As predicted from the previous page, this Retention Map for HPS™ C18 columns show exponential increases in retention times as the % organic composition of the mobile phase is reduced (% aqueous increases) while the pH of the eluent is changed to basic. This produces little to no useful resolution of analytes at near neutral and in acidic pH. This is typical ARP and can be utilized to aid your method development of organic bases on typical HPLC reverse phase materials.

But now look at the Map for UDC Cholesterol™ Column.

At all pH’s these target basic analytes elute very differently (an atypical ARP manner) compared to HPS™ C18. Below a critical acetonitrile % the retention is ARP whilst above it is ANP.

How does this happen?

This behavior can be explained by a transition of the stationary phase from a state of being less polar than the mobile phase, to a state where it is more polar that the mobile phase at a particular acetonitrile percent composition of the mobile phase.

Acetonitrile Is A Key Factor.

This transition in ARP to ANP does not usually occur with this and other Cogent TYPE-C™ phases if MeOH is the organic solvent as this is a more polar solvent than acetonitrile, thus the stationary phase remains the least polar from zero to 100% methanol.

One of the major differences between conventional type-B, polar HPLC columns and TYPE-C™ HPLC columns are these atypical reverse phase retention profiles.

The negligibly low silanol activity of TYPE-C™ phases often negates the problems associated with polar type B columns, thus allowing unique and previously difficult separations to be accomplished isocraticaly. The problems discussed on this and previous pages of the type B columns apply to TYPE-C™ as well if the chromatography is performed at a non-optimal pH.

For example; at pH 2.18 and 6.80 the three basic drugs (tricyclic antidepressants) shown are not well resolved. Also the width and symmetry are poor. But at pH 8.00 sharp, symmetric peaks are achieved with the potential for baseline resolution. Examination of the pH results also reveals that UDC Cholesterol™ does not exhibit stablility hysteresis when changing pH.

Orthogonal Analysis

Thus orthogonal confirmatory analysis is possible on the same column, plus the option exists to accomplish resolution by ARP with high % aqueous or ANP with low % aqueous ( better suited to prep applications ) by merely changing the pH of the eluent.