Chromatography Lab
Part A: Primary Colors
Background: In science, it is often useful to know if a material is composed of a pure substance
or if it is a mixture. Paper chromatography is a technique that is widely used to separate mixtures.
If the markers are a mixture, you can expect that as the solvent travels up the chromatography
paper, different molecules will dissolve in the solvent and travel up the paper at different rates.
This will lead to the formation of different color bands on the paper, one band for each substance
in the mixture.
Objectives: To separate colors found in markers and to calculate the Rf value of primary colors.
Materials: Filter paper, solvent, 3 beakers/ cups, markers (3 per group), pencil, ruler, calculator
Filter paper
Hypothesis (Which markers are made of a mixture of colors? What colors?)
_____________________________________________________________
_____________________________________________________________
Dependent Variable: _______________________________
Independent Variable: ______________________________
Procedure:
Pencil line
1. Measure 3cm from the bottom of the filter paper and draw a line across the width in pencil
(refer to picture above). Repeat 3 times.
2. Each group will have 3 markers: a primary color (red, yellow or blue) a secondary color
(orange, green, purple) and the dark color (black or brown).
3. On the starting line, dab one color at a time, about 10 times each. Use one color per piece
of filter paper.
4. In a beaker, add a small amount of solvent (about 1 - 2 mL).
5. Place filter paper in beaker and let sit 10 - 15 minutes or until the solvent reaches 1-2 cm
from the top of the filter paper. Do not allow the pigment to touch the solvent. While you
wait, start part B.
6. After 10-15 minutes or when the solvent is about 1 cm from the top of the paper, remove
the paper and immediately mark where the solvent traveled to with a pencil.
7. With your pencil, mark a line by each pigment.
8. Record what colors were in each of the markers that were used.
9. Measure each of the 3 primary colors in cm from the starting line to where the pigment
ended. Record in Table 2.
10. Each pigment will move a distance that is proportional to the distance moved by the
solvent. This is expressed as the Rf (Reference front) value. For example, suppose that a
pigment moves 75 mm while the solvent moves 100 mm. Then
Rf =
distance of pigment from origin
distance of solvent front from origin
Rf =
75 mm
100 mm
=
0.75
11. Calculate Rf values for each of the pigment bands you have identified and record in Table
2.
Data for Part A:
Table 1 - Colors
Color of Marker
Colors used in each marker
Table 2- Pigment Separation
Primary Color
Distance pigment
traveled (cm)
Red
Distance solvent
traveled (cm)
Yellow
Blue
Graphing- Graph each primary color based on the Rf Value.
Conclusion Questions:
1. What colors were in each marker?
2. Which primary color had the highest Rf value?
3. Rank the primary colors from highest to lowest for their Rf values.
Rf Value=
Pigment/solvent
Part B: Leaf Chromatography
Background:
In this activity, you will use paper chromatography to determine if a green leaf contains a mixture
of pigments. Four pigments are widely found in leaves: carotene, xanthophyll, chlorophyll a, and
chlorophyll b. Use the pigment key to identify what colors each pigment reflects.
Pigment Key:
Chlorophyll a= blue-green
Chlorophyll b = olive green/ yellow-green
Xanthophyll = yellow
Carotene = orange yellow
Objectives:


To separate pigments found in plants.
To calculate the Rf value of plant pigments
Hypothesis (If we use chromatography paper to separate plant pigments, what will the paper
look like? Are plant made of multiple pigments?): ______________________________________
_______________________________________________________________________________
Dependent Variable: _______________________________
Independent Variable: ______________________________
Materials: Filter paper, solvent, 1 beaker/ cup, spinach leaf, pencil, ruler, calculator, coin
Procedure:
1. Measure 3cm from the bottom of the filter paper and draw a line across the width in pencil.
2. In a beaker, add a small amount of solvent (about 1 - 2 mL.)
3. Use a coin to extract the pigments from spinach leaf cells by placing a small section of leaf
on the top of the pencil line. Use the ribbed edge of the coin to crush the leaf cells. Be sure
the pigment line is on top of the pencil line. Use a back and forth movement exerting firm
pressure throughout (refer to picture above).
4. Place paper in beaker and let sit 10 - 15 minutes or until the solvent reaches 1-2 cm from
the top of the filter paper. Do not allow the pigment to touch the solvent.
5. After 10-15 minutes or when the solvent is about 1 cm from the top of the paper, remove
the paper and immediately mark where the solvent traveled to with a pencil.
6. With your pencil, mark a line by each pigment.
7. Measure the distance each pigment migrated from the bottom of the pigment origin to the
bottom of the colored band and record in Table 3. You should be able to observe 4
pigment bands.
Data for Part B:
Table 3: Separation of Leaf Pigments
Color
Name of Pigment Distance
(use pigment key) pigment
traveled (cm)
Distance
solvent traveled
(cm)
Rf Value=
Pigment/solvent
Example of Chromatography:
Analysis Questions:
1. Why is chromatography useful?
2. What would the chromatography paper look like if the leaf pigment was not a mixture of
multiple pigments?
3. Why are most plants green (be specific)?
4. Why do leaves change colors in the fall?
5. Draw, label (solvent and all pigments) and color what your filter paper looks like.