Surface chemistry of the adsorbent is the major factor affecting RP HPLC retention. If the surface of silica is densely covered by bonded ligands, then we will have only dispersive interactions, and this type of packing material will show "pure" reversed-phase retention dependencies. This is especially true for polar or ionizible compounds.
Changes of the retention of dodecanoic acid with eluent composition on the column packed with the most densly bonded adsorbent (Prodigy-ODS2, 150x4.6 mm). Brown - 100% MeCN, blue - 99/1 MeCN/Water, green - 95/5 MeCN/Water, red - 90/10 MeCN/Water. LC/MS detection of molecular ion at m/z=200
Retention behavior of dodecanoic acid is similar to what we had seen for alkylbenzenes. Decreasing of the acetonitrile content in the eluent is decreasing the competition of the eluent with the analyte for the place on the adsorbent surface. The less the amount of acetonitrile, the higher the retention.
Dodecanoic acid is a polar and ionizible compound, but its retention dependence does not show any specific (polar or ionic) interactions with silica surface covered with C18 ligands. Coverage for this adsorbent (Prodigy-ODS2) is 3.3 Ámole/m2 or two C18 groups per 100┼2. Note, that all peaks are symmetrical which is also an indication of only dispersive interactions in the column.
Chromatograms in the next picture show the retention behavior of the same solution of dodecanoic acid on Hypersil-C1 reversed-phase column (150x4.6 mm).
Changes of the retention of dodecanoic acid with eluent composition on the column packed with Hypersil-C1. Brown - 100% MeCN, blue - 99/1 MeCN/Water, red - 95/5 MeCN/Water. LC/MS detection of molecular ion at m/z=183+200+201
Bonding density of trimethylsilane ligands on silica surface for Hypersil-C1 is 4.9 Ámole/m2 or 2.9 groups per 100 ┼2 which is higher than on Prodigy-ODS, but ligands are 18 times shorter. As we know from the silica surface chemistry, the original fully hydroxilated surface of silica gel has from 5 to 8 -OH groups per 100┼2. Thus, only half of available -OH groups react with trimethylclorosilane. The other half are still available and accessible for analyte molecules due to the small size of bonded organic moiety.
In the picture above dodecanoic acid is very strongly retained from pure acetonitrile; it did not show up in almost 25 min. Retention decreases with an increase in the water content in the eluent. This behavior is typical for the normal phase retention. So, we can say that for the very low water content the polar interactions with the surface prevail on this adsorbent.
Peak shape also noticeably changes with changing eluent composition. At 1% water in the eluent, the peak of dodecanoic acid has long tail, but 5% of water, the peak is almost symmetrical. 5% water appears to be enough to suppress the polar interactions of dodecanoic acid with residual silanols.
Peak tailing at 1% o water content has a more complicated nature. In the "Theory" chapter, we discussed the basic retention equation.
According to that retention is proportional to the surface area and to the derivative of
the component adsorption isotherm. If the adsorption isotherm is convex, the peak will
have a tail.