The Application Notebook
Volume 0, Issue 0
Grapefruit juice was analyzed by gradient UHPLC for naringin - a flavanoid that gives it its bitter taste. A ZORBAX Rapid Resolution HD (RRHD) StableBond-C18 2.1 x 150 mm, 1.8 µm column resolved over 60 peaks including naringin in 7 min, and was re-equilibrated in just 3 min by operating at a high flow rate (0.8 mL/min).
John W. Henderson Jr., Judy Berry, and Maureen Joseph, Agilent Technologies, Inc.
Grapefruit juice was analyzed by gradient UHPLC for naringin — a flavanoid that gives it its bitter taste. A ZORBAX Rapid Resolution HD (RRHD) StableBond-C18 2.1 x 150 mm, 1.8 μm column resolved over 60 peaks including naringin in 7 min, and was re-equilibrated in just 3 min by operating at a high flow rate (0.8 mL/min). The high pressure (965 bar) is well within the 1200 bar operating range of the RRHD column and 1290 Infinity LC.
Gradient reversed phase HPLC with long (150 mm) 1.8 μm columns are an option for analyzing complex matrices with numerous analytes, such as grapefruit juice. Sufficiently flushing the column with strong solvent and re-equilibrating with weaker starting conditions is necessary but can be time consuming. Rapid Resolution High Definition (RRHD) 1.8 μm, columns withstand pressures up to 1200 bar, so doubling or quadrupling the flow rate is reasonable, and proportionally reduces the total analysis time including re-equilibration.
See figures for method parameters. A grapefruit was hand squeezed and its juice filtered through a 0.22 μm RC syringe filter directly into an autosampler vial for analysis.
Figure 1 shows the analysis of filtered grapefruit juice using a mobile phase gradient with an initial isocratic hold, a linear ramp in organic solvent to elute peaks of interest, a high organic flush, and a re-equilibration to starting conditions. The 2.1 × 150 mm, 1.8 μm column offers high efficiency even at a high 0.8 mL/min flow rate and a system pressure range of 480 to 965 bar, well within the 1200 bar limit of the RRHD column and Agilent 1290 Infinity LC. More than 60 peaks in grapefruit juice were resolved in 7 min, including the main peak of interest, naringin. The amount of naringin was 0.12 mg/mL juice.
Figure 2 superimposes chromatograms of six injections. Retention time RSD for naringin is 0.03% and RSD for peak area is 0.83, indicating that 3 min is sufficient for flushing with strong solvent and re-equilibration to initial gradient conditions. The benefit of an RRHD 2.1 × 150 mm column UHPLC is clearly demonstrated: a re-equilibration time of just 3 min versus 10 min — the time needed to flush and re-equilibrate (5 column volumes each) a 2.1 × 150 mm column with 3.5 or 5 μm particles operated at 0.2 mL/min.
The high efficiency and resolving power of sub 2 μm RRHD columns and the ability to operate at high flow rates under UHPLC conditions, make these columns a valuable tool for rapid gradient analysis of complex samples.
Agilent Technologies, Inc.
2850 Centerville Road, Wilmington, DE 19808
tel. (800)227-9770; fax (302)633-8901