Analysis of Carotenoids

Significance of the topic

Carotenoids are ubiquitous pigments in foods and biological materials that play important roles in nutrition, color quality and oxidative stability. Reliable chromatographic separation and detection of carotenoids — including structural and geometric isomers — is essential for food quality control, compositional analysis, authenticity testing and research into bioavailability. Reversed-phase C30 stationary phases have become a preferred solution for resolving structurally similar carotenoids due to enhanced shape selectivity and improved isomer separation compared with conventional C18 phases.

Objectives and overview of the application

This application demonstrates a robust reversed-phase liquid chromatography method for separation and identification of eight common carotenoids. The method aims to provide clear resolution of xanthophylls (oxygenated carotenoids) and hydrocarbon carotenes, to support analysis of food coloring and carotenoid composition in green and yellow vegetables and other matrices.

Methodology and chromatographic conditions

The method uses a gradient reversed-phase separation with a C30-type stationary phase optimized for carotenoids. A multi-solvent gradient (methanol, acetonitrile, tetrahydrofuran and water) is employed to progressively elute polar xanthophylls first and then nonpolar carotenes. Detection is by photodiode array at 450 nm, the approximate absorption maximum for many carotenoids, enabling spectral confirmation and peak purity assessment.
Key method parameters (summary):

• Column: Shim-pack SR-C30, 250 mm × 4.6 mm I.D., 5 µm.
• Mobile phases: A = methanol, B = acetonitrile, C = tetrahydrofuran (THF), D = water.
• Gradient program: initial composition ≈ 65% A / 0% B / 25% C / 10% D (hold 5 min); ramp to ≈ 25% A / 30% B / 45% C / 0% D (25–35 min); quick return to initial and re-equilibration until 45 min. Total run time 45 min.
• Flow rate: 1.0 mL/min; injection volume: 5 µL; column temperature: 40 °C.
• Detection: PDA at 450 nm (full spectra available for peak identification).

Used instrumentation

• LC system: Shimadzu i-Series LC-2070C 3D.
• Column: Shim-pack SR-C30, 250 × 4.6 mm, 5 µm (P/N 227-31218-04).

Main results and discussion

The method successfully separated eight carotenoids commonly analyzed in food matrices: astaxanthin, lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene. The SR-C30 phase provided improved isomer discrimination and sharper peak shapes relative to more generic reversed-phase chemistries, allowing reliable identification by retention time and spectral data from the PDA detector. The gradient composition (use of THF together with methanol and acetonitrile) enhances elution strength for highly nonpolar carotenes like lycopene and β-carotene while retaining early elution of more polar xanthophylls.

Practical observations:

• Use of THF is advantageous for eluting late, nonpolar carotenoids but requires attention to solvent compatibility and safe handling due to THF’s volatility and peroxide formation risk.
• PDA detection at 450 nm offers high sensitivity for carotenoids; spectral scans support discrimination of coeluting or closely eluting isomers.
• Run time and re-equilibration ensure reproducible retention but can be adjusted (shortened or optimized) depending on required throughput and matrix complexity.

Benefits and practical applications of the method

• High-resolution separation of geometric and structural carotenoid isomers useful for food compositional analysis, nutritional labeling and quality control of natural colorants.
• Applicable to monitoring carotenoid profiles in vegetables, fruits, supplements and formulated food products, enabling discrimination between similar pigments and detection of synthetic additives or degradation products.
• PDA spectral information increases confidence in peak identity without immediate need for mass spectrometry, reducing analytical cost for routine assays.

Future trends and opportunities

• Coupling SR-C30 separations with high-resolution mass spectrometry to provide definitive identification, including positional isomers and oxidized degradation products.
• Development of faster UHPLC C30 methods or core–shell C30 phases to reduce run time while maintaining isomer separation.
• Green chemistry approaches: replacing THF with less hazardous solvents or using solvent-minimizing gradients and microflow formats.
• Integration of automated sample preparation (SPE, online extraction) and chemometric tools for deconvolution of complex food matrices and quantitation in regulatory workflows.
• Use of isotopically labeled internal standards for more accurate quantification in complex matrices and routine QA/QC.

Conclusion

The described reversed-phase LC method using a Shim-pack SR-C30 column and a methanol/acetonitrile/THF/water gradient provides reliable separation and detection of a broad range of carotenoids. The approach is well suited for detailed compositional analysis and quality control in food and related industries, offering robust isomer separation, spectral confirmation via PDA and practical adaptability for both routine and research applications.

Reference

• Shimadzu Application News 01-01081 (JP). Shimadzu Corporation, First Edition: Sep. 2026; © Shimadzu Corporation 2026.