# ISOLATION AND IDENTIFICATION OF SECONDARY METABOLITES

19.2.1 THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is an analytical method to separate and also isolate the compounds from a mix, it is a useful procedure to purify a compound isolated from a plant extract.

The test sample is deposited near one end of a glass plate which has been previously coated with a thin layer of adsorbent 0.1 mm thick (stationaiy phase). The sheet is then placed in a closed cuvette containing one or more mixed solvents (eluent or mobile phase). As the solvent mixture rises by capillarity through the adsorbent, there is a differential partition of the products present in the sample between the solvent and the adsorbent (Si02 or Al,03).

The relationship between the distances traveled by the solute and the eluent from the origin of the plate is called front retainer (Rf), and has a constant value for each compound under particular conditions (adsorbent solvent, temperature, etc.), also for determining the degree of purity and track a chemical reaction.

The test sample is deposited near one end of a glass plate which has been previously coated with a thin layer of adsorbent 0.1 mm thick (stationaiy phase). The sheet is then placed in a closed cuvette containing one or more mixed solvents (eluent or mobile phase). As the solvent mixture rises by capillarity through the adsorbent, there is a differential partition of the products present in the sample between the solvent and the adsorbent (SiO, or Al,03).

The relationship between the distances traveled by the solute and the eluent from the origin of the plate is called front retainer (Rf), and has a constant value for each compound under particular conditions (adsorbent solvent, temperature, etc.). To calculate the Rf of a compound, measure the distance from spot origin to the center of the spot and the following equation applies:

Rf = distance traveled by the compound (X)/ distance traveled by the eluent (Y)

Calculation front retention (Rf) eluents: to choose the eluents to be used in chromatography, it is necessary know the polarity of each solvent, in phytochemical analysis, are used, solvents from lower to higher polarity. Samples are deposited on the plate as spots, with a capillary tube: a point of extract of plant diluted, a point of a fraction obtained from a column. A small (3x7 cm) TLC plate takes just few minutes to run.

The analysis is qualitative, and it will show, how many compounds are present in the extract, how many in each fraction of the column, and how pure is a compound obtained from each fraction. For example: If it is eluted a thin layer chromatography of a sample of diluted extract or fraction of a chromatographic column or an insoluble precipitate or plant material, and so on.

First, dissolve a small quantity of sample in the adequate solvent and apply a sample with a capillary tube, approximately 1 cm from the lower end of the chr omatographic plate, allow dry and place the plate in a glass vessel containing a mix of benzene: acetone 9:1. For example, if the chromatographic plate is 5 x 10 cm use a glass container of 250 mL with a lid and add 10 mL of eluent, in this case it would be: 9 mL benzene (B) and 1 mL of acetone (A), (0.5 cm before the upper end of the plate remove the plaque and dry) observe under ultraviolet light and spray with cobalt chloride solution (different developers subsequently described). If the sample in the TLC plate (Figs. 19.1 and 19.2), did not move up, then another solvent system is used: chloroform-methanol 9:1. In case the sample was not displaced from the application point in chromatographic plate, a third system of eluent is used; Butanol-acetic acid-water, in the ratio 7:2:1, if higher polarity is required, increase water volume, and decrease acetic acid, 7:1:2 and so on.

Chromatography is an effective isolation technique because the compounds on a mix travel different distance in the plate.

Each compound on a mix has a specific area on the plate because of its polarity and each one can be scraped away, to be analyzed and chemically identified. (Lewis, 1989) Revelators, most of the chromatographic plates have a fluorescent indicator that allows the active compounds be viewed under ultraviolet light (254 rnn).

For compounds that do not absorb UV light, for chromatogram display, it is required to use a developer that reacts with adsorbates producing colored compounds:

FIGURE 19.1 TLC plate and cuvette.

FIGURE 19.2 Thin layer chromatography with different elution systems 19.2.1.1 COBALT CHLORIDE

In 800 mL of distilled water add 20 g of CoCl, and 100 mL of concentrated sulfuric acid, gradually added sliding by the wall of the flask then add water until 1 L. This solution is sprayed to reveal a TLC.

FIGURE 19.3 Plant extracts.

19.2.1.2 DRAGENDORFF REAGENT (MODIFIED) (SPECIFIC FOR ALKALOIDS)

Solution A is prepared dissolving 0.85 g of bismuth subnitrate in a mixture of 10 mL of glacial acetic acid and 40 mL of water. Solution B: 8.0 g of potassium iodide are dissolved in 20 mL of distilled water. Both solutions (A and B) are mixed just before the test: 5 mL of solution A and 5 mL Solution В is added 20 mL of glacial acetic acid, add distilled water to 100 mL (Dominguez, 1988)

• 19.2.1.3 EHRLICH (FURAN RINGS)
• 30 mg p-dimethylamino benzaldehyde are dissolved in 20 mL of ethanol, this reagent is used as revealing agent in chromatography by placing it in a camera with HC1 for few minutes and compounds in TLC containing furan ring, will show purple, blue, or violet stains.

19.2.1.4 POLYOLS

Dissolve 200 mg vanillin in 50 mL of ethanol and at the time to be used is mixed with a solution of 3% perchloric acid (НСЮ4) in water, this sand sprays the plate and 3 or 4 min allowed to diy. Pale blue stains will appear changing to pink and lilac color changes to gray.

19.2.1.5 MARQUIS (ALKALOIDS)

It is a mixture of sulfuric acid and formaldehyde in a ratio of 10:1.

• 19.2.1.6 QUINONES
• 100 mg of methylene blue are dissolved in 20 mL of ethanol, acetic acid (half milliliter), and 100 mg of zinc powder. Shake closed bottle and spray chromatography plate with that solution if color stains appear in few minutes indicating the presence of quinones.
• 19.2.1.7 SESQ UITERPENLACTONES

Reagent is a mix of methanolic solution saturated with hydroxylamine, added with hydrochloric acid mixed with 2N sodium hydroxide (1:1), first spray with this solution the plate and then with a second reagent, which is prepared, mixing a solution of feme chloride (FeCl3)l% in methanol with 2N hydrochloric acid in a ratio (1:1).

19.2.1.8 SUGARS

Mixing 0.93 g of aniline with 1.66 g of o-phthalic anhydride and dissolved in 100 mL of butanol. The chromatographic plate is then sprayed and heated to 110°C, the recommended eluents for chromatography are: isopropanol-acetic acid- water in a ratio of 3:1:1 or butanol-water-acetic acid 7:2:1 (Dominguez, 1988).

• 19.2.1.9 ESSENTIAL OILS
• 30 mg of vanillin in 3 mL of ethanol then add 3 mL of water and 5 mL of concentrated sulfuric acid.
• 19.2.1.10 FLAVONOIDS
• 100 mg p-nitro aniline is dissolved in 15 mL of distilled water by adding 3 mL of hydrochloric acid and just to be used add 80 mg of sodium nitrite (for norhidroguaiaretic acid, NADG, orange or red spot appears reddish).
• 19.2.1.11 CARBONYL GROUP
• 3 g of 2,4-dinitrophenylhydrazine was solved in 30 mL of 85% phosphoric acid, heated to accelerate dissolution, dilute with 19.8 mL of absolute ethanol and filter the solution.
• 19.2.1.12 KELLER-KILL1AN1 TEST (CARDIAC GLYCOSIDES)

Solution A is prepared pouring 0.1 g of 3, 5 dinitrobenzoic acid in 10 mL of methanol, solution B: 0.5g of potassium hydroxide dissolved in 10 mL of methyl alcohol. In a test tube, we placed 20 mg of extract in methanol and added three drops of solution A and then three drops of solution В a reddish color appears (Garcia Gonzalez, 1992).

19.2.2 COLUMN CHROMATOGRAPHY

Column chromatography is a method used for separation, isolation and purification of various organic compounds in solid or liquid state.

The column for chromatography can be small or big depending on the quantity of vegetal material to be placed on it.

In column chromatography, it is very important to control the flow of solvent through the column to obtain the correct separation of fractions of the mix.

The stationary phase material must be moistened with mobile phase and packed correctly in the column to avoid empty spaces. The plant extract or sample to be separated is placed on the top of packed stationary phase. The mobile phase is added into the column over the sample. A collecting flask is placed at the bottom of column near the end to collect each fraction. The mobile phase go down through stationary phase (silica or alumina) leading the fractions elute out to be collected in a flask placed below. When the mobile phase descends, each compound of the extract travels at different speed through the stationary phase. This depends by the affinity of organic compounds with both stationary and mobile phases. The compounds of the extract more related to solvent polarity (mobile phase) descend fast and drop into the flask first. The fractions or compounds on the fraction more related to stationary phase will descend slow and reach bottom late. Column chromatography is a useful teclmique to isolate the compounds present in a mixture. Liquid chromatography using columns is a very useful procedure because almost every organic mixture can be separate using the specific polarity of the mobile phase, not only with single solvents but solvents mixed in calculated proportions.

The nonpolar or less polar compounds in a mixture will be the first compounds or fractions to drop into the collector flask, the more polar fractions or compounds are retained in the stationary phase, being necessary to use more polar solvents or mixes of two or more solvents of middle to high polarity. Retention tune is the time a compound or fraction takes to travel through the column, and is specific for each compound, under the same conditions.

Eluents and agents use in chromatography column will be selected from those with better resolution on thin layer chromatography. The separation of each fraction of the extract or mixture can be monitored by the color developed in the descent of the extract through the adsorbent, this can be observed in form of colored rings at different heights of the column. The elution process can take more time depending the amount of mixture or extract, the column diameter, the mobile phase used and even if the mobile phase drops by gravity or accelerated using nitrogen gas or vacuum (flash chromatography). Crystallization is used for the purification of the obtained compounds and the criteria to follow considering that can be done with pure solvents or mixtures of them provided that they comply with the following requirements: to be very soluble at high temperature, impurities more soluble in cold temperature than solute, solvent volatile to be removed easily from crystals, and finally does not react with the solute.