Fluorescence-labelling for analysis of protein in starch using asymmetrical flow field-flow fractionation (AF4)

  • Yoo, Yeongsuk (Department of Chemistry, Hannam University) ;
  • Choi, Jaeyeong (Department of Chemistry, Hannam University) ;
  • Zielke, Claudia (Department of Food Technology, Engineering and Nutrition, Lund University) ;
  • Nilsson, Lars (Department of Food Technology, Engineering and Nutrition, Lund University) ;
  • Lee, Seungho (Department of Chemistry, Hannam University)
  • Received : 2017.01.05
  • Accepted : 2017.02.08
  • Published : 2017.02.25


Starch is a mixture of amylose (AMY) and amylopectin (AMP) which are different in physical properties such as molar mass (M), rms radius ($R_g$) and hydrodynamic diameter ($d_H$). The rheological and functional properties of starch are influenced by various factors including the molecular size, molar mass distribution (MD) and the concentration ratio of AMY and AMP. It is also important to analyze proteinaceous material in starch as they affect the flavor and texture of food to which starch is added. In this study, asymmetrical flow field-flow fractionation (AF4) was employed for separation and quantitation of AMY and AMP in starches (Amaranth, potato, taros and quinoa). AF4 was coupled with a multi-angle light scattering (MALS) and a refractive index (RI) detector for determination of the absolute M, MD and molecular structure. It was found that AMP has the M and $R_g$ ranging $3.7{\times}10^7{\sim}6.5{\times}10^8g/mol$ and 84 ~ 250 nm, respectively. Also the existence of branch was confirmed in higher M. In addition, proteinaceous material in starch was analyzed by AF4 coupled with a fluorescence detector (FS) after fluorescence-labeling. AF4-FS with fluorescence-labelling showed a potential for investigation on existence of proteinaceous material and the interaction between proteinaceous material and polysaccharide in starch.


Supported by : Hannam University


  1. K. G. Wahlund, M. Leeman, and S. Santacruz, Anal. Bioanal. Chem., 399(4), 1455-1465 (2011).
  2. E. Chiaramonte, L. Rhazi, T. Aussenac, and D. R. White, J. Cereal Sci., 56(2), 457-463 (2012).
  3. N. Lindeboom, P. R. Chang, and R. T. Tyler, Starch/ Staerke, 56(3-4), 89-99 (2004).
  4. L. Nilsson, Food Hydrocoll., 30(1), 1-11 (2013).
  5. A. Kilara and T. Y. Sharkasi, Crit. Rev. Food Sci. Nutr., 23(4), 323-395 (1986).
  6. V. B. Tolstoguzov, In 'Understanding and Controlling the Microstructure of Complex Foods', pp. 185-206, 2007.
  7. C. H. Remsen and J. P. Clark, J. Food Process Eng., 2(1), 39-64 (1978).
  8. W. E. Moore and J. L. Carter, J. Texture Stud., 5(1), 77-88 (1974).
  9. S. Emami, L. G. Tabil, R. T. Tyler, and W. J. Crerar, J. Food Eng., 82(4), 460-465 (2007).
  10. M. B. Cardoso, D. Samios, and N. P. Silveira, Starch-Starke, 58(7), 345-352 (2006).
  11. R. W. Kerr, F. C. Cleveland, and W. J. Katzbeck, J. Am. Chem. Soc., 73(1), 111-117 (1951).
  12. J. D. Timpa, 'Characterization by size exclusion chromatography with refractive index and viscometry: complex carbohydrates-cellulose, starch and plant cell wall polymers', Polymeric Materials Science and Engineering, Proceedings of the ACS Division of Polymeric Materials Science and Engineering, 272-273 (1993).
  13. Z. P. Stojanovic, K. Jeremic, S. Jovanovic, W. Nierling, and M. D. Lechner, Starch-Starke, 61(3-4), 199-205 (2009).
  14. K. Muneeruddin, J. J. Thomas, P. A. Salinas, and I. A. Kaltashov, Anal. Chem., 86(21), 10692-10699 (2014).
  15. P. Hong, S. Koza, and E. S. P. Bouvier, J. Liq. Chromatogr. Rel. Technol., 35(20), 2923-2950 (2012).
  16. K. G. Wahlund and J. C. Giddings, Anal. Chem., 59(9), 1332-1339 (1987).
  17. A. Litzen and K. G. Wahlund, J. Chromatogr. A, 476(C), 413-421 (1989).
  18. K. G. Wahlund and A. Litzen, J. Chromatogr. A, 461(C), 73-87 (1989).
  19. A. Litzen and K. G. Wahlund, Anal. Chem., 63(10), 1001-1007 (1991).
  20. A. Litzen, Anal. Chem., 65(4), 461-470 (1993).
  21. M. Van Bruijnsvoort, K. G. Wahlund, G. Nilsson, and W. T. Kok, J. Chromatogr. A, 925(1-2), 171-182 (2001).
  22. S. You, S. G. Stevenson, M. S. Izydorczyk, and K. R. Preston, Cereal Chem., 79(5), 624-630 (2002).
  23. S. Lee, P.-O. Nilsson, G. S. Nilsson, and K.-G. Wahlund, J. Chromatogr. A, 1011(1-2), 111-123 (2003).
  24. H. Dou, B. Zhou, H. D. Jang, and S. Lee, J. Chromatogr. A, 1340, 115-120 (2014).
  25. D. Perez-Rea, B. Bergenståhl, and L. Nilsson, Anal. Bioanal. Chem., 407(15), 4315-4326 (2015).
  26. P. Roger, B. Baud, and P. Colonna, J. Chromatogr. A, 917(1-2), 179-185 (2001).
  27. W. J. Kim, H. E. Chul, S. T. Lim, J. H. Han, S. G. You, and S. Lee, Bull. Korean Chem. Soc., 28(12), 2489-2492 (2007).
  28. A. Rolland-Sabate, P. Colonna, M. G. Mendez-Montealvo, and V. Planchot, Biomacromolecules, 8(8), 2520-2532 (2007).
  29. H. Dou, E. Magnusson, J. Choi, F. Duan, L. Nilsson, and S. Lee, Food Chem., 192, 228-234 (2016).
  30. J. Alftren, J. M. Peñarrieta, B. Bergenståhl, and L. Nilsson, Food Hydrocoll., 26(1), 54-62 (2012).
  31. L. A. Bello-Perez, P. Roger, B. Baud, and P. Colonna, J. Cereal Sci., 27(3), 267-278 (1998).
  32. J. J. Kirkland, C. H. Dilks Jr, S. W. Rementer, and W. W. Yau, J. Chromatogr. A, 593(1-2), 339-355 (1992).
  33. L. A. Bello-Perez, O. Paredes-Lopez, P. Roger, and P. Colonna, Cereal Chem., 73(1), 12-17 (1996).
  34. S. H. Yoo and J. L. Jane, Carbohydr. Polym., 49(3), 307-314 (2002).
  35. C. Yang, B. Meng, M. Chen, X. Liu, Y. Hua, and Z. Ni, Carbohydr. Polym., 64(2), 190-196 (2006).
  36. M. M. Millard, W. J. Wolf, F. R. Dintzis, and J. L. Willett, Carbohydr. Polym., 39(4), 315-320 (1999).
  37. H. J. He, R. P. Yu, T. Zhu, Z. B. Gu, and H. Xu, Guang Pu Xue Yu Guang Pu Fen Xi, 26(9), 1636-1639 (2006).