Flash Purification of δ-tocopherol and Subsequent HPLC Analysis for Sample Purity

July 2, 2010
L. Lloyd

S. Ball

K. Mapp

The Application Notebook

The Application Notebook, The Application Notebook-07-02-2010, Volume 0, Issue 0
Page Number: 41–42


L. Lloyd, S. Ball and K. Mapp, Varian Inc., Church Stretton, Shropshire, UK.


Following the recent surge in popularity of flash chromatography as a rapid purification technique, two options now exist for small- to large-scale purifications of crude samples. HPLC is a high performance method using small particle media, which is suited to the purification of complex samples where high purity is required. Flash is a low-pressure technique using larger particle media and is suited to less complex samples or where lower purities are acceptable.

An analytical HPLC system was used for method development of a normal phase purification of δ-tocopherol. Flash chromatography was chosen as the purification technique, followed by subsequent screening of the collected fractions by HPLC to assess sample purity.

Tocopherols are a series of organic compounds consisting of various methylated phenols. They occur in alpha, beta, gamma and delta forms, determined by the number of methyl groups on the chromanol ring. Due to their limited UV absorption, tocopherols require an alternative to UV detection — ELSD.

The Varian 971-FP instrument uses an evaporative light scattering detector (ELSD) as an additional method of detection. Data are shown to demonstrate how a separation developed on a standard HPLC system can be scaled up for purification on the 971-FP flash instrument.

Figure 1: Structure of d-tocopherol.

Methods and Materials

Sample preparation

For method development, a solution containing 0.2 mg/mL of δ-tocopherol in heptane was used. Further 2 mg/mL and 10 mg/mL concentrations were also required for the purification stages.

Analytical HPLC

An analytical HPLC system consisting of a single isocratic pump, 7725i Rheodyne valve (with 200 µL sample loop) and Varian 385-LC ELS detector was used. The operating conditions were as follows:

Instrument: Isocratic HPLC with 385-LC ELSD

Column: SepTech ST60-10 Si, 10 µm 250 × 4.6 mm (pn: A8061250X046)

Sample conc.: 10 µL of 0.2 mg/mL solution of δ-tocopherol

Eluent: 90:10 heptane:ethyl acetate

Flow rate: 1 mL/min

Flash purification

Instrument: 971-FP with 385-LC ELSD

Column: SF15-12g Si 35 (pn: AX1371)

Sample conc.: 1000 µL of 10 mg/mL solution of δ-tocopherol

Eluent: 90:10 heptane/ethyl acetate

Flow rate: 6 mL/min

Fraction analysis

The fractions collected from the δ-tocopherol purification were analysed by HPLC using the SepTech ST60-10 Si column.

Results and Discussion

Figure 2 shows the chromatogram obtained from a 10 µL injection of a 0.2 mg/mL solution of δ-tocopherol onto the SepTech ST60-10 Silica HPLC column. This gave an analytical loading of 0.002 mg on-column.

Figure 2: Analysis of d-tocopherol on a SepTech ST60-10 Si column with ELS detection showing the analytical load, 0.002 mg.

A second injection, 100 µL of a 2 mg/mL solution, was made to demonstrate a preparative load (× 100) on the same column (Figure 3). This revealed some additional impurities in the sample that were not previously apparent.

Figure 3: Analysis of d-tocopherol on a SepTech ST60-10 Si column with ELS detection at increased sample load to show impurity profiles, 0.2 mg.

The flash purification was done on the 971-FP flash instrument, was done on by direct injection of the δ-tocopherol. Figure 4 shows the resulting chromatogram.

Figure 4: Flash purification of d-tocopherol on an SF15-12g Si 35 SuperFlash column with ELS detection. The coloured bands indicate the fractions collected.

During the flash purification run, four fractions were collected (as highlighted by the colored bands in Figure 4). 50 µL of each fraction were analysed using the HPLC analytical method. Figure 5 shows an overlay of their HPLC profiles.

Table 1

Table 1 summarizes the fraction qualities and shows δ-tocopherol was isolated with 100% purity.

Figure 5: HPLC analysis of the four fractions from the flash purification.


The results demonstrate that chromatographic separations can be transferred between HPLC and flash systems, saving both time and money.

A Varian 385-LC ELS detector can be used in combination with both techniques to give increased sensitivity for method development and fraction analysis.

These data represent typical results.

For further information, contact your local Varian sales office.

SepTech, SuperFlash, Varian and the Varian Logo are trademarks or registered trademarks of Varian, Inc. in the US and other countries. Rheodyne is a registered trademark of the IDEX Health & Science Group. ©2010 Varian Inc.

Figure 5: HPLC analysis of the four fractions from the flash purification.

Varian Inc.

Essex Road, Church Stretton, Shropshire SY6 6AX

tel. +44 1694 723581 fax +44 1694 722171

E-mail: flashpur@varianinc.com

Website: www.varianinc.com