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.
L. Lloyd, S. Ball, and K. Mapp, Varian, Inc.
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.
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 utilizes 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.
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.
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
Sample Conc: 10 μL of 0.2 mg/mL solution of δ-tocopherol
Eluent: 90:10 heptane:ethyl acetate
Flow Rate: 1 mL/min
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
The fractions collected from the δ-tocopherol purification were analyzed by HPLC using the SepTech ST60-10 Si column.
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.
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.
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.
During the flash purification run, four fractions were collected (as highlighted by the colored bands in Figure 4). 50 μL of each fraction were analyzed using the HPLC analytical method. Figure 5 shows an overlay of their HPLC profiles.
Table I summarizes the fraction qualities and shows δ-tocopherol was isolated with 100% purity.
Table I: Composition of purification fractions
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.
Table I: Composition of purification fractions Varian, Inc.
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