This application note uses Agilent ZORBAX RRHD columns with UHPLC/MS to compare Morphine’s HILIC and RPLC. Both methods use acetonitrile/ammonium acetate buffer mobile phase for isocratic elution. The results show that HILIC mode allows for more sensitive MS detection due to more efficient spraying and desolvation in the ESI-MS source, as a result of the volatile acetonitrile rich mobile phase used.
This document compares the performance of Hydrophilic Interaction Chromatography (HILIC) and Reversed-Phase Liquid Chromatography (RPLC) for the analysis of morphine and three of its metabolites. The study utilizes Agilent ZORBAX RRHD columns, specifically the HILIC Plus and Eclipse Plus C18, coupled with an Agilent 1290 Infinity LC System and an Agilent 6410A Triple Quadrupole Mass Spectrometer.
The analysis demonstrates that HILIC mode provides more sensitive mass spectrometer detection compared to RPLC for these polar analytes. This improvement is attributed to the use of a highly organic mobile phase in HILIC, which facilitates more efficient spraying and desolvation in the ESI-MS source, resulting in a more intense signal and reduced baseline noise.
| Column 1 | Agilent ZORBAX RRHD HILIC Plus, 2.1 x 100 mm, 1.8 ym |
|---|---|
| Column 2 | Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 x 100 mm, 1.8 pm |
| Column 3 | Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 3.5 jim |
| Column 4 | Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 5 pm |
| Temperature | 25°C |
| Flow rate | 0.4 or 1.0 mL/min |
| Injection volume | 2 pL |
HILIC allows for more sensitive MS detection due to more efficient spraying and desolvation in the ESI-MS source caused by the volatile acetonitrile-rich mobile phase.
The highly efficient sub-2 ym UHPLC column improves MS sensitivity by a factor of 5 compared to a traditional 5 ym column.