Sample of Lab report of isolation of β-carotene from spinach

PURPOSE STATEMENT

The experiment aimed at isolating Beta-carotene from spinach by solvent extraction and purifying it through the chromatography column. The results were confirmed by Thin Layer Chromatography (TLC). Solvent extraction is a method which separates substances in a mixture using a solvent. The solvent is considered for the extraction based on the chemical and physical properties of all substances present in the mixture. The compound of interest in the mixture should have greater solubility in the solvent. The solvent chosen for the extraction should not mix with the compound of interest in the mixture. In this experiment, acetone was selected as the solvent for extraction. Acetone is a polar solvent, therefore, dissolves polar and ionic compounds in the mixture. 

                  Chromatography consists of mobile phase and stationary phase. Stationary phase can be solid or liquid supported. Mobile phase is always a gas or liquid. Mobile phase moves together with components in the mixture through the stationary phase. Chromatography separates substances into their constituents depending on their interaction with the stationary phase. Therefore, this interaction makes the components of the mixtures move at different rates through the chromatography column hence enhance their separation. The column contained gel silica (silicon (IV) oxide). The mobile phase, in this experiment,  was a hexane solution. Since silicon (IV) oxide is a polar compound, therefore, polar compounds come out of the column last while non-polar compounds elute from the column first. Thin Layer Chromatography plate, contains a silicon (IV) oxide, and thus functions in the same way as column chromatography. 

EXPERIMENTAL

0.5 grams spinach, 0.5 grams anhydrous magnesium sulfate and 1 gram of sand were put in a mortar then grounded for a minute. The grounded mixture was transferred into a test tube, followed by the addition of 3 milliliters of acetone. The suspension was thoroughly mixed with a spatula and then left to rest undisturbed for a while. The column chromatography was prepared as required and equilibrated by running hexane solution through it.  After 15 minutes of soaking, a few drops of the extract were collected as crude extract reference and the remaining extract was transferred into a 50ml beaker. The crude extract was re-dissolved in 1ml hexanes and swirled to make sure much of it dissolved. The extract solution was then added to the prepared column, followed by addition of small amount of hexanes directly to the column to push the extract through the column.  As the yellow band was being pushed down the column, the mobile phase was made more polar by changing it to nine to one hexane to acetyl acetate. The yellow component was collected as it eluted out of the column.

                  Three dots were marked on TLC paper using a pencil at 1-centimeter mark of the paper for the standard Beta-carotene, the yellow extract, and crude extract. After all the dots dried briefly, the paper was placed in TLC chamber composed of 70% hexane and 30% acetone.

RESULTS

The solvent front on TLC paper was at 10cm mark. Distance traveled by standard Beta-carotene, yellow extract, and crude extract were as follow; 9.5cm, 9.4cm, and 9.7cm respectively.

DISCUSSION

Beta-carotene is non-polar compound hence; it took less time interacting with the column and eluted out of the column first. When the column was made more polar the movement of the yellow band through the column doubled. The distances traveled by yellow extract and the standard -carotene were almost the same on the TLC paper, for instance, 9.5cm and 9.4cm respectively. In the experiment, magnesium sulfate was used to absorb some of the water in the spinach. The sand acted as an abrasive.

Conclusion

The yellow extract was indeed Beta-carotene as confirmed by Thin Layer Chromatography. It showed a yellow distinctive color on the TLC plate and its Rf value (0.94) was close to standard Beta-carotene (0.95). This experiment is important for teaching students on the techniques of Thin Layer Chromatography and solvent extraction.