## 3 Results
### Optimal Conditions for HPLC
#### 3.1.3 Influence of the Stationary Phase
At basic pH of sodium bicarbonate the effect of free silanol groups, which could cause unwanted additional interaction is weaker, because the basic analytes became uncharged and contribute lower to electrostatic interactions [33]. Figure 3: Comparison of peaks widths (**A**), plate numbers/m (**B**) and tailing factors (**C**) for OATD-02 and related substances on different columns. Retention in reversed-phase liquid chromatography (RP-LC) can be described by the analyte-stationary phase interactions: hydrophobicity, steric resistance, hydrogen bonding of basic or acidic analytes to, respectively, acidic or basic sorbent sites, and ion-interaction. The mechanism of retention in the reversed phase is primarily hydrophobic interactions. If hydrophobicity could be the only stationary phase property which affected selectivity, a plot of log k for one column vs. another column would give a straight line, with all the points on the line [34]. For further comparison of the retentivity of two C18 columns, the data of log k for the Waters XSelect CSH-C18 column with ion-exchange properties plotted vs. log k for the Zorbax Extend-C18 column were illustrated in Figure 5. The slope of this plot depends on the hydrophobicity of the CSH-C18 column compared to the Zorbax Extend-C18 column. There are deviations from this plot, which reflect differences in column selectivity for the four described compounds. These dependencies support that retention is influenced not only by hydrophobicity, but especially for charged analytes. Figure 4: Plots of the logarithms of the relative retention \(\alpha\) for (**A**) OATD-02/ OAT-9014 and (**B**) OAT-9016/ OATD-02 vs. the logarithms of the retention factors of (**A**) OATD-02 and (**B**) OAT-9016 for different stationary phases. The Waters XSelect CSH-C18 column (2.5 \(\upmu\)m, 100 mm \(\times\) 3 mm) was chosen as the optimal column for the best peak shapes (tailing factor < 1.5), retentions and column efficiency for the OATD-02 and its impurities. The OATD-02 sample solution was injected at different mobile phases and different HPLC conditions using different stationary phases, flow rates, and mobile phases. The best obtained HPLC chromatographic condition was summarized in Table 3. \begin{table}
\begin{tabular}{l l} \hline
**Parameters** & **Results** \\ \hline Column & Waters XSelect CSH C18 2.5 \(\upmu\)m, 100 mm \(\times\) 3 mm \\ \hline Mobile phase A & 10 mM NaHCO\({}_{3}\) (840 mg/L) in H\({}_{2}\)O \\ \hline Mobile phase B & 10 mM NaHCO\({}_{3}\) (840 mg/L) in H\({}_{2}\)O:ACN (20:80) \\ \hline Flow rate & 0.5 mL/min \\ \hline Mobile phase C & 350 \(\upmu\)M alizarin in MeOH \\ \hline Post-column reagent flow rate C & 0.5 mL/min \\ \hline Column temperature & 30 \({}^{\circ}\)C \\ \hline Detection & Fluorescence, \(\lambda_{\text{ex}}\)= 470 nm, \(\lambda_{\text{em}}\)= 580 nm \\ \hline Injection volume & 5 \(\upmu\)L \\ \hline Sample solvent & water \\ \hline Sample concentration & 1 mg/mL \\ \hline Run time & 23 min \\ \hline \multirow{7}{*}{Gradient} & Time & Conc. \%B \\ \cline{2-3}  & 0 min & 0 \%B \\ \cline{2-3}  & 2-4 min & 6 \%B \\ \cline{2-3}  & 5–9 min & 10 \%B \\ \cline{2-3}  & 18 min & 100 \%B \\ \cline{2-3}  & 18.1–23 min & 0 \%B \\ \hline \end{tabular}
\end{table} 
Table 3: The best chromatographic conditions for OATD-02 separation. Figure 5: Retentivity comparison of two C18 columns; log k for especially Waters XSelect CSH-C18 column plotted vs. log k for the Zorbax Extend-C18 column.