تويت
أفيدوني بالإجابة على الأسئلة ضروري
Experiment
Separation of Plant Pigments
Purpose: To investigate the separation of plant pigments in spinach leaves by chromatography.
Discussion:
Chemists resort to many techniques to isolate pure substances from natural products. In this
experiment the method of separation by chromatography is employed. The solvent moving
through a piece of paper carries the soluble colored substances. Some of the substances bind
more strongly to the paper than others. The strongly bound substances are not carried as far as
the weakly bound substances: the result is a separation of the compounds. In spinach leaves
the pigmented substances are: chlorophyll a (blue green), chlorophyll b (olive green),
xanthophylls (yellow brown), and carotene (yellow orange).
Procedure:
I. Extraction of Pigment
A. With a mortar and pestle grind 4 clean dry spinach leaves with 10 mL of 95% ethanol
and a pinch of fine sand. After 5 minutes of grinding, add 10 more mL of 95% ethanol,
mix will, and allow the mixture to stand for 10 minutes.
B. Filter the contents of the mortar with a wad of glass wool in a funnel. Squeeze the glass
wool by pressing it against the funnel with a spatula. If the filtrate (liquid) is not clear,
filter it again.
II. Chromtographic Separation
STOPPER
T-hook
TEST TUBE
PAPER (Stationary
phase)
SPOT (Substance to
be separated)
SOLVENT (Mobile
phase)
A. Prepare four strips of chromatography paper as shown in the diagram.
B. Mark a dot very lightly with pencil 3 cm from the bottom of the first piece of
chromatography paper.
Place a tiny spot of spinach extract on the pencil mark by dipping a capillary tube into
the spinach extract and then touching the capillary tube to the pencil mark on the first
strip of chromatography paper.
Let the spot dry, then apply another tiny drop of spinach extract to
the same spot.
Repeat until the spots have been added 4 times.
Repeat the same procedure as above to the 2nd piece of chromatography paper.
To the 3rd piece of chromatography paper mark with a dot of permanent ink 3 cm from
the bottom of paper.
To the 4th piece of chromatography paper mark with a dot of washable ink 3 cm from the
bottom of paper.
C. To a large test tube add the solvent (10% acetone – 90% petroleum ether) to
approximately 2 cm in depth.
Hook the top of the first chromatography paper to the T hook in the stopper.
Place the paper into the test tube being careful that the spot does not touch the liquid.
The spot must be above the surface of the liquid.
Replace the stopper, taking care not to tilt the test tube.
The liquid should not wash over the spot.
D. Observe the paper periodically. Take care that the solvent front does not travel to the
top of the paper.
Remove the paper before the solvent reaches the top,.
Draw a line along the top of the solvent front.
Air dry the paper.
Since the spots fade quickly, draw around the spots as soon as the paper is dry.
E. Repeat the above procedure with the 2nd piece of chromatography paper.
F. Repeat the above procedure with the 3rd and 4th piece of chromatography paper.
However, since the ink does not fade, you will not have to draw a circle around the spots
after the paper is dry.
G. Staple the chromatograms to the front of your laboratory report in the space provided.
For the 1st and 2nd chromatogram, those with the spinach, identify each of the colored
spots.
H. For the 1st and 2nd chromatogram measure the distance traveled by each spot and
calculate the average distance and Rf value for each spot.
Chemistry 12A Name ____________________
REPORT SHEET
SEPARATION of PLANT PIGMENTS
Identify each of the colored spots by writing the names of the pigmented substances on the
chromatogram and staple the chromatogram below:
In chromatography, the solvent always moves as fast or faster than the substances being eluted.
For comparison of various substances, chemists frequently calculate the Rf value for each substance.
The Rf value simply expresses the distance a component travels as a fraction of the total distance
traveled by the solvent:
Rf = distance traveled by the substance
distance moved by the solvent
Thus, if a substance moves 5.0 cm, while the solvent front moves 20.0 cm, the Rf would be 0.25:
Rf = 5.0 cm = 0.25
20.0 cm
Data obtained form Chromatogram:
Distance traveled by:
spot 1
spot 2
average
solvent front
chlorophyll a
chlorophyll b
xanthophylls
carotene
Calculation of Rf values:
chlorophyll a
chlorophyll b
xanthophyll
carotene
QUESTIONS:
1. What is the mobile phase in the chromatography carried out in this experiment?
2. What is the stationary phase in this experiment?
3. What are the substances being separated?
4. Why must a pencil rather than a pen be used for marking the chromatogram?
5. Why must the container be covered during chromatography?
6. Why must the solvent level be below the spot of solution when the chromatogram is placed
in the elution jar?
7. Why must the elution of the solutions be stopped before the solvent front rises to the top of
the paper?
Separation of Plant Pigments
Purpose: To investigate the separation of plant pigments in spinach leaves by chromatography.
Discussion:
Chemists resort to many techniques to isolate pure substances from natural products. In this
experiment the method of separation by chromatography is employed. The solvent moving
through a piece of paper carries the soluble colored substances. Some of the substances bind
more strongly to the paper than others. The strongly bound substances are not carried as far as
the weakly bound substances: the result is a separation of the compounds. In spinach leaves
the pigmented substances are: chlorophyll a (blue green), chlorophyll b (olive green),
xanthophylls (yellow brown), and carotene (yellow orange).
Procedure:
I. Extraction of Pigment
A. With a mortar and pestle grind 4 clean dry spinach leaves with 10 mL of 95% ethanol
and a pinch of fine sand. After 5 minutes of grinding, add 10 more mL of 95% ethanol,
mix will, and allow the mixture to stand for 10 minutes.
B. Filter the contents of the mortar with a wad of glass wool in a funnel. Squeeze the glass
wool by pressing it against the funnel with a spatula. If the filtrate (liquid) is not clear,
filter it again.
II. Chromtographic Separation
STOPPER
T-hook
TEST TUBE
PAPER (Stationary
phase)
SPOT (Substance to
be separated)
SOLVENT (Mobile
phase)
A. Prepare four strips of chromatography paper as shown in the diagram.
B. Mark a dot very lightly with pencil 3 cm from the bottom of the first piece of
chromatography paper.
Place a tiny spot of spinach extract on the pencil mark by dipping a capillary tube into
the spinach extract and then touching the capillary tube to the pencil mark on the first
strip of chromatography paper.
Let the spot dry, then apply another tiny drop of spinach extract to
the same spot.
Repeat until the spots have been added 4 times.
Repeat the same procedure as above to the 2nd piece of chromatography paper.
To the 3rd piece of chromatography paper mark with a dot of permanent ink 3 cm from
the bottom of paper.
To the 4th piece of chromatography paper mark with a dot of washable ink 3 cm from the
bottom of paper.
C. To a large test tube add the solvent (10% acetone – 90% petroleum ether) to
approximately 2 cm in depth.
Hook the top of the first chromatography paper to the T hook in the stopper.
Place the paper into the test tube being careful that the spot does not touch the liquid.
The spot must be above the surface of the liquid.
Replace the stopper, taking care not to tilt the test tube.
The liquid should not wash over the spot.
D. Observe the paper periodically. Take care that the solvent front does not travel to the
top of the paper.
Remove the paper before the solvent reaches the top,.
Draw a line along the top of the solvent front.
Air dry the paper.
Since the spots fade quickly, draw around the spots as soon as the paper is dry.
E. Repeat the above procedure with the 2nd piece of chromatography paper.
F. Repeat the above procedure with the 3rd and 4th piece of chromatography paper.
However, since the ink does not fade, you will not have to draw a circle around the spots
after the paper is dry.
G. Staple the chromatograms to the front of your laboratory report in the space provided.
For the 1st and 2nd chromatogram, those with the spinach, identify each of the colored
spots.
H. For the 1st and 2nd chromatogram measure the distance traveled by each spot and
calculate the average distance and Rf value for each spot.
Chemistry 12A Name ____________________
REPORT SHEET
SEPARATION of PLANT PIGMENTS
Identify each of the colored spots by writing the names of the pigmented substances on the
chromatogram and staple the chromatogram below:
In chromatography, the solvent always moves as fast or faster than the substances being eluted.
For comparison of various substances, chemists frequently calculate the Rf value for each substance.
The Rf value simply expresses the distance a component travels as a fraction of the total distance
traveled by the solvent:
Rf = distance traveled by the substance
distance moved by the solvent
Thus, if a substance moves 5.0 cm, while the solvent front moves 20.0 cm, the Rf would be 0.25:
Rf = 5.0 cm = 0.25
20.0 cm
Data obtained form Chromatogram:
Distance traveled by:
spot 1
spot 2
average
solvent front
chlorophyll a
chlorophyll b
xanthophylls
carotene
Calculation of Rf values:
chlorophyll a
chlorophyll b
xanthophyll
carotene
QUESTIONS:
1. What is the mobile phase in the chromatography carried out in this experiment?
2. What is the stationary phase in this experiment?
3. What are the substances being separated?
4. Why must a pencil rather than a pen be used for marking the chromatogram?
5. Why must the container be covered during chromatography?
6. Why must the solvent level be below the spot of solution when the chromatogram is placed
in the elution jar?
7. Why must the elution of the solutions be stopped before the solvent front rises to the top of
the paper?