HPLC Retention of Polar Compounds and Separation of Polar Compounds
The reverse-phase mode of HPLC is the technique of choice to solve many separation problems. One of the limitations of reverse-phase columns is the lack of retention of highly polar compounds on conventional stationary phases. Traditionally, the ion-pairing reagents have to be employed to achieve the separation of these compounds. In its turn, the use of ion-paring reagents also has its limitations, i.e. artifacts when using gradient elution, incompatibility with Mass Spectrometry (MS), Evaporating Light Scattering Detection (ELSD), preparative chromatography, and more complex mobile phase preparations.
None of these limitations exist for Primesep® mixed-mode stationary phases, which are suitable for the separation of polar and non-polar compounds at both analytical and preparative scales in isocratic and gradient modes. These stationary phases allow for a great degree of flexibility in the separation of a broad range of analytes on one stationary phase platform. They use simple mobile phases that are compatible with multiple detection modes. Retention comes from two available interactions – reverse phase (due to hydrophobic interaction) and ion-exchange (due to electrostatic interaction).
If compounds are converted in a non-ionized form, it is typically less hydrophilic than the charged one. Charged functionalities act as an extreme case of polarity. . Therefore, charged analytes are un-retained by hydrophobic interactions alone. Ionizable compounds are much more hydrophobic in their neutral state than their ionized form and can often be retained by reversed-phase in this state. This concept is frequently employed to retain carboxylic acids by reversed-phase under acidic conditions (pH below 3) where they are neutral. However, for bases, the mobile phase pH must be above 11 for the compounds to be neutral and therefore hydrophobic enough for retention under reversed-phase conditions. Operation under these conditions required special phases with hybrid particles that are stable at a high pH.
Retention profiles of the different ionizable and neutral compounds at different pH (using only reverse phase mechanism) show no common pH where all the different molecules can be retained. Because of that, it is always difficult to analyze samples containing acidic, basic, and zwitterionic compounds in a single HPLC analysis.
In contrast, Primesep mixed mode columns with cation exchange properties provide retention for all classes of charged and neutral compounds in acidic/neutral pH region. Retention profile vs. pH is different for different molecules. In order to produce efficient separations for polar compounds containing ionizable groups, a significant concentration of a buffer (1-50 mM) should be present in the mobile phase. The buffer serves a dual role – it controls the pH of the separation system and it provides competitive ions for ion-exchange mechanisms.
Obelisc R column
Obelisc R has reversed-phase character and can be used in traditional, reversed-phase type applications. Due to the presence of ionic groups and a long hydrophobic chain, Obelisc R offers additional retention and tuning that is not available with traditional reversed-phase columns. Typical mobile phases used with Obelisc columns are based on acetonitrile, water, and the mass spec compatible buffers ammonium formate (pH 3) and ammonium acetate (pH 5). If it is necessary to detect in low UV (<220 nm) then phosphate buffer is recommended.