**5.4. Column chromatography**

Column chromatography was used to separate the compounds. For this purpose, 1 g of sample was taken as a result of the rotations of the extracts and placed in a column (measures 20 cm long, 3 cm in diameter), prepacked with 30 g suspended in hexane. Silica gel was used to complete the separation of the components of the sample.

have in these fractions. Column chromatography also helps us to know the polarity of our compounds present in the sample, and therefore it is necessary to make several different solvent systems, this is vitally important because the good or bad purification of our compounds will depend on it. For thin-layer chromatography, the following components were used: A stationary phase: Silica gel 60F 254 Merk 0.25 mm thick, with a ceric sulfate developer solution, and the following solvent systems were tested: chloroformmethanol (7:3) (9:1) (1:9) (5:5), hexane-ethyl acetate (9:1) (5:5) (1:9), methanol-acetone (4:6) (9:1) (3:9), chloroform-ethyl acetate (3:7), methanol-hexane (3:7), chloroform-acetone (9:1), and ternary systems were also tested: ethyl acetate-chloroform-methanol (2:7:1), acetone-

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In **Figure 9**, the separation of components of the fruit extract is observed, where at least six components are distinguished, the solvent mixture was hexane/ethyl acetate in a 9:1 ratio. **Figure 10** shows the results for the leaf extract, where at least three main components are identified.

In **Figure 11**, other plates are shown chromatography's with a different mixture of solvents, without success in the separation of components. Observing the results of the chromatography, we know that most of our compounds have an intermediate polarity, this based on our solvent system that is the best for the separation of compounds. Another interesting fact about our results is that we have several compounds with very similar characteristics, this is deductible because although you can see spots individually (each corresponding to a differ-

chloroform-methanol (2:7:1).

**Figure 9.** Chromatographic plates of the fruit extract.

ent compound) they are too close together.

Three solutions were prepared at different proportions of hexane and ethyl acetate. The first solution containing 40 ml of hexane plus 10 ml of ethyl acetate, the second 20 ml of hexane plus 20 ml of ethyl acetate, and the third 10 ml of hexane plus 40 ml of ethyl acetate. To pack the column, 30 g silica gel were mixed with 100 ml of hexane, then poured into the column. Hexane is passed through the column to have a uniform packing. Then it was proceeded to repeat this operation several times to achieve a homogeneous packing. 3 ml of hexane was left on the surface of the silica, and then the sample was prepared. It is homogenized with 2 g of silica and rotavapor for a few minutes to evaporate solvent residues, and is added little by little to the column, leaving at least 1 ml of hexane remaining on the surface. Elute with the prepared solutions. Collect in a flask each fraction of the column (different color layers), evaporate each of the fractions in a rotavapor under reduced pressure and at a temperature of 50°C. Seven fractions of each of the columns were obtained for both the fruits and leaves. These were analyzed by thin-layer chromatography and HPLC.
