Chromatography06

8/7/2019 Chromatography06

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Extraction and Fractionation of Plant Pigments from Spinach Leaves

When we see a green leaf from a plant what we actually see is light reflected off the surface of the leaf, specifically, the green wavelengths of light. During Autumn, or

when leaves begin to die, we see different colors from the same leaves. Actually, leaves

contain many different pigments – the compounds that absorb and reflect differentwavelengths of light. In lab today we will extract the pigments from leaves of spinach,

then separate the different pigments that are present using a process called paper chromatography. Then, based on the separation of pigments, we will try to distinguishexactly which pigments are present.

A few hints for today’s lab:

The solvent we will use to separate the pigments is petroleum ether:acetone. Thissolvent is very hydrophobic, so any pigments that are very “greasy” or

hydrophobic themselves will interact with the solvent (move with it). The more

hydrophilic pigments will not react well with the solvents, but will react well with

the chromatography paper (not move well with the solvent). Handle all thesolvents carefully.

The chromatography paper itself is very polar. It is made of cellulose, which

contains sugars with –OH groups that form hydrogen bonds with oxygen atoms of

the pigments.

EXTRACTION OF PIGMENTS

1. Obtain about 2-3 nice dark green spinach leaves (or coleus, or beet leaves). Remove

and discard the major veins of the leaf and save the rest of the tissue.2. Use a razor or scissors to cut the leaf into tiny pieces. Add the pieces to a chilled

mortar and pour about 15ml of ice-cold acetone onto the leaf pieces. Add a pinch of sand to the mortar. You will find these materials in the freezer .3. Grind this mixture with the pestle for about a minute.

4. Pour the ground-up contents of the mortar into a 50 ml Erlenmeyer flask, label the

flask, and affix the stopper.5. Swirl the flask vigorously for 30 seconds or so, then place the flask into the

refrigerator for 10 minutes. Allow the pulp to settle.

6. While you are waiting , prepare the paper and jars for the rest of the experiment as

described below. (Step 1 of PAPER CHROMATOGRAPHY)7. Retrieve your flask from the fridge. Don’t disturb the pulp at the bottom of the

flask .

PAPER CHROMATOGRAPHY

1. Use the large jars as chromatography tanks. Place a small piece of filter paper into the

jar so that the paper rests against the wall of the jar. This small piece of paper will helpsaturate the atmosphere of the jar with vapors from the solvent. Gently pour petroleum

ether:acetone solvent into the jar to a depth of about 1 cm. Cover the jar tightly with foil.

2. Obtain a long sheet of chromatography paper. Do not handle the paper with your

fingers. Oils from your skin will alter how the solvent reacts with the paper and affect



your results. One member of your team should wear a pair of latex gloves to handle the

paper.

3. Use a fine pencil to mark a thin light pencil line across the wide dimension of the paper 2cm from the bottom. Your pigment extract will be applied along this pencil line.

4. Dip the end of a glass capillary tube into the pigment solution in the flask and quickly

streak the capillary tube across the paper along the pencil line. Leave about a 1cm sidemargin on the edges of the paper where no pigment is applied. Your instructor will

demonstrate this technique for you.

Allow the solvent to dry completely before reapplying more extract. 5. Repeat streaking the paper with extract until you have a dark green line on your paper.

This may take 6-10 applications. Take your time – the narrower your line of pigment extract the better results you will get.

6. Let the paper dry completely, then curl it into a tube and staple the edges at each endto form a cylinder. Do not handle with your fingers, and do not overlap the ends

when you staple them together. Make sure the pigment line is at the bottom end of the

cylinder, and is visible.

7. Get your jar, remove the small strip of paper, and place your cylinder into the jar, withthe pigment end of the tube in the solvent. Cover tightly with foil right away. Do not

move the jar once the paper has been placed into the solvent.8. Allow the pigments to separate for 10-15 minutes, or until there is a clean

separation of four to five bands of different pigments. Let the solvent run until it is

about 1cm from the top of the paper. Do not let the solvent run off the top of the

paper. When the solvent has reached 1cm from the top remove the paper from the jar and immediately mark the solvent front with a pencil. Allow the paper to dry

completely.

9. Once dry, delineate each of the colored bands with a pencil, and record the color andthe distance each band moved from the starting line or origin (the pencil line you drew at

the beginning). Describe the colors as specifically as you can (olive green, grass green,

yellow orange, etc). Record the distance of the solvent front from the origin.10. Calculate the R f values for each of your pigments

R f = distance traveled by pigment / distance traveled by solvent

DATA ANALYSIS

For each of your pigments you have an R f value and a description of the color of

each band. Pigments may be identified by their color and the R f values.Chlorophyll a is blue green

Chlorophyll b is olive-green

Beta-carotene is yellow-orangeVioloxanthin is yellow

Lutein is yellow

(Violoxanthin and lutein are both xanthophylls pigments and might overlap)



1. Use your chromatogram to fill in the following table:

Pigment / solvent band Distance

traveled (cm)

R f

2. What do the R f values indicate about the relative attraction of the pigments to the more

polar paper or the more non-polar (hydrophobic) solvent? Review the introduction to this

lab if necessary…

3. From your chromatogram, what is the order in which the pigments separated?

Chlorophyll a _____

Chlorophyll b _____

Beta-carotene _____ Lutein _____

Violoxanthin _____

4. Is each band one pure pigment? Explain.

HAND IN THIS SHEET AT THE END OF LAB WITH YOUR CHROMATOGRAM.ONE SHEET PER GROUP.

NAMES

_____________________________ ___________________________

_____________________________ ___________________________

_____________________________ ___________________________

Chromatography06

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