Both Obelisc columns are manufactured by us (SIELC Technologies), so I will only cover technical aspects of the columns. Both columns are mixed-mode columns: Obelisc N is HILIC/ion-exchnage column and Obelisc R is reversed-phase/ion-exchange column
1. Obelisc N is HILIC/normal phase column which works the same way as other HILIC column but has a better retention control and longer retentionfor polar analytes. The column has two ionizable groups attached to the surface of silica gel. Both groups are on the same molecule and they are separated by a long hydrophilic linker. The presence of ionizable groups helps you change polarity of the phase (by changing ionization state of these groups) and increase or decrease ionic interactions with you ionizable analytes. Pure hydrophilic analytes with no ionizable groups will retain based on HILIC/normal phase mechanism. Hydrophilic Ionizable compounds (basic and acidic) will be retain based on HILIC/normal phase and ion-exchange mechanisms. The option of having ionic interaction helps you achieve a new level of selectivity and also, in some cases, reduce amount of CAN in the mobile phase from 90% to 70-80% with out losing retention. Reduced amount of ACN will help solubility of hydrophilic compounds in HILIC/NP mobile phases. You will need to play with the amount of ACN, buffer concentration and buffer pH to achieve the desired selectivity. It sounds complex but in real life it is very easy, you just need to figure out how buffer and buffer pH changes ionization state of compounds and how it affects ion-exchange portion of interaction on the column. The good starting point for your particular compounds would be ACN/water=75/25 with 10 mmol of ammonium formate pH-3 (or 0.1% of formic/acetic acid). In case of Obelisc N column you don’t need to have 50-100 mmol buffer to get long retention. If your retention is too short, you can gradually increase ACN concentration (5% increments) or play with pH (ammonium acetate vs. ammonium formate).
2. Obelisc R is a reverse phase column with both basic and acidic groups on the surface. Both groups are on the same molecule and are separated by hydrophobic linker. Hydrophobic ionizable compounds will retain based on reverse phase and ion-exchange mechanisms (both cation-exchange and anion-exchange). Hydrophilic basic and acidic compounds will retain based on cation-exchange and anion-exchange mechanisms. You can change acetonitrile concentration as well as buffer concentration and buffer pH. Buffer pH will change ionization state of stationary phase to the great extend. For example, in case of basic compounds going from pH 3 to pH 5 will increase retention time 5-20 folds. Retention time for all compounds can be adjusted independently. Selectivity will also change with the choice of buffer. Good starting point for method development ic 50% CAN with 20 mmol ammonium formate pH 3. Based on the retention at these conditions you might need to change acetonitrile content, buffer concentration or buffer pH
Before you start working with a column, I would suggest to inject compounds without the column and measure peak areas for all analytes. This peak area should be the same (5% error is okay) when compounds are analyzed with the column.
We are always ready to help you with your method development. Please provide me with your email and I will send you charts and graphs for method development on Obelisc columns or other mixed mode columns produced by SIELC)
Regards,
Vlad
