Determination of the Mobile Phase in Low-pressure Column Chromatography Using Thin-layer Chromatography to Purify Astaxanthin

Abstract

Astaxanthin, a compound that functions as an antioxidant, can be extracted from numerous sources such as the microalgae Haematococcus pluvialis, and can be purified using a low-pressure column chromatography (LPCC). The selection of the mobile phase in the LPCC process can be performed using thin-layer chromatography (TLC). TLC is the simplest way to qualify astaxanthin from microalgae extract and it has been widely applied in pharmaceutical analysis. The working principle of TLC is “like dissolves like”, where polar compounds dissolve in polar solvents, while non-polar compounds dissolve in non-polar solvents. The standard astaxanthin solution and the microalgae extract samples were spotted onto the activated TLC plates at the bottom. TLC plates were placed in a chamber which was filled with a solvent mixture (eluent) below the astaxanthin and sample spots. The standard astaxanthin solution and the sample spots were then eluted along with the eluent. The microalgae extract samples were subsequently degraded into several parts. The part that has the same height as the standard astaxanthin means the astaxanthin compound. In this study, based on the TLC analysis conducted by comparing the distributions of the standard astaxanthin with those of compounds found in the microalga extract, we observed that the eluent with a ratio of n-hexane:acetone of 3:1 (v/v) with a retardation factor (Rf) 0.3548 could effectively separate astaxanthin from the extract, thus making it suitable for use in the LPCC process.