Column Chromatography Lab Report

瀀瀀ract The purpose of Isolation of Bixin from Annatto Seeds using Column Chromatography was to separate and purify macroscopic amounts of pure bixin compound from annatto seed ex-tract.1 A mixture of bixin, methylbixin, and norbixin were extracted from annatto seeds. Bixin was then isolated from the mixture through the use of the column chromatography technique. The isolated bixin was characterized through Thin Layer Chromatography (TLC) analysis, which displayed an Rf value of 0.317, which was similar to the Rf value of the bixin standard, thus con-firming that the product was indeed isolated bixin. The isolated bixin product was a dark red sol-id after rotary evaporation. The starting mass of the annatto seeds was 1.00 g, and the final …show more content…
The column was carefully clamped vertically. 3% etha-nol in DCM (10 mL) was added to the column and a small amount was drained to check for and eliminate any air bubbles. The slurry of suspended silica gel was poured into the column using a glass powder funnel. An addition 33.0 mL of 3% ethanol in DCM was added to the column to rinse out the Erlenmeyer flask and powder funnel. The solvent was pipetted into the column in a circular motion to get the remaining silica gel off of the walls of the column. The stopcock of the column was opened and the solvent was collected in another Erlenmeyer flask until the solvent level in the column was 1 cm above the silica gel. The collected solvent was a yellow color and therefore not reused. The annatto extract in the 50mL round bottom flask was dissolved in 10% ethanol in DCM (2 mL). The annatto extract was then concentrated by repeatedly pipetting the solution until all of the extract was completely dissolved and concentrated. The dissolved extract was then gently added in a circular motion on the wall of the column and became a dark red band on top of the silica gel. The stopcock was opened and the solvent drained until the top of the sili-ca gel became a yellow color. Solvent was added 2 pipette fulls at a time, and then drained into an Erlenmeyer flask until the solvent on top of the silica gel was almost clear. …show more content…
The extraction with 10% ethanol in DCM was repeated four times, as opposed to one large extraction, because multiple extractions produces a greater yield of extracted product as explained by the partition coefficient.2 To deter-mine which solvent was best to use to run the column chromatography, TLC was used. Three TLC plates containing annatto extract, a more concentrated spot of annatto extract, and 1 stand-ard were run in three different solutions of ethanol in DCM. It was determined that 3% ethanol in DCM was best to use to run the column because the Rf value should be between 0.25 to 0.50.1 The Rf values on the TLC plate in 3% ethanol in DCM were .446 for all three lanes. The Rf val-ues on the TLC plate in plain DCM were .017, .051, and .000, indicating that it would be diffi-cult to get the compound to elute off the column with this solvent.1 The Rf values on the TLC plate in 10% ethanol in DCM were .864, .847, and .864, indicating that separation would not oc-cur with this solvent.1 After the column was packed, the sample was concentrated and loaded onto the column. The sample formed a dark red band about 1 cm thick on top of the silica gel. If the sample had not been loaded in a narrow band, there would have been reduced separation, leading to broad bands after separation occurred making it difficult to