Skip to main content
SpringerLink
Log in

Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

The on-line combination of comprehensive two-dimensional liquid chromatography (LC × LC) with the 2,2′-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC × RP-LC–ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC × RP-LC, good performance for the ABTS assay was obtained. The combination of LC × LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC–ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics.

On-line HILIC×RP-LC–ABTS analysis of cocoa proanthocyanidins

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Kusznierewicz B, Piasek A, Bartoszek A, Namiesnik J (2011) Phytochem Anal 22:392–402

    Article  CAS  Google Scholar 

  2. Szajdek A, Borowska EJ (2008) Plant Foods Hum Nutr 63:147–156

    Article  CAS  Google Scholar 

  3. Iriti M, Faoro F (2009) In: Watson RR (ed) Complementary and alternative therapies and the aging population: an evidence-based approach. Elsevier Inc., San Diego

    Google Scholar 

  4. Shahidi F, Wanasundara PKJPD (1992) Crit Rev Food Sci Nutr 32:67–103

    Article  CAS  Google Scholar 

  5. Koşar M, Dorman HJD, Başer KHC, Hiltunen R (2004) J Agric Food Chem 52:5004–5010

    Article  Google Scholar 

  6. Dudonné AS, Vitrac X, Coutière P, Woillez M, Mérillon J-M (2009) J Agric Food Chem 57:1768–1774

    Article  Google Scholar 

  7. Joubert E, Manley M, Botha M (2008) Phytochem Anal 19:169–178

    Article  CAS  Google Scholar 

  8. Spranger I, Sun B, Mateus AM, de Freitas V, Ricardo-da-Silva JM (2008) Food Chem 108:519–532

    Article  CAS  Google Scholar 

  9. Huang D, Ou B, Prior RL (2005) J Agric Food Chem 53:1841–1856

    Article  CAS  Google Scholar 

  10. Magalhaes LM, Segundo MA, Reis S, Lima JLFC (2008) Anal Chim Acta 613:1–19

    Article  CAS  Google Scholar 

  11. Karadag A, Ozcelik B, Saner S (2009) Food Anal Meth 2:41–60

    Article  Google Scholar 

  12. Moon J-K, Shibamoto T (2009) J Agric Food Chem 57:1655–1666

    Article  CAS  Google Scholar 

  13. Laguerre M, Decker EA, Lecomte J, Villeneuve P (2010) Curr Opin Clin Nutr Metab Care 13:518–525

    Article  CAS  Google Scholar 

  14. Gülçin I (2012) Arch Toxicol 86:345–391

    Article  Google Scholar 

  15. Niederländer HAG, van Beek TA, Bartasiute A, Koleva II (2008) J Chromatogr A 1210:121–134

    Article  Google Scholar 

  16. Malherbe CJ, de Beer D, Joubert E (2012) Int J Mol Sci 13:3101–3133

    Article  CAS  Google Scholar 

  17. Re R, Pellegrini N, Proteggente A, Apnnal A, Yang M, Rice-Evans C (1999) Free Radic Biol Med 26:1231–1237

    Article  CAS  Google Scholar 

  18. Koleva II, Niederländer HAG, van Beek TA (2001) Anal Chem 73:3373–3381

    Article  CAS  Google Scholar 

  19. Floegel A, Kim D-O, Chung S-J, Koo SI, Chun OK (2011) J Food Comp Anal 24:1043–1048

    Article  CAS  Google Scholar 

  20. Martysiak-Lrowska D, Wenta W (2012) Acta Sci Pol Technol Aliment 11:83–89

    Google Scholar 

  21. Li F, Zhang L-D, Li B-C, Yang J, Yu H, Wan J-B, Wang Y-T, Li P (2012) Free Radic Res 46:286–294

    Article  CAS  Google Scholar 

  22. Bushey MM, Jorgenson JW (1990) Anal Chem 62:161–167

    Article  CAS  Google Scholar 

  23. Davis JM, Giddings JC (1985) Anal Chem 57:2168–2177

    Article  CAS  Google Scholar 

  24. Davis JM, Giddings JC (1985) Anal Chem 57:2178–2187

    Article  CAS  Google Scholar 

  25. Giddings JC (1990) In: Cortes HJ (ed) Multidimensional chromatography: techniques and applications. Marcel Dekker, New York

    Google Scholar 

  26. Kalili KM, de Villiers A (2009) J Chromatogr A 1216:6274–6284

    Article  CAS  Google Scholar 

  27. Kalili KM, de Villiers A (2010) J Sep Sci 33:853–863

    Article  CAS  Google Scholar 

  28. Beelders T, Kalili KM, Joubert E, de Beer D, de Villiers A (2012) J Sep Sci 35:1808–1820

    Article  CAS  Google Scholar 

  29. Kalili KM, Vestner J, Stander MA, de Villiers A (2013) Anal Chem 85:9107–9115

    Article  CAS  Google Scholar 

  30. Kalili KM, de Villiers A (2013) J Chromatogr A 1289:58–68

    Article  CAS  Google Scholar 

  31. Kalili KM, de Villiers A (2013) J Chromatogr A 1289:69–79

    Article  CAS  Google Scholar 

  32. Kalili KM, Cabooter D, Desmet G, de Villiers A (2012) J Chromatogr A 1236:63–76

    Article  CAS  Google Scholar 

  33. Kuhlmann O, Krauss G-J (1997) J Pharm Biomed Anal 16:553–559

    Article  CAS  Google Scholar 

  34. Kucera P, Umagat H (1983) J Chromatogr 255:563–579

    Article  CAS  Google Scholar 

  35. Lestremau F, Wu D, Szücs R (2010) J Chromatogr A 1217:4925–4933

    Article  CAS  Google Scholar 

  36. Giddings JC (1984) Anal Chem 56:1258A–1270A

    Article  CAS  Google Scholar 

  37. Pannala AS, Rice-Evans C (2001) Methods Enzymol 335:266–272

    Article  CAS  Google Scholar 

  38. Kivilompolo M, Hyötyläinen T (2007) J Chromatogr A 1145:155–164

    Article  CAS  Google Scholar 

  39. Kivilompolo M, Oburka V, Hyötyläinen T (2008) Anal Bioanal Chem 391:373–380

    Article  CAS  Google Scholar 

  40. Dugo P, Cacciola F, Donato P, Airado-Rodríguez D, Herrero M, Mondello L (2009) J Chromatogr A 1216:7483–7487

    Article  CAS  Google Scholar 

Download references

Acknowledgments

KMK and AdV gratefully acknowledge Stellenbosch University, Sasol and the National Research Foundation (NRF, Grant 70995 to AdV) for funding. SDS and TVH gratefully acknowledge the Agency for Innovation by Science and Technology in Flanders (IWT) for financial support. Dalene de Beer is thanked for advice on the antioxidant assays and Edmund Luckay (IWBT) for the donation of the grape sample.

Author information

Authors and Affiliations

  1. Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

    Kathithileni M. Kalili & André de Villiers

  2. Separation Science Group, Department of Organic Chemistry, Ghent University, Krijgslaan 281S4, 9000, Ghent, Belgium

    Seppe De Smet, Tim van Hoeylandt & Frédéric Lynen

Authors
  1. Kathithileni M. Kalili
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seppe De Smet
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tim van Hoeylandt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Frédéric Lynen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. André de Villiers
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to André de Villiers.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kalili, K.M., De Smet, S., van Hoeylandt, T. et al. Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants. Anal Bioanal Chem 406, 4233–4242 (2014). https://doi.org/10.1007/s00216-014-7847-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-7847-z

Keywords

Search

Navigation