Organic modifier effect chromatographic

In almost all of the transformations described above it has been assumed that both vinyl groups of protoporphyrin-IX (19) have been affected. Obviously, by judicious control of stoichiometry it is possible in many cases to effect only partial transformations of protoporphyrin-IX in which only one vinyl group has been modified. Under these circumstances a mixture of the 3-vinyl and 8-vinyl derivatives results, and in a number of situations these isomers can be isolated, separated and characterized. In order to purify almost all of the compounds shown in Scheme 8, it is usually necessary to esterify the propionic acid groups on the 13- and 17-positions so that the products can be effectively chromatographed, usually on alumina or silica. Indeed, in many instances the reactions are carried out on the dimethyl ester derivatives, and this enables normal organic solvents to be employed. 

Because different forms of analyte usually show different affinity to the stationary phase, secondary equilibria in HPLC column (ionization, solvation, etc.) can have a significant effect on the analyte retention and the peak symmetry. HPLC is a dynamic process, and the kinetics of the secondary equilibria may have an impact on apparent peak efficiency if its kinetics is comparable with the speed of the chromatographic analyte distribution process (kinetics of primary equilibria). The effect of pH of the mobile phase can drive the analyte equilibrium to either extreme (neutral or ionized) for a specific analyte. Concentration and the type of organic modifier affect the overall mobile phase pH and also influence the ionization constants of all ionogenic species dissolved in the mobile phase. 

Note that these equations assume that to is independent of an approximation that is not always valid. The internal porosity may depend on the organic modifier concentration, particularly at low or moderate values of this concentration because the organic solvent may swell the alkyl-bonded layer of RPLC packing materials. It is easy to account for this effect in numerical solutions of chromatographic models [12-14], not in algebraic solutions. 

---