Table 1.
The consecutive steps of separation by open column chromatography using silica-9 as a stationary phase. Approximate volumes of eluents and runtime are given for the following conditions: (i) 20 g of dry silica-9 suspended in hexane: acetone (90:10 v/v) was applied to the column; (ii) glass chromatography column with filter disc, 15 mm; (iii) evaporated carotenoid fraction after saponification of pigment extract obtained from 2 × 109 cells was dissolved in hexane: acetone (90:10 v/v) and applied to the column; (iv) chromatography was carried out at room temperature
| Eluent | Description | Product |
|---|---|---|
| Hexane: acetone (90:10 v/v) | Clearly separated band migrates down through the column for approximately 25 min and approximately 40 mL of eluent is used. Three to four millilitres of this fraction is collected. | β,β-Carotene and its derivatives |
| Hexane: acetone (80:20 v/v) | After the change of eluent, a hardly visible band (more intensive in the case of Dtx purification) migrates for about 30 min and approximately 50 mL of eluent is used. A volume of 6–8 mL of this fraction is collected. | Cryptoxanthin-epoxide |
| Third band is very intense and in half-length of column, it splits into two bands, which become better separated with time. From the second fraction, migration of the third one takes about 50 min and approximately 80 mL of eluent is used. A volume of 14–20 mL of this fraction is collected; in the case of Dtx purification, their colour is very intensive. | Diatoxanthin | |
| Hexane: acetone (70:30 v/v) | Clearly marked band migrates for about 20 min since Dtx is collected and approximately 40 mL of eluent is used. A volume of 15–22 mL of this fraction is collected. | Diadinoxanthin |
| Acetone | Acetone results in migration of all remaining fractions, which are visible as four blurred bands collected together in a volume of 17–25 mL. Their migration takes about 30 min and approximately 60 mL of eluent is used. | Fucoxanthin derivatives |