Analysis of Fat Soluble Vitamins Using a Thermo Scientific Accucore XL C18 4 μm HPLC Column

Significance of the topic

Accurate measurement of fat soluble vitamins in foods and supplements is essential for nutritional quality control and regulatory compliance. These vitamins occur at trace levels amid complex matrices and are sensitive to light, oxygen, and temperature. High-performance liquid chromatography (HPLC) at ambient temperature provides reliable separation and detection while preserving compound stability.

Objectives and study overview

This study evaluates the performance of the Thermo Scientific Accucore XL C18 4 µm HPLC column for simultaneous analysis of six fat soluble vitamins. Key goals include comparing resolution, efficiency, and run time against a conventional 5 µm fully porous C18 column under identical chromatographic conditions.

Methods and instrumentation

Standards of vitamins K1, K2, E, E acetate (500 µg/mL each), D2 (1500 µg/mL), and D3 (25 µg/mL) were prepared in 80:20 acetonitrile:methanol. Separation used isocratic elution at 80:20 (v/v) acetonitrile:methanol, 30 °C column temperature, 1 mL/min flow, 5 µL injections, and UV detection at 280 nm. A later run at 1.5 mL/min assessed throughput gains.

Instrumental setup

• HPLC system: Thermo Scientific Dionex UltiMate 3000 RSLC
• Column under test: Accucore XL C18, 4 µm solid core, 150 × 4.6 mm
• Reference column: Fully porous C18, 5 µm, 150 × 4.6 mm
• Mobile phase: Acetonitrile:methanol 80:20 v/v
• Detection: UV at 280 nm

Main results and discussion

The 4 µm Accucore XL column achieved baseline resolution (Rs > 2.5) for all six vitamins, including a 30 % enhancement in the critical D2/D3 pair (Rs = 2.5 vs 1.92). Efficiency (HETP) increased on average by 82 % compared to the 5 µm column, with backpressure rising only from 47 to 62 bar. Raising flow rate to 1.5 mL/min shortened run time by approximately 3 minutes while maintaining an Rs of 2.3 for D2/D3 and a pressure under 100 bar.

Benefits and practical applications

Using the Accucore XL C18 column delivers sharper peaks, lower detection limits, and higher throughput without modifying standard HPLC setups. Laboratories can achieve robust quantification and identification of fat soluble vitamins in food, supplement, and quality control environments.

Future trends and potential applications

Advancements may include pairing superficially porous columns with mass spectrometry for enhanced sensitivity, exploring green solvent systems, and applying core-enhanced technology to other challenging analyte classes. High-throughput workflows and automation can further streamline routine vitamin analysis.

Conclusion

The Accucore XL C18 4 µm column offers substantial gains in resolution and efficiency over conventional 5 µm phases under identical conditions. Improved peak separation, minimal pressure increase, and faster runs underscore its suitability for routine fat soluble vitamin assays.