Page 51 - Application Handbook - Liquid Chromatography

Application No.L501
News

The conditions used for SFE are shown in Table 1. We
investigated column selection, chose the Shim-pack Area
UCX-SIL analytical column, optimized each analytical
condition for online SFE-SFC analysis, then performed
analysis using the conditions shown in Table 2. 200000

Table 1 SFE Conditions for α-Tocopherol
100000
Offline SFE: r = 0.9998
2
Extraction Vessel : 5 mL
Extraction Solvent : CO2
Flowrate : 5 mL/min 0
Temperature : 40 ˚C 0.0 0.5 1.0 1.5 2.0 Conc. (mg/mL)
Back Pressure : 15 MPa
Extraction Time : 15 min
(Static 2 min → Dynamic 3 min) × 3 times Fig. 4 Linearity for Standard α-Tocopherol Obtained by SFC
Trap & Pressure Down Conditions
Trap Column : Shim-pack VP-ODS (50 mm L. × 4.6 mm I.D.)
Temperature : 60 ˚C Table 3 Repeatability for Standard α-Tocopherol Obtained by
Pressure Down Time : 10 min (15 - 25 min)
SFC (n=6)
Recovery Conditions No Retention Time (min) Area Height
Elution Solvent : Hexane
Flowrate : 2 mL/min Average 7.242 127,338 19,682
Temperature : 60 ˚C RSD (%) 0.057 0.573 0.274
Fraction Time : 3.5 min (25 - 28.5 min)
n SFE Evaluation of α-Tocopherol in a Commercial Table 4 shows the repeatability of the quantitative
Supplement α-tocopherol result obtained by repeated SFE treatment,
For the α -tocopherol extract obtained through offline and α -tocopherol recovery relative to the theoretical
SFE, we performed SFC under the conditions shown in value (7.4 mg). Fig. 5 shows the overlaid chromatograms
Table 2 then evaluated the extraction procedure. Extract for α-tocopherol. Good recovery and repeatability was
was mixed with hexane to make up 10 mL before being confirmed after just one extraction, showing that offline
used for SFC analysis. A representative SFC chromatogram SFE is effective for vitamin E compound extraction.
is shown in Fig. 3.

Table 2 SFC Conditions for α-Tocopherol Table 4 Repeatability and Recovery of α-Tocopherol in a
Commercial Supplement Using SFE
SFC Conditions:
Column : Nacalai COSMOSIL Cholester No Conc. (mg/mL) Recovery (%)
(250 mm L. × 4.6 mm I.D., 5 µm)
Flowrate : 3 mL/min 1 0.776 104.46
Modifier : IPA 2 0.780 105.00
Gradient : 2 % (0 min) - 20 % (10 min) - 50 % (10 - 12 min) 3 0.772 103.92
Temperature : 40 ˚C
Back Pressure : 15 MPa 4 0.790 106.35
Injection Volume : 2 µL 5 0.761 102.44
6 0.758 102.04
Average 0.773
mAU RSD (%) 1.549
20
15
10
5
0 mAU
0.0 2.0 4.0 6.0 8.0 min 20

Fig. 3 SFC Analysis of α-Tocopherol Obtained by SFE from a
Commercial Supplement
10
First, we used a standard product to evaluate the
suitability of the α-tocopherol SFC conditions used for
evaluation of offline SFE. Fig. 4 shows the linearity in 0
the sample concentration range of 0.5 µg/L to 2.0 µg/L,
and Table 3 shows the repeatability at a concentration 6.0 7.0 8.0 min
of 1.0 µg/L. Good linearity and sufficient repeatability in
terms of retention time, peak area and peak height Fig. 5 Overlaid Chromatograms for α-Tocopherol After SFE
were obtained.

First Edition: Jan. 2016

For Research Use Only. Not for use in diagnostic procedures.
The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.
The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject
to change without notice.
www.shimadzu.com/an/ © Shimadzu Corporation, 2016