Stevia Analysis (Part 2 of 2)
Separation with diverse HILIC phases
Zimmermann et al. tested diverse HILIC phases (Hydrophilic Interaction Chromatography)  optimizing the separation of stevioside and rebaudioside A under RP conditions. Thereby, the invers retention behavior of HILIC chromatography to RP chromatogaphy was advantageous. Amongst others the phases Luna® HILIC (Phenomenex), NUCLEODUR® HILIC (MACHEREY-NAGEL), KinetexTM HILIC (Phenomenex) and TSKgel Amide-80 (Tosoh) are investigated. The two first-mentioned phases are based on fully porous silica. KinetexTM HILIC is manufactured from core-shell silica and the phase from Tosoh is made from a polymeric support. Ammonium formate – acetonitrile is used as MS-suitable eluent. Its composition and buffer concentration, as well as the flow rate were optimized for the respective column.
Technical data of columns and optimized HPLC conditions
|Column||Luna HILIC|| NUCLEODUR
|Particle size [µm]||3||3||2.6||3|
|Pore size [Å]||200||110||100||100|
|Eluent||ammonium formate buffer – acetonitrile|
|Buffer conc. [mM]||5||10||5||5|
|Eluent comp. [v/v]||11:89||14:86||7.8:92.2||17.5:82.5|
|Flow rate [mL/min]||0.68||0.60||0.66||0.45|
|Injection vol. [µL]||1.0|
|Detection||LC-MS/MS (EI negative)|
Chromatograms of HILIC columns (Zimmermann et al. )
The elution sequence is the same for all columns: Rub rubusoside, Sbi steviolbioside, Dul A dulcoside A, Reb B rebaudioside B, Ste stevioside, Reb C rebaudioside C, Reb A rebaudioside A (more details in app # 125740)
Due to the invers retention behavior of the HILIC phases, a good separation of the main compounds stevioside and rebaudioside A is achieved. In consequence of its distinct better selectivity and peak symmetry seven steviol glycosides can be separated on NUCLEODUR® HILIC within 6 min. The other columns require 30 % more time.
The elution sequence is the same for all columns: Rub rubusoside, Sbi steviolbioside, Dul A dulcoside A, Reb B rebaudioside B, Ste stevioside, Reb C rebaudioside C, Reb A rebaudioside A (more details in appl. no. 125740)
Rapid separation with an RP phase, based on core-shell silica
In core-shell technology a sol-gel process is used to build a shell of porous silica around a core of solid silicon dioxide. The diffusion path of the eluent through the relatively thin porous layer is shortened considerably compared to the diffusion path of a fully porous silica particle. Shorter diffusion paths allow a rapid mass transfer of the analyte between the core-shell particles and the eluent.
The pictured app # 125621 shows the separation on an RP 18 modified core-shell silica phase.
The core-shell phase NUCLEOSHELL® RP 18 (MACHEREY-NAGEL) separates nine steviol glycosides in 7 min under RP conditions with UV detection, whereas classical RP 18 phases need around 20 min for this separation.
RP 18, as well as HILIC phases are very suitable for the separation of steviol glycosides from the herbal sweetener stevia. NUCLEOSHELL® RP 18 separates nine steviol glycosides with high peak symmetry; NUCLEODUR® HILIC facilitates a good and rapid separation of the main compounds stevioside and rebaudioside A.
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 Pezutto, J.M. et al., Proc Natl Acad Sci U.S.A. 82 (April 1985), 2478–2482
 VERORDNUNG (EU) Nr. 1131/2011 DER KOMMISSION vom 11. November 2011 zur Änderung von Anhang II der Verordnung (EG) Nr. 1333/2008 des Europäischen Parlaments und des Rates hinsichtlich Steviolglycosiden. Amtsblatt der Europäischen Union, L 295/205 (12. November 2011)
 Chaturvedula, V.S.K. et al., Phytochemistry Letters, Vol. 4 (3), (September 2011), 209–212
 Madan, S. et al., Stevia rebaudiana Bertoni – a review, Indian J Nat Prod Resour 1(3), (2010), 267–286
 Zimmermann, B.F., et al., Separation of steviol glycosides by hydrophilic liquid interaction chromatography, Food Anal. Methods, DOI 10.1007/s12161-011-9229-x, (published online 13. April 2011)
Stevia image: © fovito #24754835, www.fotolia.com
Stevioside structure: Yikrazuul, ISBN 3-540-40291-8 (2010)