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Thin Layer Chromatographic Analysis of Food Colorants from Three Morphotypes of Annatto (bixa Orellana L.)

Hari Pada Seal
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

Mohammad Amdad Ali
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

Md. Usuf Ali
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

Mosammat Hasina Akhter
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

Fowzia Sultana
Department of Poultry Science, Faculty of Animal Husbandry, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

Abstract/ Executive Summary:

This article describes a simple solvent extraction method for the extraction of colorants from the three morphotypes such as, (Morphotype-1 (M₁), Morphotype-2 (M₂), and Morphotype- 3 (M₃) of Annatto (Bixa orellana L.) seeds, and their separation, vivid, and qualitative demonstration by thin-layer chromatography. Several solvent systems (hexane, chloroform, acetone, ethanol, and a mixed-solvent having composition of CHCl₃/C₂H₅OH/CH₃COOH (80:2:1)) were applied for extraction of colored components. It was observed that a large portion of colorants was extracted by chloroform. Its effluent was deep brick red in color and transparent. Furthermore, various carrier solvent systems (Benzene-Ethyl acetate) were used to separate the components from the extracts. Carrier solvent system with the ratio of 7:3 was found as superior solvent for chloroform extracts. Three colored-spots were observed for all morphotypes. Among them, the first one was yellow colored having very low polarity and the second and third spots were both redbrick colored having medium and higher polarity respectively. In addition, for M₁ no colorless-spot was observed in low and medium polar systems, revealing that the amount of wax and gum were minimum in the extract and superior morphotype among the three.

Keywords: Solvent Extraction, Seed Coat, Bixa orellana, Natural Colour, Rf Value

Location: Bangladesh Agricultural University (BAU).

Start Date:

End Date:

Program Area: Postharvest and Agro-processing

Commodity: Bread

Objectives:

To assessment of three morphotypes of Annatto in order to develop a simple inexpensive process for extracting the colorants.

Methodology:

The matured-fruit-capsule of Annatto (Bixa orellana L.) of three morphotypes: morphotype-1 (M1), morphotype-2 (M2), and morphotype-3 (M3) were collected from Botanical Garden of Bangladesh Agricultural University (BAU). The fruit-capsules were dried in sunlight for 30 hours and seeds were separated out from the capsules. After that, the seeds were dried in sunlight for another 30 hours and preserved in a refrigerator at 20?C for further analysis. To extract the colorants, 100 g of seeds were soaked in 200 ml particular organic solvent for 30 minutes and stirred abrasively by a glass rod for one hour (Islam, 2008). Extracting solvents were selected based on their polarity, viz. hexane (Polarity: 0), chloroform (Polarity: 4.1), acetone (Polarity: 5.1), and ethanol (Polarity: 5.2) (Finar, 2000). In addition, a mixed-solvent system of CHCl3/C2H5OH/CH3COOH (80:2:1) also was employed. Finally, it was filtered by Whatman Grade-114 filter paper to prepare the stock solution for Thin Layer Chromatographic (TLC) analysis.The chromoplate were prepared by dispersing a suspension of finely powdered silica gel adsorbent (50 g silica gel thoroughly mixed and swelled in 125 ml of water) on a glass strip (EMerck India limited, Mumbai, 400018) of suitable size (5 cm × 1 cm × 0.3 cm). A very thin uniform layer of silica was formed on glass strip with the aid of a spreader. It was then dried in an oven at 105±5 °C for 12 hours. For obtaining a chromatogram, the chromoplate was spotted with a minute drop of annatto extract with the help of a capillary tube, and then placed vertically in a solvent tank. Development of the chromatogram occurred by capillary movement of the solvent up the silicaadsorbent layer. When solvent front reached at the upper end of the TLC plate, it was marked and the plate was taken out and allowed to dry. If the components were colored, then the spots were readily located. To ascertain the presence of colorless spots, the TLC plate was transferred to iodine chamber. Both colored and colourless spots on the TLC plate were recorded and Rf values were calculated. The Rf value depends on the relative polarities of the carrier solvent and spotcomponents. It is defined as the ratio of the distance travelled by a component to the distance travelled by the solvent front. Rf value indicates the polarity of the spot-components in the TLC plate. Similar polarities of carrier solvent and spot-components leading higher Rf value and vice-verse.

Source of Information: ISSN: 2224-0616 Int. J. Agril. Res. Innov. & Tech. 2 (1): 7-12, June, 2012 Available online at http://www.ijarit.webs.com

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Three coloring constituents in each morphotypes (M₁, M₂, and M₃) of Annatto were observed. Among the three pigments, one was associated with high Rf value, i.e., very low polarity and yellow in color. The rest of the two pigments were brick red and orange colored associated with medium and low Rf value, respectively. Moreover, after exposing in iodine vapor, most of the chromatogram indicated the presence of colorless components. Studies from Rf values of colorless components revealing that one was low polar colourless components and other two colorless compounds were of medium polarity. Furthermore, chloroform extract effluent was very deep brick red in color and transparent. On depending of the chromatogram, mixed solvent systems of Bz/EtOAc of 7:3 was superior carrier solvent for separating the dyes from the chloroform extracts. Thus, dyes separated from the extract can be used in coloring the food, medicine, cosmetic and other purpose.

Knowledge Management: Journal