High Resolution of Complex Lipids (Triglycerides) Using the Agilent 1290 Infinity LC and ZORBAX RRHT and RRHD 1.8 µm Columns

Michael Woodman, Agilent Technologies, Inc.

The typical HPLC resolution is shown by a separation of complex triglycerides in vegetable oil. Using a 24-min gradient and a 3.0 mm × 150 mm, 1.8 μm column, the analysis time of 35 min is typical; however, resolution is insufficient for good compositional investigation of the mixture. The separation of the main components is shown in Figure 1.

Figure 1

The high resolution of the system is further demonstrated by separation on a much longer column, using more of the power range of the system. At 0.29 mL/min, incorporating a shallow gradient condition and an RRHD, 2.1 mm × 400 mm, 1.8 μm column, the separation is dramatically improved. The separation of the main components is shown in Figure 2. Subambient column temperature control, a standard feature of the Agilent Thermostatted Column Compartment, has significant advantages for many difficult isomer separations, including enantiomeric separations, and for shape-selective separations such as polycyclic aromatic hydrocarbons.

Figure 2

Configuration

• G4220A 1290 Infinity Binary Pump with Integrated Vacuum Degasser

• G4226A 1290 Infinity Autosampler

• G1316C 1290 Infinity Thermostatted Column Compartment

• G4212A 1290 Infinity Diode Array Detector

Conclusions

The high resolution and pressure capability of the system allows one to use high efficiency 2.1 mm id columns, generating approximately 97,000 theoretical plates and having approximately 400% lower solvent consumption compared to 4.6 mm id columns. With nearly three times higher efficiency, run time was increased by only about 80%. The low flow rate and high resolution facilitate the interfacing of the separation to high resolution TOF and QTOF mass spectrometers to produce high confidence peak identification and compositional information.

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