduced velocity might be represented as

v = u.d_p/ D_M

where, D_M is the diffusion coefficient of solute in the mobile phase. It might be considered as the ratio of the time needed to displace solute molecules a distance equal to one particle diameter to the time needed for the similar displacement through molecular diffusion. It expresses the balance among mass transport through diffusion or molecular motion across a single particle. Substituting the value of u (= L/t_m), reduced velocity may be expressed as

v = L.d_p/t_m.D_M

Thus, the complete equation for the dependence of the reduced plate height may be represented as modified van Deemter equation

h =   B/v  +  A.v^0..33  +  C.v

where, B = 1.2 for solid core (pellicular) packing and 2.0 for completely porous column packing. Also, A = 0 for well packed column and C = 0.05 for porous particles decreasing to 0.003 for pellicular particles. There is no theory accurately elaborates the dispersion from flow in homogeneity in the mobile phase. A logarithmic plot of Eq. (8.4) is shown in Figure. The reduced plate height has a minimum value in the range 2-3 for intermediate region of velocities where reduced velocity is 3 -5.