PHYSICAL AND CHEMICAL METHODS OF EVALUATION FOODS

EXTRACTION AND SEPARATION OF PIGMENTS to Top

Vegetables derive their color from pigments inherent in the plant. The types and proportions of the different pigments are the primary determinants of the vegetable's color. The objective of this experiment is to use paper chromatography to e,.tamine the different pigments in three leafy vegetables of varying depths of green color. The leafy vegetables that will be used are spinach (dark green), leaf lettuce (medium green), and Belqian endive (light green).

Materials: spinach 1(-)(-) ml petroleum ether leaf lettuce 5 ml n-bllkriol Belqian endive ml acetone 6 -test tubes --7 6" test tubes stoppers for test tubes Q'@as-@ stirring rod scissors Whatman #1 filter paper melting point tube straight pins medicine dropper Volumetric flask.

Methods:

Extracting

1. Cut a piece of green leaf approximately (5 cm) square. Drop it into a small amount of boiling water and boil for minutes. Remove leaf from the water and pat it dry between pieces of papertoweling.

2. Combine 7 ml of petroleum ether and 1(-) ml of acetone. With scissors shred the leaf into the test tube and mak:e sure that the pieces are covered with solvent.

4. Let stand a few minutes until most of the pigments are dissolved and the leaf has lost most of its color. Stir once or twice with the glass stir-ring rod.

5. Decant the liquid into another test tube. Add 1(-) ml of Water, stopper, and invert the test tube several times. Let stand until 2 phases separate.

6. With a medicine dropper transfer the upper dark green layer to a clean test tube. Add I(D ml of water, stopper, and invert the test tube Several times- Again let stand until the phases separate.

7. Repeat the washing as in step 6. The pigments are now in the Petroleum ether phase and water has removed most of the acetone. 8. If the Pigments do not separate on the chroaiatogram, repeat the washing procedure.

B.Chromatographic Separation of Pigments

1. Prepare the developing fluid. Pipette 1 ml of n-butanol into a 10(-) ml volumetric flas@@. and Ma@@e to Volume with petroleum ether.CAUTION: Keep away from flame!

Four aliquots of the developer into a 6" (15 cm) test tube toa depth of 1/@@" (I.?- cm). Stopper with cor@.-s.

2. Cut several strips 6" (15 cm) long by (l.'-? cm) wide from Whatman #1 filter paper. Mal::e a -fold 1/2" (1.@@ cm) from one end of each strip, and with a pencil place a dot in the center and 1/@-" cm) from the other end of the strip.

4. With a melting point tube apply a small drop of the petroleum ether extract of the pigments to the filter paper strip at the position mar@:-.L-d by the dot. Keep the spot small (@-,/16" or 0.4 cm in diameter) . Mak-:e 4 to 8 applications. Practice spotting with a scrap of filter paper.

5. With a straight pin attach the strip of filter paper at the center of the crease to the bottom of the cor@::. Mal.-.e Sure the strip h@inqs straight.

6. Lower the strip into one of the test tubes which contains developer. The developing fluid should reach the end of the filter strip, but it should not touch the spot of pigments.

7. Let the chromatoqram develop until the pigments are separated.

8. Appearinq on the chromatoqrams of extracts from most green leaves are carotenes which move with the solvent front. Blue-green chlorophyll a moves ahead of the olive green chlorophyll b. A faint grey band of pheophytin a may be observed in front of chlorophyll a. One or two spots or bands of yellow .,anthophylls may appear on the chromatoqram, too.

REFERENCES to Top

From Charley, Helen, 198@@, "Food Study Manual," --7rd Ed., John Wiley and Sons, New York.

´ ´ Updated: Wednesday, October 31, 2007. ´ Oregon State University.
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