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Development of Vitamin E Analysis in Palm Oil
Significant reduction of analytical time compared with normal phase HPLC analysis.
Reduction of organic solvent consumption compared with normal phase HPLC analysis.
Supercritical Fluid Chromatograph - Nexera
TM
UC
Introduction
Palm oil is made from oil palm and mainly produced in Asia.
Palm oil contains various functional natural compounds,
which include tocopherol homologues known as Vitamin E.
Currently, normal phase HPLC is widely used for quantitative
analysis of tocopherol homologues in palm oil. However,
this separation mode requires a long time more than 15 min
and consumes much organic solvent. Here, we introduce the
analysis method for tocopherol in palm oil using Nexera UC,
which is Shimadzu supercritical fluid chromatograph (SFC),
and compare the analytical time and organic solvents
consumptions against conventional HPLC analysis.
Analytical method of tocopherol
homologues by normal phase HPLC
Fig. 1 shows the chemical structure of tocopherol
homologues. Tocopherol has main four types of
homologues, and they are required to measure for quality
control in palm oil manufacturing. Tocopherol homologues
in palm oil are typically analyzed in normal phase HPLC that
uses much n-hexane. Table 1 and Fig.2 show analytical
conditions and chromatogram of tocopherol homologues
by normal phase HPLC analysis.
Column : Shim-pack
TM
CLC-SIL
*1
(250 mm× 4.6 mm I.D., 5 μm)
Mobile phase : n-hexane/2-propanol=100:0.5
Flow rate : 2 mL/mn
Column temp. : 40 ̊ C
Injection Vol. : 20 μL
Vial : SHIMADZU LabTotal™ for LC 1.5 mL, Glass
＊2
Detection : PDA 292 nm
Table 1 Analytical Conditions（HPLC）
1*
P/N: 228-17872-92
2*
P/N: 227-34001-01
About Supercritical fluid chromatography
Supercritical Fluid Chromatography (SFC) is one of the
chromatography systems with supercritical fluid (i.e.,
supercritical carbon dioxide) as mobile phase. The low cost
of carbon dioxide (purity: 99.9 %) can significantly trim the
total running cost of analyses. Generally, SFC shows high
separation than typical HPLC and shortens analytical time.
Furthermore, SFC can replace normal phase HPLC because
supercritical carbon dioxide is similar to n-hexane.
Method development of analytical
conditions of tocopherol homologues by
SFC
Table 2 shows the analytical conditions for SFC, and fig 3
shows the acquired chromatogram. We created the
calibration curves for alpha-tocopherol in the range from
0.8 mg/L to 500 mg/L. The calibration curves for other
tocopherol homologues were created in the range from 0.8
mg/L to 100 mg/L. Repeatability, linearity of the calibration
curves, and LOD (limit of detection) are summarized in
Table 3.
Column : Shim-pack UC-X NH2
*3
(250 mm× 2.1 mm I.D., 5 μm)
Mobile phase : A: CO2
B: Methanol
Flow rate : 4 mL/mn
Time program : B conc. 1.5 %(0-2.5 min)→ 20 % (2.51-2.9 min)
→1.5 %(2.91-3.0 min)
Column temp. : 40 ̊ C
Injection Vol. : 5 μL
Vial : SHIMADZU LabTotal™ for LC 1.5 mL, Glass
＊2
BPR setting : 15 MPa, 50 ̊ C
Detection : PDA 292 nm (reference 500 nm)
Table 2 Analytical Conditions（SFC）
*3
P/N: 227-30522-02
alpha beta gamma delta
R1 CH3 CH3 H H
R2 CH3 H CH3 H
R3 CH3 CH3 CH3 CH3
Fig. 1 Chemical structures of tocopherol homologues
Tocopherol
alpha beta gamma delta
%RSD
(peak area, n=6 (100 mg/L))
0.24 0.37 0.21 0.65
Linearity of calibration curve 0.9999 0.9999 0.9999 0.9999
LOD
(mg/L, calculated values)
0.4 0.6 0.6 0.7
0.0 0.5 1.0 1.5 2.0 2.5 min
0
5
10
15
mV
alpha-tocopherol
beta-tocopherol
gamma-tocopherol
delta-tocopherol
Fig. 2 Chromatogram of tocopherols by normal phase HPLC
0.0 2.5 5.0 7.5 10.0 12.5 min
0
10
20
30
40
mV
alpha-tocopherol
beta-tocopherol
gamma-tocopherol
delta-tocopherol
Fig. 3 Chromatogram of tocopherol homologues by SFC
Table 3. Repeatability, linearity, and LOD of tocopherol homologues
Kosuke Nakajima
1,2
Mei hui
3
1
Shimadzu Corporation,
2
Shimadzu (Asia Pacific) Pte Ltd.,
3
Shimadzu Malaysia Sdn. Bhd.
