Characterization of reversed-phase liquid chromatographic columns containing positively charged functionality

To date, the most commonly used column characterization databases do not determine the relative positive charge associated with new generation RP columns, or they fail to successfully discriminate between RP columns of purportedly low level positive and neutral characters. This paper rectifies this in that it describes a convenient and robust chromatographic procedure for the assessment of the low levels of positive charge on a range of RP columns. The low degree of positive charge was determined by their electrostatic attraction towards the negatively charged 4-n-octylbenzene sulfonic acid (4-OBSA) relative to their retention of the hydrophobic marker toluene (Tol).

The new parameter (_α_4-OBSA/Tol) was determined for 15 commercially available RP-LC columns. When this was combined with existing Tanaka parameters it was possible to guide the chromatographer towards similar columns as “backup / equivalent phases” or dissimilar columns for exploitation in method development strategies. It should be noted that under certain chromatographic conditions the retention mechanism(s) may be too complex to allow direct location of a “backup / equivalent” column(s).

The α_4-OBSA/Tol results indicate that even the new generation neutral alkyl phases may exhibit a small degree of positive charge at low buffer concentrations. Mobile phases containing low % MeCN were demonstrated to promote mixed mode (anionic exchange / hydrophobic) retention whereas at high % MeCN anionic exchange retention dominated.

The measure of electrostatic repulsion between positively charged columns and positively charged bases was assessed by evaluating the relative retention of a range of bases and neutral analytes. The greatest electrostatic repulsion was observed with hydrophilic bases. While there was no correlation between the positive charge associated with the phases assessed by electrostatic attraction or repulsion, the columns could be broadly divided into three subsets (i.e., significant positive character, medium to low positive character and insignificant positive character).

Finally, the results were used to highlight the usefulness of the column characterization database containing the new anionic exchange retention parameter (_α_4-OBSA/Tol) for the selection of an equivalent column possessing a low level of positive character in the analysis of a real-life biopharmaceutical application.