Effect of chain length on performance of reverse phase:

In general, mobile phases with no or little polarity are used with polar bonded phases. For non-polar bonded phases, however, mobile phases are selected from solvents with high polarities. Thus, the mobile phase composition is adjusted to change the overall retention time and/or to pull specific peak pairs apart as illustrated above. Solvent optimization involving four solvents have also been described. Thus, the separation of solutes with different functional groups may be improved by the use of ternary mobile phases for the precise control of mobile phase-eluent strength in conjunction with solvent programming. More sophisticated automated methods of mobile phase optimization are commercially available. Most software packages are designed to use one of the two alternative approaches.

A linear hydrocarbon chain is a popular bonded phase where alkyl group may have a variety of chain length, usually a methyl (C-1), ethyl (C-2), octyl (C-8) or octadecyl (C-18).  The effect of chain length of the alkyl group upon performance of siloxane column in reverse phase chromatography resulting in better resolution is illustrated in Figure. It is observed that poor resolution is observed with methyl group but it becomes better with octyl and still better with octadecyl group. Thus, longer chains

Figure: Effect of chain length on performance of reverse phase siloxane column packed with 5 µm particles. Peaks;1. Uracil, 2. Phenol, 3. Acetophenone, 4. Nitrobenzene, 5. Methyl benzoate, 6. Toluene

produce more retentive packings. On the basis of this observation, it may be concluded that octadecyl packing can be used for application where maximum retention is required. In order to emphasize this point further, a comparison of chromatograms obtained on columns of octadecyl and octyl packings under the same mobile phase conditions of methanol/water (50:50) is presented in Figure. Mixture of sample represents a variety of functional groups. Thus, bonded octyl packings represent a good compromise for the separation of compounds with low to high polarity and samples with wide ranging polarities. It seems that bonded alkyl phases permit rapid analyses and rapid reequilibration when the mobile phase is altered as in solvent programming.