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HPLC separations of stevia ingredients
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The herbal sweetener stevia was allowed as food additive
in the EU in December 2011. Thus, the analysis of stevia, a mixture of various
steviol glycosides, has become more important.
HPLC separations of steviol glycosides with a classical RP
18 phase based on porous silica, diverse HILIC phases and a modern RP 18 phase
on core-shell silica are presented below.
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Introduction |
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Stevia is a mixture of sweeteners
extracted from the plant Stevia
rebaudiana, which is native to South America. Its sweeting power is 300-fold
stronger than sugar and for hundreds of years well-known to the natives in the
border area of Paraguay and Brazil [1]. In 1887, the Swiss botanist M. G.
Bertoni discovered the plant and gave it the name Stevia rebaudiana Bertoni. Already in 1920 stevia was cultivated in
plantation. The leaves were used to sweeten tea and coffee. Further
investigations as sugar substitute were made in England in World War
II. In Japan, cultivation in greenhouses were carried out in 1954, and in the
1970s stevia was allowed as sugar substitute. Due to several studies about
possible health risks from stevia [2], stevia products were prohibited in the
USA in 1991. Already since 1986 stevia leaves and stevia glycosides were also
imported to Europe. Due to insufficient studies about a harmlessness to health,
they were withdrawn from sale in 2001. After evaluations of further studies by
the WHO, no indications for mutagene effects on humans have been found until
now. In 2008 authorization or partial authorization are granted in Australia,
New Zealand and the USA. On December 2nd, 2011 an EU regulation
became valid, which allows a processing of the natural stevia sweetener steviol
glycoside in food and drinks [3].
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Chemical structure of stevioside
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Stevia sweetener mostly consists
stevioside and nine to twelve steviol glycosides in relation to the species
[4]. The sweetness results mainly from stevioside and rebaudioside A. Further
sweet ingredients are rebaudiosides C, D, E and F, as well as ducloside A.
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Separation with a classical RP phase |
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HPLC methods are preferred for an
analytical separation of steviol glycosides, even if methods with capillary
electrophorese (CE) and High Performance Thin Layer Chromatography (HPTLC) have
been published [5].
A separation of steviol glycosides
from the commercially available stevia product „Daforto Stevia Basic“ is shown in application no.
125622 in the MN application database. The HPLC column used is
packed with a base deactivated RP 18 phase based on porous silica. Acetonitrile
– phosphate buffer at pH 2.6 is used as eluent in an isocratical run. Detection
is performed with an UV detector at 210 nm. With this classical RP application
nine steviol glycosides can be separated in 20 min (chromatogram and more details).
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Separation with diverse HILIC phases
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Zimmermann et al. tested
diverse HILIC phases (Hydrophilic Interaction Chromatography) [6] 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.
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Technical data of columns and
optimized HPLC conditions
| Column |
Luna HILIC
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NUCLEODUR
HILIC
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Kinetex HILIC
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TSKgel
Amide-80
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Length [mm]
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150
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125 |
150 |
150
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ID [mm]
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3
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2
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2.1
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2
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Particle size [µm]
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3
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3
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2.6
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3
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| Pore size [Å]
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200
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110
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100
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100
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| Eluent |
ammonium formate buffer – acetonitrile
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Buffer conc. [mM]
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5
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10
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5
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5 |
Eluent comp. [v/v]
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11:89
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14:86
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7.8:92.2
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17.5:82.5
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Flow rate [mL/min]
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0.68
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0.60
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0.66 |
0.45 |
Temperature [°C]
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40 |
Injection vol. [µL]
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1.0 |
| Detection |
LC-MS/MS (EI negative)
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Chromatograms of HILIC columns (Zimmermann
et al. [6])
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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)
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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.
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Rapid separation with an RP phase, based on core-shell silica
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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 application no.
125621 shows the separation on an RP 18 modified core-shell silica phase.
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Separation of nine steviol glycosides with an RP 18 core-shell
phase
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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.
Furthermore it could be shown that detection with MS (appl. no. 125720) or ELSD (appl. no. 125730) is also possible using an eluent of
acetonitrile – 0.1 % formic acid in water.
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Conclusion
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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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References
[1] Naturwiss. Rundschau 27 (1974), 231
[2] Pezutto, J.M. et al., Proc Natl Acad Sci U.S.A. 82 (April 1985), 2478–2482
[3] 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)
[4] Chaturvedula, V.S.K. et al., Phytochemistry Letters, Vol. 4 (3), (September 2011), 209–212
[5] Madan, S. et al., Stevia rebaudiana Bertoni – a review, Indian J Nat Prod Resour 1(3), (2010), 267–286
[6] 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)
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Image credits
Stevia image: © fovito
#24754835, www.fotolia.com
Stevioside structure: Yikrazuul, ISBN 3-540-40291-8 (2010)
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