Shodex Column Recommendation for Oligomer and Lipid Class Analysis in Omega-3 Fish Oils

Record: 798

Category: Column Selection
Product: KF-800, ODP-50, VT-50 2D
Sample: Omega-3 Fish Oils

Question
A customer wants to improve their method for analyzing oligomers in fish oil rich in omega-3 fatty acids. They currently follow the AOCS/EP method for oligomers and partial glycerols in fish oils and are using three TSKgel G1000Hxl columns in series, but still have poor resolution between lipid classes.

They would like to separate polymers and lipid classes such as free fatty acids (FFA), triglycerides (TG), diglycerides (DG), monoglycerides (MG), and ethyl esters.
They also mentioned the European Pharmacopeia (version 7.0) method for oligomers in fish oil rich in omega-3 acids as a potential reference.

Can we suggest suitable Shodex columns to improve their method?

Answer
Yes — this type of analysis involves two complementary chromatographic modes:

1. Size Exclusion Chromatography (SEC) for oligomers and high–molecular-weight fractions, and
2. Reversed Phase Chromatography (RP) for separation of lipid classes.

For the oligomer analysis, the closest Shodex equivalent to TSKgel G1000Hxl is the KF-800 series.
We recommend using a pore-graded combination such as KF-802.5 + KF-802 + KF-801, which follows the USP-NF / EP method for omega-3 fatty acid oligomers.

This configuration broadens the separation range and provides better resolution between polymerized components, partial glycerides, and triglycerides compared to using three identical pore sizes in series.

Analysis of Oligomers in Omega-3 Free Fatty Acids According to USP-NF – KF-802.5 + KF-802 + KF-801

For the lipid-class separation, we recommend the RSpak ODP-50 column.
This polymer-based reversed-phase column provides stable retention for triglycerides, diglycerides, monoglycerides, and free fatty acids using organic mobile phases such as acetonitrile/isopropanol or methanol/MTBE.
It is highly reproducible and suitable for both UV and ELSD detection.

Analysis of Unsaturated Fatty Acids in Supplements – ODP-50 4D
Unsaturated Fatty Acids – ODP-50 4D

In summary:

• Use KF-802.5 + KF-802 + KF-801 for the SEC step (oligomer and polymer profiling).
• Use ODP-50 for the RP step (class separation of FFA, MG, DG, TG, ethyl esters).
  This dual approach aligns with the EP/USP workflow and provides higher resolution than the current setup.

Additional Comments

If the customer wants to compare separation selectivity or evaluate LC–MS compatibility, additional studies using C18U-2D (RP) and VT-50 2D (HILIC) for fatty acids are available:
Comparisons of Two Separation Modes on Saturated Fatty Acid LC–MS Analysis – C18U-2D vs VT-50 2D

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