Lab11. 1 Principles of Chromatography
Background
Color additives are an integral part of our lives. From foods and drinks to cosmetics, pharmaceuticals, and toiletries,
color additives make products appetizing or appealing. When we look at yellow candy, we think lemon. When we see
red, we visualize and taste strawberry or cherry. The color orange reminds us of the fruit, and green summons the taste
of lime or mint. These colors enhance the taste experience with artificial flavorings that have no natural color. The Food
and Drug Administration (FDA) strictly regulates the dyes that go into our foods, drugs, and cosmetics. Once approved,
they are given names and numbers. These names begin with FD&C, which is an abbreviation for the Food, Drug, and
Cosmetic Act of 1938 that gives the FDA the power to oversee safety in these areas. FD&C Yellow No. 5, known as
tartrazine, is the only dye that manufacturers are required to indicate on labels. In a small percentage of people it causes
an allergic reaction.
An analysis of a mixture requires that the components be separated and studied individually. With many mixtures,
one of the most effective methods of separating components is to partition them between mobile and stationary phases
in a process known as chromatography.
Two methods are paper chromatography and thin-layer chromatography, or TLC. In each of these techniques, there
is a mobile solvent phase and a stationary solid phase (paper or silica gel-coated plate). As the solvent migrates up the
stationary phase via capillary action, it comes in contact with an applied dot of a sample to be analyzed. The solvent
dissolves the dot into the mobile phase which continues to move up the stationary phase. If the sample contains
multiple chemicals, they separate along the way, due to differences in polarity. Chromatography paper and the silica gel
on the TLC plates are highly polar. The more polar of the dissolved substances are more strongly attracted by the
stationary phase, and they migrate shorter distances before attaching to the surface. Nonpolar substances remain
longer in the mobile phase and travel farther before attaching. The attachment of a sample’s components to the surface
of the stationary phase is called adsorption.
For effective separations that use volatile solvents, the chromatography chamber, or developing chamber, must be
covered to create a saturated atmosphere of mobile phase vapors. Evaporation of the more volatile component of a
developing solvent can change the solvent polarity and adversely affect the separation of all the components in the
mobile phase.
One quantitative measurement used to identify a given chemical absorbed on the stationary phase is the retention
factor, Rf. This calculation is a ratio of the distance that the substance moves up the stationary phase to the distance that
the mobile phase (solvent front) travels.
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑠
𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
The Rf value is an identifying characteristics for a given substance when the same solvent system, concentration,
and type of paper or TLC plate are used.
Pre-laboratory Questions
1. What is chromatography?
2. Calculate the Rf value of a substance that traveled 5.2 cm on a TLC plate while the solvent front travelled 8.1 cm.
3. Does each spot on the developed chromatography paper or plate represent one substance?
4. In a TLC experiment, three pigments A, B, and C, were dissolved in a solvent. A is slightly polar, B is highly polar,
and C is moderately polar. List in order how these will appear on the surface of the paper or plate.
5. Read the laboratory procedures. Diagram the procedures using illustrations and/or a flow chart to describe the steps.
Ensure that your diagrams are labeled with quantities and descriptions.
Materials
For each group:
2 Dropping pipets
3 Polystyrene test tubes
6 Straight pins
Test tube Rack
2 1” Squares of Parafilm
3 Chromatography paper strips
Scissors
7 Capillary tubes 10ml graduated cylinder
2 TLC Chromatography plates
Metric Ruler
Plastic forceps
Paper Towels
Transparent Tape
Chemical Station (Shared by all groups):
FD&C Yellow No. 5 food coloring
FD&C Red No. 40 food coloring
FD&C Blue No. 1 food coloring
Strawberry Kool-Aid solution
Grape Kool-Aid solution
Orange Kool-Aid solution
100 mL of 0.1% aqueous sodium chloride Chromatography Solvent
Chlorophyll Extract
Procedure
Part 1: Paper Chromatography
1A: Separating Known FD&C Dyes
1. Obtain six strips of chromatography paper. At 1.5 cm from the bottom of each strip, draw a line with a pencil
across the paper. This line will be the starting point or origin for each applied dye.
2. On a paper towel, orient three strips parallel to each other with the pencil lines at the bottom.
3. At the chemical station, insert a capillary tube into the bottle of Red No. 40 dye and place your forefinger
over the top end of the tube to retain a small column of dye (3-4 mm).
4. On the first strip, place one small dot of the dye in the center of the pencil line by touching the paper
and briefly releasing your finger from the top of the capillary tube.
5. In the same manner, use new capillary tubes to collect and apply Blue No. 1 to the second strip
and Yellow No. 5 to the third.
6. Place six test tubes in a test tube rack.
7. Fill your graduated cylinder with 10ml of 0.1% aqueous sodium chloride.
8. Fill the NaCl pipet with 0.1% NaCl solution from your graduated cylinder and fill each test tube with 1 mL
of solution (approximately 20 – 25 drops)
9. Insert a straight pin into the center of the chromatography strip around 5 mm from the top.
This will serve as a hanger for the strip when it is placed into the test tube.
10. Carefully lower each of the strips with the applied dyes down the center of each test tube and into the solvent.
Each strip should be in the center of the tube, not touching the walls. Adjust the strip’s position by moving
the pin side to side. The spot should be above the surface of the solvent.
11. Allow the solvent front to wick up the paper and through the spot until the front reaches within a centimeter
of the top. This should take around 10-15 minutes. While your lab group waits to finish steps 12 – 17,
start section 1B.
12. Remove each of the strips, remove the pins, place the strips on a paper towel, and mark the solvent front
with a pencil before it dries.
13. With a pencil, mark the middle of the colored spots that were deposited above the origin.
14. Place the strips parallel to each other on a sheet of white paper and tape them down with transparent tape.
Label each strip with its dye name at the bottom.
15. Measure the distance the solvent front traveled. All measurements should be measured in millimeters.
Record the distance in the data table.
16. Measure the distance each colored spot moved, from the origin to the mark in the middle of the spot.
All measurements should be measured in millimeters. Record the distance in the data table.
17. Calculate the Rf value for each of the spots and record the values in the data table.
1B: Separating Unknown Kool-Aid Dyes
1. Orient the other three strips of chromatography paper parallel to each other on a paper towel
with the drawn lines at the bottom.
2. At the chemical station, insert a new capillary tube into the bottle of strawberry Kool-Aid
and place your forefinger over the top end of the tube to retain a small column of dye.
3. On the first strip, place one small dot of the dye in the center of the line by touching the paper and
briefly releasing your finger from the top of the capillary tube. Allow the dot to dry. Repeat this application
two more times, allowing the dot to dry a few seconds between reapplications.
4. Repeat steps 2 and 3 above for orange Kool-Aid on the second strip and grape Kool-Aid on the third strip,
using a new capillary tube for each type of Kool- Aid.
5. Insert a straight pin into the center of the chromatography strip around 5 mm from the top.
6. Carefully lower each of the strips with the applied dyes down the center of each test tube and into the solvent.
Do not allow the tubes to touch the walls. Adjust their position if you need to by moving the pin side to side.
7. After the solvent has climbed to within a centimeter of the top, remove the strips, remove the pins,
place the strips on a paper towel to dry.
8. Mark the solvent front with a pencil and mark the middle of each colored spot with a pencil.
9. Tape the three chromatography strips to the right of the known FD&C dye strips on the white paper.
10. Measure the distance the solvent front moved from the origin for each strip. Record in the data table.
11. Measure the distance each spot moved. Since each Kool-Aid flavor may contain more than one dye,
measure the distance each color has moved. Each Kool-Aid flavor has its own data table. Three rows are
provided in the data tables, one for each possible color.
12. Calculate and record the Rf value for each of the colors.
13. Identify the dye in each of the Kool-Aid samples. Record their identity in the data table.
14. Empty the solvent from the six tubes, rinse them with water, and allow five to dry. Fill the sixth with water
and leave it on your test tube rack.
15. Clean up your station according to your teacher’s instruction. Rinse the capillary tubes carefully. Allow them
to soak in test tube containing water.
Day 2
Part 2: Thin-Layer Chromatography
Plant Pigments in Chlorophyll Extract
1. Use a clean dropping pipet. Barely depress the bulb and fill only the stem (up to
the base of the bulb) with the chromatography solvent at the chemical station.
2. Add 35 drops of solvent to the bottom of a clean, dry test tube and place it in
the test tube rack. Cover the test tube with a square of Parafilm.
3. Repeat steps 1 and 2 for the second test tube.
4. Pick up a TLC plate, touching only the edges. With a pair of scissors,
trim 2 mm off one end. If you set the plate on the counter,
set it gel-side up. The gel-side is white, the other side is glass.
5. On the opposite end, measure 1.5 cm from the bottom and place a light pencil mark on the right edge of
the silicon dioxide gel. This will be your origin for the dye. Place this TLC plate on a paper towel.
6. Repeat steps 4 and 5 for the second TLC plate.
7. Insert the last of your capillary tubes into the chlorophyll bottle at the chemical station.
Obtain a 3 – 4mm column of liquid and place your forefinger over the top of the capillary tube.
8. On the first TLC plate, directly to the left of the pencil mark, place a small dot of the dye in the center of the gel
by briefly releasing your finger from the top of the capillary tube. Allow the dot to dry.
Repeat this application again and allow it to dry.
9. Repeat steps 7-8 for the second TLC plat.
10. Remove the Parafilm covers from both test tubes and lower the marked ends of the TLC plates into the tubes.
Reseal both tubes with the Parafilm and place them in the test tube rack.
In each tube, the solvent will wick up the plate and encounter the dot of dye.
11. Wait for the solvent to travel up the plate until it is 1 cm of the top. This may take 30 minutes.
Remove the plates and place them gel side up on a paper towel.
12. Quickly mark the solvent front on the top right edge of the plate with a pencil and indicate the middle
of each colored spot(s) with a light pencil mark on the right edge.
13. Measure the distance the solvent front moved from the origin for each plate and
record it in the data table for plate 1 and 2.
14. Measure the distance each spot moved from the origin to the middle of each spot and record it in the data table.
15. Calculate the Rf value for each pigment and record it in the data table.
16. Average the Rf values and record the figures in the data table.
17. Pigments found in plant extracts include chlorophyll a (light green), chlorophyll b (dark green),
carotene (orange), xanthophylls (yellow), and anthocyanin (red or blue).
Identify the pigments on your chromatogram and record them in the data table.
Data Table 1A: FD&C standard Dyes
FD&C Dye
Spot Color
Distance Solvent Distance Spot
Rf
Traveled (mm)
Traveled (mm)
Red No. 40
Blue No. 1
Yellow No. 5
Data Table 1B: Unknown Mixes: Koolaid Dyes
Strawberry
Spot Spot Color Distance Solvent
Distance Spot
Rf
Identity
Traveled (mm)
Traveled (mm)
1
2
3
Orange
Spot Spot Color Distance Solvent
Distance Spot
Rf
Identity
Traveled (mm)
Traveled (mm)
1
2
3
Grape
Spot Spot Color Distance Solvent
Distance Spot
Rf
Identity
Traveled (mm)
Traveled (mm)
1
2
3
Data Table 2: Plant Pigments in Chlorophyll Extract
Spot Spot
Solvent Solvent Spot
Spot
Rf
Rf
Rf
Identity
Color
Front
Front
Distance Distance Plate1 Plate2 Ave.
(mm)
(mm)
(mm)
(mm)
Plate 1 Plate 2 Plate 1
Plate 1
1
2
3
4
Laboratory Questions
1. Why must the line of origin be drawn in pencil and not ink?
2. Explain why an Rf value is a dimensionless number.
3. Why is it better to allow the solvent front to move almost to the top of a paper strip or TLC plate rather than
stopping it at the halfway point during development?
4. A student calculated an Rf value to be 1.5. Explain why this value is not possible.
5. What would happen if the sample spot on a chromatography strip or TLC plate were placed below
the solvent surface in the developing chamber?
6. How would the separation of pigments be affected on a TLC plate with a volatile mobile phase solvent
if the developing chamber were left uncovered.
7. What are the component colors for strawberry, orange, and grape Kool-Aid?
8. List the three FD&C dyes you examined in order of increasing polarity, as indicated by your chromatogram.
9. Major pigments in plants include chlorophyll a (light green), chlorophyll b (dark green), carotene (orange),
xanthophylls (yellow), and anthocyanin (red or blue).
Which of these pigments did you detect in your chromatogram of the Chlorophyll Extract?
10. Which of the pigments in the Chlorophyll Extract is the least polar?
Which of the pigments in the Chlorophyll Extract is the most polar?
(Template)
Lab11. 1 Principles of Chromatography
Pre-laboratory Questions
1. What is chromatography?
2. Calculate the Rf value of a substance that traveled 5.2 cm on a TLC plate while the solvent front travelled 8.1 cm.
3. Does each spot on the developed chromatography paper or plate represent one substance?
4. In a TLC experiment, three pigments A, B, and C, were dissolved in a solvent. A is slightly polar, B is highly polar,
and C is moderately polar. List in order how these will appear on the surface of the paper or plate.
5. Read the laboratory procedures. Diagram the procedures using illustrations and/or a flow chart to describe the steps.
Ensure that your diagrams are labeled with quantities and descriptions.
Objectives:
Procedure:
Data Table 1A: FD&C standard Dyes
FD&C Dye
Spot Color
Distance Solvent Distance Spot
Rf
Traveled (mm)
Traveled (mm)
Red No. 40
Blue No. 1
Yellow No. 5
Data Table 1B: Unknown Mixes: Koolaid Dyes
Strawberry
Spot Color Distance Solvent
Distance Spot
Rf
Identity
Traveled (mm)
Traveled (mm)
Spot Color
Orange
Distance Solvent
Distance Spot
Traveled (mm)
Traveled (mm)
Rf
Identity
Spot Color
Grape
Distance Solvent
Distance Spot
Traveled (mm)
Traveled (mm)
Rf
Identity
Data Table 2: Plant Pigments in Chlorophyll Extract
Spot No.
Spot
Solvent Solvent Spot
Spot
Rf
Rf
Rf
Identity
from Origin
Color
Front
Front
Distance Distance Plate1 Plate2 Ave.
(mm)
(mm)
(mm)
(mm)
Plate 1 Plate 2 Plate 1
Plate 1
1
2
3
4
Laboratory Questions
1. Why must the line of origin be drawn in pencil and not ink?
2. Explain why an Rf value is a dimensionless number.
3. Why is it better to allow the solvent front to move almost to the top of a paper strip or TLC plate rather than
stopping it at the halfway point during development?
4. A student calculated an Rf value to be 1.5. Explain why this value is not possible.
5. What would happen if the sample spot on a chromatography strip or TLC plate were placed below
the solvent surface in the developing chamber?
6. How would the separation of pigments be affected on a TLC plate with a volatile mobile phase solvent
if the developing chamber were left uncovered.
7. What are the component colors for strawberry, orange, and grape Kool-Aid?
8. List the three FD&C dyes you examined in order of increasing polarity, as indicated by your chromatogram.
9. Major pigments in plants include chlorophyll a (light green), chlorophyll b (dark green), carotene (orange),
xanthophylls (yellow), and anthocyanin (red or blue).
Which of these pigments did you detect in your chromatogram of the Chlorophyll Extract?
10. Which of the pigments in the Chlorophyll Extract is the least polar?
Which of the pigments in the Chlorophyll Extract is the most polar?