Catalog No. AP7661
Publication No. 7661
Separation of a Dye Mixture
Using Chromatography
AP* Chemistry Big Idea 2, Investigation 5
An Advanced Inquiry Lab
Introduction
The entire palette of artificial food colors is derived from just seven dyes certified by the FDA for use in food, drugs, and
cosmetics. How can these FD&C dyes be identified in a mixture? How do the molecular structures of the dye molecules influence
their properties, including their relative solubility or affinity for different solvents?
Concepts
• Chromatography
• Polarity
• Rf values
• Intermolecular forces
• Food chemistry
Background
The use of color additives increased dramatically in the United States in the second half of the nineteenth century. As the
economy became more industrial, demographics shifted, fewer people lived on farms, and city populations grew. People became
more dependent on mass-produced foods.
Color additives were initially used to make food more visually appealing to the consumer and, in some cases, to mask poorquality, inferior or imitation foods. For example, meat was colored to appear fresh long after it would have naturally turned
brown. Jams and jellies were colored to give the impression of higher fruit content than they actually contained. Some food was
colored to look like something else—imitation crab meat, for example. Many of the food colorings and additives were later discovered to be harmful or toxic.
In 1883, the United States Department of Agriculture (USDA) Bureau of Chemistry began regulating the food industry
to help ensure a safe food supply. Food coloring regulation is just one example of the agency’s efforts. Food colorants were
being added to food with little or no health testing. To propagate the food safety effort, in 1906 the USDA hired a consultant,
Dr. Bernard Hesse, to determine colorants that would be safe to consume in food. In 1907, the number of synthetic food dyes
approved for use in the United States was reduced from 695 to just seven. As additional data was collected through consumer
reports and laboratory testing, more dyes were eliminated or restricted. Only two of the original dyes from 1907 are still accepted
for use today. Five others were added between 1907 and 1971. In total, only seven dyes color all U.S. food today. All of the
FD&C-approved food dyes are charged, water-soluble organic compounds that bind to natural ionic and polar sites in large food
molecules, including proteins and carbohydrates.
Chromatography is one of the most useful methods of separating and purifying organic compounds. There are many different
types of chromatography but most depend on the principle of adsorption. The two important components of chro­matography are
the adsorbent and the eluent. Adsorbents are usually solid materials that will attract and adsorb the materials to be separated. The
eluent is the solvent, which carries the materials to be separated through the adsorbent.
Chromatography works on the concept that the compounds to be separated are slightly soluble in the eluent and will spend
some of the time in the eluent (or solvent) and some of the time on the adsorbent. When the components of a mixture have varying
affinities for the eluent, they can then be separated from one another. The polarity of the molecules to be separated and the polar­
ity of the eluent are very important. Changing the polarity of the eluent will only slightly affect the solubility of the molecules but
may greatly change the relative attraction for the adsorbent. Affinity of a substance for the eluent versus the adsorbent allows molecules to be separated by chromatography.
*AP is a registered trademark of the College Board, which was not involved in the production of, and does not endorse, this product.
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IN7661
121613
Paper chromatography is often used as a simple separation technique. In paper chromatography, the adsorbent is the paper
itself, while the eluent can be any number of solvents. When the paper is placed in a chromatography chamber, the eluent moves
up the strip by capillary action. Organic molecules that are “spotted” onto the paper strip separate as they are carried with the eluent at different rates. Those molecules that have a polarity closest to the polarity of the eluent will move up the strip the fastest.
The choice of the eluent is the most difficult task in chromatography. Choosing the right polarity is critical because this determines the level of separation that will be achieved. Different samples will spend varying amounts of time interacting with the
paper and the solvent. Through these different interactions, the samples will move different distances along the chromatography
paper. The distance a sample moves along the chromatography paper is compared to the overall distance the solvent travels—this
ratio is called the Rf value or rate of flow.
Experiment Overview
The purpose of this advanced inquiry lab is to investigate the factors that influence the separation of food dyes using paper
chromatography. The investigation begins with a baseline activity comparing the separation or resolution of three FD&C dyes,
Red No. 40, Blue No. 1, and Yellow No. 5, using two solvents. Reviewing the evidence provided by the cooperative class data
leads to the selection of a solvent for further study. In the guided-inquiry section of the lab, students will design an experiment
to identify a solvent that will give maximum resolution of a mixture of dyes. The results may be applied to study the connection
between structure and mobility of food dyes. An investigation into the composition of colored candy shells may be incorporated
as an optional extension activity.
Pre-Lab Questions
Rf for A
4.8
Rf = —— = 0.86
5.6
2.8 cm
4.8 cm
2. Calculate the Rf value for the spot in sample B using
sample A as an example.
5.6 cm
1. Figure 1 is a sample paper chromatogram for three
samples: A, B and C. Label the drawing with the following items: the stationary phase, the mobile phase
and the solvent front.
3. Sample C gave two spots on the paper chromatogram.
What does this tell you about the composition of the
sample?
4. Based on the Rf values of samples A and B, what can
you conclude about the intermolecular attractions both
samples have for the eluent and the paper?
A
B
C
Figure 1.
Materials
FD&C food dye mixtures, 1 mL
Erlenmeyer flasks, 250-mL, 2
Isopropyl alcohol solution, CH3CHOHCH3, 2%
Graduated cylinder, 25-mL
Sodium chloride solution, NaCl, 2%
Pencil
Unknown dye mixtures
Ruler
Water, distilled or deionized
Toothpicks
Beaker, 50-mL
Wash bottle
Beakers, 100-mL, 2
Watch glasses, 2
Chromatography paper strips
–2–
IN7661
© 2013 Flinn Scientific, Inc. All Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Separation of a Dye Mixture Using Chromatography, Catalog No.
AP7661, from Flinn Scientific, Inc. No part of this material may be reproduced or transmitted in any form or by any means, electronic or mechanical, including, but not limited to photocopy, recording, or any information storage and retrieval system, without permission in writing from Flinn Scientific, Inc.
Safety Precautions
Isopropyl alcohol is a flammable liquid and is slightly toxic by ingestion or inhalation. Use proper exhaust ventilation to keep
airborne concentrations low. The FD&C dyes are slightly hazardous by ingestion, inhalation, and eye or skin contact. Red No.
40 may be absorbed through skin and Yellow No. 5 may be a skin sensitizer. All dyes are irritating to skin and eyes. Avoid contact
with eyes, skin, and cloth­ing. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash
hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.
Introductory Activity
1. Position the chromatography paper strip so it is 152 mm tall and 19 mm wide. Note: Handle the paper by the edges so the
analysis area is not accidentally compacted or contaminated.
2. Using a ruler and a pencil, draw a faint line 15 mm from the bottom of the paper across the width of the strip. Measure 9.5
mm from the edge and place a dot on the line. This is the starting point for the sample.
3. Using the same ruler, measure 20 mm from the top of the strip and fold across the width of the strip. This will allow the
strip to hang on the lip of the flask.
4. Repeat steps 2 and 3 for a second paper strip.
5. Obtain the dye mixture.
6. Using a clean toothpick, spot the chromatography strip by placing a toothpick into the dye mixture solution and then
touching the tip of the toothpick gently onto the designated pencil dot. Allow the sample to dry. Repeat the procedure two
to three more times. Note: This step is necessary to increase the concentration of the sample but do not allow the size of
the spot to increase.
7. Repeat step 6 for the second chromatography strip.
8. While the samples are drying, obtain two 250-mL Erlenmeyer flasks and watch glasses to cover the tops of the flasks.
9. Pour 20 mL of the assigned 2% chromatography solvent into each flask. Cover the flasks with the watch glasses.
10. Once the chromatography paper is dry, remove the watch glass from the top of the flask. Carefully hang the chromatography strip into the flask with the sample end down. Do not get any solvent on the upper portion of the strip. The sample
spots must remain above the level of the solvent. If the solvent level is too high, the samples will dilute into the solvent!
11. Carefully place the watch glass back on the top of the flask. Allow the chromatogram to develop. Record observations of
the dye sample as the solvent travels up the paper and the chromatogram develops.
12. Repeat steps 10 and 11 using the other chromatography strip and flask.
13. When the chromatography solvent is within 1–2 cm of the fold in the chromatography strip, stop the run by removing the
strip from the flask.
14. With a pencil, lightly draw a line to mark the distance the solvent traveled. This is called the solvent front.
15. Measure the distance from the pencil line at the bottom of the strip to the solvent front. Record this distance in millimeters
in an appropriate data table.
16. With a pencil, trace the shape of each dye band or spot to mark its location on the chromatography strip. This should be
done immediately because the color and brightness of some spots may fade over time.
17. Measure and record the distance in millimeters that each dye band or spot traveled. Measure from the line at the bottom of
the paper to the center of each band or spot.
18. Repeat steps 13–17 for the other chromatograms.
Analyze the Results
Compile the class data and calculate the average Rf value for each dye in both solvents. Compare general observations regarding the separation using the different solvents, including developing time, color spreading, and direction of travel.
–3–
IN7661
© 2013 Flinn Scientific, Inc. All Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Separation of a Dye Mixture Using Chromatography, Catalog No.
AP7661, from Flinn Scientific, Inc. No part of this material may be reproduced or transmitted in any form or by any means, electronic or mechanical, including, but not limited to photocopy, recording, or any information storage and retrieval system, without permission in writing from Flinn Scientific, Inc.
Guided-Inquiry Design and Procedure
Form a working group with other students and discuss the following questions.
1. Examine the structures of the FD&C Red No. 40, Blue No. 1 and Yellow No. 5 dyes. What are the similarities and differences in the structures of the three dyes?
2. In the Introductory Activity, the developing solvents were 2% sodium chloride aqueous solution and 2% isopropyl alcohol aqueous solution. Draw separate molecular diagrams of how sodium chloride and isopropyl alcohol would interact in
water. Identify the types of intermolecular attractions within each diagram.
3. Based on the diagrams and intermolecular attractions identified in Question 2, predict and compare the nature of intermolecular attractions experienced by the FD&C Red No. 40, Blue No. 1 and Yellow No. 5 dyes with the two solvents.
4. Chromatography paper, and paper in general, is highly hydrophilic. Paper is made from a natural polymer called cellulose,
which is a long chain of glucose molecules. Glucose is a cyclic structure with a number of —OH groups around the ring.
a. Predict and explain the types of intermolecular forces that would occur between paper and water. How do these interactions account for the hydrophilic nature of paper?
b. Explain the types of intermolecular interactions that would occur between the FD&C Red No. 40, Blue No. 1 and
Yellow No. 5 food dyes and the paper.
5. Write a detailed step-by-step procedure using dilutions of more concentrated solvents to investigate the effect of concentration on dye separations in various unknown dye mixtures.
6. Include all materials, glassware and equipment that will be needed, safety precautions that must be followed, the concentrations of the solvents, etc.
7. Review additional variables that may affect the reproducibility or accuracy of the experiment and how these variables can
be controlled.
8. Carry out the experiment and record the results in an appropriate data table.
Analyze the Results
Compile the data within your group. Identify the optimal solvent tested by your group. Propose an explanation for why the
chosen solvent was best.
Opportunities for Inquiry
As noted in the Background section, FD&C food dyes are used in a wide range of food products, most notably the outer
shells of candies. Candy may be placed in 5–6 drops of water. Stir the candy until the color dissolves. Repeat with two more candies. This is the color sample. Design an experiment to determine the composition of the dye mixture in the candy shell.
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IN7661
© 2013 Flinn Scientific, Inc. All Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Separation of a Dye Mixture Using Chromatography, Catalog No.
AP7661, from Flinn Scientific, Inc. No part of this material may be reproduced or transmitted in any form or by any means, electronic or mechanical, including, but not limited to photocopy, recording, or any information storage and retrieval system, without permission in writing from Flinn Scientific, Inc.
AP Chemistry Review Questions
Integrating Content, Inquiry and Reasoning
1. Hydrocarbons are nonpolar compounds containing carbon and hydrogen atoms. The properties of three hydrocarbons are
summarized below.
Methane
Octane
Eicosane
CH4
C8H18
CH3(CH2)18CH3
Natural gas
Gasoline
Lubricant (grease)
Gas, bp –161 °C
Liquid, bp 126 °C
Solid, mp 37 °C
a. How do the attractive forces between molecules change in the transition from the gas to the liquid to the solid state?
b. Based on its properties, which compound has the strongest attractive forces? The weakest attractive forces?
c. Write a general statement describing how the size of a molecule influences the strength of London dispersion forces
between molecules.
2. Dyes are organic compounds that can be used to impart bright, permanent colors to fabrics. The affinity of a dye for a fabric depends on the chemical structures of the dye and fabric molecules and also on the interactions between them. Three
common fabrics are wool, cotton and nylon. Wool is a protein, a naturally occurring polymer made up of amino acids with
ionized (charged) side chains. Cotton is a naturally occurring polymer made up of glucose units with hydrophilic groups
surrounding each glucose unit. Nylon is a synthetic polymer made of hydrocarbon repeating chains joined together by
highly polar amide (–CONH–) functional groups.
Structure for STUDENT VERSION
a. The chemical structure of methyl orange is drawn below. Identify the groups in the dye that will bind to ionic and polar
sites in a fabric.
¯O 3S
.. ..
N
N
CH3
N
CH3
H
b. Complete the following “If/then” hypothesis to explain how the structure of a fabric will influence the relative color
intensity produced by methyl orange.
Figure 2. Methyl Orange
“If a fabric contains more ionic and polar groups in its structure, then the intensity of the dye color due to methyl
orange should (increase/decrease), because _____________________________________________________________
______________________________________________________________________________________________.”
c. Using this hypothesis, predict the relative color intensity that would be produced by methyl orange on cotton, nylon and
wool. Rank the fabrics from 1 = lightest color to 3 = darkest color.
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IN7661
© 2013 Flinn Scientific, Inc. All Rights Reserved. Reproduction permission is granted only to science teachers who have purchased Separation of a Dye Mixture Using Chromatography, Catalog No.
AP7661, from Flinn Scientific, Inc. No part of this material may be reproduced or transmitted in any form or by any means, electronic or mechanical, including, but not limited to photocopy, recording, or any information storage and retrieval system, without permission in writing from Flinn Scientific, Inc.
Structure for TEACHER VERSION
Teacher’s Notes
Investigation 5—Separation of a Dye Mixture Using Chromatography
Part I. Lab Preparation
Page No.
Part II. Teacher Guidance Page No.
• Materials Lists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
• Curriculum Alignment. . . . . . . . . . . . . . . . . 7–8
• Estimated Time Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
• Lab Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
• Pre-Lab Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
• Teaching Tips. . . . . . . . . . . . . . . . . . . . . . . . . . 9
• Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
• Disposal Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Part III. Sample Data, Results, and Analysis
• Answers to Pre-Lab Questions . . . . . . . . . . . . . . . . . . . . . . . 10
• Sample Data for Introductory Activity . . . . . . . . . . . . . . 10–11
Part IV. Supplemental Materials
• Chemical Structures of
FD&C Food Dyes. . . . . . . . . . . . . . . . . . . . . . 15
• Answers to Guided-Inquiry Discussion Questions . . . . . 11–12
• Sample Data for Guided-Inquiry Activity . . . . . . . . . . . . 12–13
• Answers to AP Chemistry Review Questions . . . . . . . . . 13–14
Part I. Lab Preparation
Materials Included in Kit (for 24 students working in pairs)
Food Dye FD&C Blue No. 1, 2 g
Food Dye FD&C Yellow No. 6, 2 g
Food Dye FD&C Blue No. 2, 2 g
Isopropyl alcohol, CH3CHOHCH3, 70%, 250 mL
Food Dye FD&C Green No. 3, 1 g
Sodium chloride solution, NaCl, 20%, 500 mL
Food Dye FD&C Red No. 3, 2 g
Chromatography paper strips, 200
Food Dye FD&C Red No. 40, 2 g
Toothpicks, 250
Food Dye FD&C Yellow No. 5, 2 g
Additional Materials Required (for each lab group)
Water, distilled or deionized
Graduated cylinder, 25-mL
Beaker, 50-mL
Wash bottle
Beakers, 100-mL, 2
Watch glasses, 2
Erlenmeyer flasks, 250-mL, 2
Additional Material Required (for Pre-Lab Preparation)
Water, distilled or deionized
Graduated cylinders, 10-mL, 25-mL, 100-mL
Balance, 0.01-g precision
Spatulas, 7
Beaker, 100-mL
Stirring rods, 7
Beakers, 250-mL, 7
Volumetric flask, 500-mL
Time Required
This laboratory activity can be completed in two 50-minute class periods. It is important to allow time between the
Introductory Activity and the Guided-Inquiry Activity for students to discuss and design the guided-inquiry procedures. Also,
all student-designed procedures must be approved for safety before students are allowed to implement them in the lab. Pre-Lab
Questions may be completed before lab begins the first day, and the data compilation and calculations may be completed after the
lab or as homework.
–6–
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IN7661
Teacher’s Notes continued
Pre-Lab Preparation
1. To prepare 100 mL of individual dye solutions, add 0.5 g of each FD&C solid dye to a separate beaker with 100 mL of
distilled or deionized water. Mix thoroughly.
2. To prepare the dye mixture for the Introductory Activity, combine 10 mL of Red No. 40, Blue No. 1 and Yellow No. 5 dye
solutions in one beaker.
3. To prepare 500 mL of 2% sodium chloride solution, fill a 500-mL volumetric flask one-third to one-half full with distilled or deionized water. Add 50 mL of 20% NaCl solution and dilute with distilled or deionized water to the mark.
Mix thoroughly.
4. To prepare 500 mL of 2% isopropyl alcohol solution, measure 14.3 mL of 70% isopropyl alcohol solution. Pour this into a
500-mL volumetric flask and dilute with distilled or deionized water to the line. Cover and mix thoroughly before dispensing.
5. See the Lab Hints section for suggestions for unknown mixtures of dyes.
Safety Precautions
Isopropyl alcohol is a moderate fire risk and is slightly toxic by ingestion or inhalation. Use proper exhaust ventilation to
keep airborne concentrations low. The FD&C dyes are slightly hazardous by ingestion, inhalation, and eye or skin contact. Red
No. 40 may be absorbed through skin and Yellow No. 5 may be a skin sensitizer. All dyes are irritating to skin and eyes. Avoid
contact with eyes, skin, and clothing. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron.
Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current
Material Safety Data Sheets for additional safety, handling, and disposal information.
Disposal
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and
review all federal, state and local regulations that may apply, before proceeding. Excess dye solutions and sodium chloride solu­
tion may be stored for future use or rinsed down the drain with excess water according to Flinn Suggested Disposal Method
#26b. Small quantities of excess isopropyl alcohol solutions may be rinsed down the drain with excess water according to Flinn
Suggested Disposal Method #18a.
Part II. Teacher Guidance
Alignment to AP Chemistry Curriculum Framework
Enduring Understandings and Essential Knowledge
atter can be described by its physical properties. The physical properties of a substance generally depend on the spacing
M
between the particles (atoms, molecules, ions) that make up the substance and the forces of attraction among them. (2A)
2A3: Solutions are homogenous mixtures in which the physical properties are dependent on the concentration of the solute
and the strengths of all interactions among the particles of the solutes and solvent.
orces of attraction between particles (including the noble gases and also different parts of some large molecules) are imporF
tant in determining many macroscopic properties of a substance, including how the observable physical state changes with
temperature. (2B)
2B2: Dipole forces result from the attraction among the positive ends and negative ends of polar molecules. Hydrogen bonding is a strong type of dipole-dipole force.
2B3: Intermolecular forces play a key role in determining the properties of substances, including biological structures and
interactions.
Learning Objectives
2.7 The student is able to explain how solutes can be separated by chromatography based on intermolecular attractions.
2.10 The student can design and/or interpret the results of a separation experiment (filtration, paper chromatography, column
chromatography, or distillation) in terms of relative strength of interactions among and between the components.
2.13 The student is able to describe the relationships between the structural features of polar molecules and the forces of
attraction between particles.
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IN7661
Teacher’s Notes continued
Science Practices
1.4 The student can use representations and models to analyze situations or solve problems qualitatively and quantitatively.
4.2 The student can design a plan for collecting data to answer a particular scientific question.
4.3 The student can collect data to answer a particular scientific question.
5.1 The student can analyze data to identify patterns or relationships.
5.2 The student can refine observations and measurements based on data analysis.
5.3 The student can evaluate the evidence provided by data sets in relation to a particular scientific question.
6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices.
6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.
Lab Hints
• Enough chromatography paper strips are included for 12 groups of students to develop 16 chromatograms each. Extra
strips are provided in case of mistakes.
• Students should avoid over-handling the chromatography strips. Oil from the skin can interfere with the capillary action
that draws water through the paper.
• In the Introductory Activity portion of the lab, it is recommended that half of the groups use 2% sodium chloride solution
as the developing solvent and the other half use 2% isopropyl alcohol as the developing solvent. This split will allow for
adequate testing of both solvents, providing enough data for students to compare benefits and drawbacks of both solvents.
• Good technique is important to achieve clean separations in paper chromatography. Common sources of student error
include “overloading” the paper by placing too much dye on the initial spot and the band broadening that occurs because
the initial spot is too large.
• Suggestions for unknown dye mixtures are below. The blue, red and yellow mixtures are suggested for identification of
dyes found in commercial food products. Mix equal volumes of each dye desired to create a mixture.
Purple: Combine Blue No. 1 and Red No. 3, or Blue No. 2 and Red No. 3, or Blue No. 1 and Red No. 40.
Orange: Combine Red No. 40 and Yellow No. 6.
Green:Combine Blue No. 2 and Yellow No. 6; or Blue No. 1 and Yellow No. 6; or Blue No. 1, Yellow No. 5 and
Green No. 3; or Blue No. 2, Yellow No. 6 and Green No. 3.
Blue:
Combine Blue No. 1 and Blue No. 2.
Red:
Combine Red No. 3 and Red No. 40.
Yellow: Combine Yellow No. 5 and Yellow No. 6.
• It is critical to allow enough time for the development of the chromatography paper. The chromatography paper must be
left in the chromatography chamber long enough for the solvent to be drawn up near the top of the strip. Do not stop the
development until the solvent front nears the top of the strip. Underdevelopment will lead to incomplete separation. Do not
allow the solvent front to move off the paper, however.
• The developing solvent and dyes will continue to move even after the paper strip is removed from the solvent. It is necessary for students to mark the solvent front and positions of the dye bands or spots immediately after the strip is removed
from the flask.
• As students plan their investigation in the inquiry portion, they must remember to run a control or baseline trial. The
2% sodium chloride and 2% isopropyl alcohol solutions are convenient baseline runs because those were used in the
Introductory Activity.
–8–
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IN7661
Teacher’s Notes continued
Teaching Tips
• See the last page of the Teacher’s Notes for chemical structures of the seven FD&C food dyes. Provide these to the students as they work through the Guided-Inquiry Design portion of the lab.
• When the food dyes are dissolved in water, the sodium salts of the sulfonate, oxide and carboxylate groups dissociate to
give negatively charged side groups.
• The test solvents used in the Introductory Activity have different polarities. The isopropyl alcohol solution decreases the
polarity of pure water because isopropyl alcohol is only slightly polar. The three-carbon chain is mostly nonpolar and the
oxygen in the alcohol group draws electrons to itself giving rise to a partial negative charge. The sodium chloride solution
increases the polarity of pure water. In addition to having the highly polar water molecules, the solution contains dissociated sodium and chloride ions.
• The best solvent is not necessarily the solvent that provides the largest Rf values. The optimal solvent will provide the
greatest ratios of Rf values among the dyes. This means the separation or resolution of the component dyes relative to each
other is maximized.
• Expand the students’ chromatography repertoire by separating dye components through column chromatography with the
Liquid Chromatography—Student Laboratory Kit available from Flinn Scientific, Catalog No. AP9093.
–9–
© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
Teacher’s Notes continued
Part III. Sample Data, Results, and Analysis
Answers to Pre-Lab Questions (Student answers will vary.)
1. Figure 1 is a sample paper chromatogram for three samples: A, B and C. Label the drawing with the following items: the
stationary phase, the mobile phase and the solvent front.
2. Calculate the Rf value for the spot in sample B using sample A as an example.
Mobile phase (solvent)
Solvent front
Rf for A
4.8
Rf = —— = 0.86
5.6
4.7 cm
2.8 cm
5.6 cm
4.8 cm
{
A
B
Rf for B
2.8
Rf = —— = 0.50
5.6
Rf for sample C (two spots)
2.8
4.7
Spot 1. Rf = —— = 0.50 Spot 2. Rf = —— = 0.84
5.6
5.6
C
Stationary phase (paper)
3. Sample C gave two spots on the paper chromatogram. What does this tell you about the composition of the sample?
Sample C is a mixture of at least two components, A and B.
4. Based on the Rf values of samples A and B, what can you conclude about the intermolecular attractions both samples have
for the eluent and the paper?
The Rf value of sample A is larger than sample B. Sample A has a stronger affinity (attraction) for the eluent (solvent) than
the paper, so it traveled farther with the solvent. Sample B has a stronger attraction for the paper than the solvent, so it
traveled a shorter distance.
Sample Data for Introductory Activity
Observations for 2% Sodium Chloride Solution
2% NaCl Solution
Solvent Front Distance
Red No. 40 Distance
Blue No. 1 Distance
Yellow No. 5 Distance
Trial 1
110 mm
9 mm
71 mm
28 mm
Trial 2
106 mm
13 mm
85 mm
34 mm
Trial 3
104 mm
8 mm
77 mm
21 mm
Rf Red No. 40
Rf Blue No. 1
Rf Yellow No. 5
Trial 1
0.081
0.65
0.25
Trial 2
0.12
0.80
0.32
Trial 3
0.077
0.74
0.20
Average Rf value
0.093
0.73
0.26
2% NaCl Solution
Total time for chromatograms to develop ranged from 25–30 minutes. Three separate dye spots were visible: blue on top,
yellow in the middle, and red on the bottom. Each spot was fairly spread out, with the largest band being the Blue No. 1 dye. All
three dyes traveled the same direction in a straight line (not curved).
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© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
Teacher’s Notes continued
Observations for 2% Isopropyl Alcohol Solution
2% Isopropyl Alcohol
Solvent Front Distance
Solution
Red No. 40 Distance
Blue No. 1 Distance
Yellow No. 5 Distance
Trial 1
106 mm
72 mm
101 mm
100 mm
Trial 2
101 mm
72 mm
94 mm
92 mm
Trial 3
102 mm
76 mm
95 mm
93 mm
Rf Red No. 40
Rf Blue No. 1
Rf Yellow No. 5
Trial 1
0.68
0.95
0.94
Trial 2
0.71
0.93
0.91
Trial 3
0.75
0.93
0.91
Average Rf value
0.71
0.94
0.92
2% Isopropyl Alcohol
Solution
Total time for chromatograms to develop ranged from 25–30 minutes. Two separate dye spots were visible: blue and yellow
on top, and red on the bottom. The Blue No. 1 and Yellow No. 5 colors overlapped resulting in a blue front followed by a green
line then a yellow tail end. The blue-yellow color band traveled straight for a portion of the time, and then the edges began trailing
while the middle continued at a “faster” rate. The shape of the blue-yellow color band was a crescent. The top of the Red No. 40
color spot was near the middle of the crescent.
Answers to Guided-Inquiry Discussion Questions
1. Examine the structures of the FD&C Red No. 40, Blue No. 1 and Yellow No. 5 dyes. What are the similarities and differences in the structures of the three dyes?
Answer to Discussion Question # 2
All three dyes, Red No. 40, Blue No. 1 and Yellow No. 5, have sulfonate (—SO3¯) functional groups. However, Blue No.
1 has the most sulfonate groups (three); Red No. 40 and Yellow No. 5 both have two. Blue No. 1 also has a positively
charged nitrogen atom and Yellow No. 5 has a carboxylate group (—CO2–). Red No. 40 and Yellow No. 5 both have double
bonded nitrogen atoms near the middle of the structures and a single —OH group. Red No. 40 has an —OCH3 group and
a methyl group on the leftmost benzene ring. Blue No. 1 is the largest molecule of the three dye molecules.
2. In the Introductory Activity, the developing solvents were 2% sodium chloride aqueous solution and 2% isopropyl alcohol aqueous solution. Draw separate molecular diagrams of how sodium chloride and isopropyl alcohol would interact in
water. Identify the types of intermolecular attractions within each diagram.
Isopropyl alcohol in water
Sodium chloride in water
Isopropyl alcohol in water
Sodium chloride in water
H3C
H
O
H
Na
O
H
H
H
O
Cl¯
+
H
Cl¯
H
H
+
Na
H
O
H
H
CH3
O
H
O
O
H
O
O
H
H3C
Ion-dipole
interactions
Ion–dipole
interactions
H
H
CH3
Hydrogenbonding
bondinginteractions
interactions
Hydrogen
– 11 –
© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
Teacher’s Notes continued
3. Based on the diagrams and intermolecular attractions identified in Question 2, predict and compare the nature of intermolecular attractions experienced by the FD&C Red No. 40, Blue No. 1 and Yellow No. 5 dyes with the two solvents.
In the sodium chloride solution, all three dye molecules would experience ion–dipole interactions due to the charged sulfonate groups, although the overall strength of these interactions will vary due to the number of groups on each molecule.
Similarly, each dye molecule will experience hydrogen bonding between water and the functional groups. Blue No. 1
would experience the strongest interactions because it has the most charged side groups—three SO3– groups and a positively charged nitrogen atom. Red No. 40 and Yellow No. 5 would experience weaker ion–dipole and hydrogen bonding
interactions with the sodium chloride solution because the molecules have fewer charged side groups. Yellow No. 5 experienced a stronger interaction with the sodium chloride solution because it has a charged carboxylate group in addition to
two charged sulfonate groups. Red No. 40 only has two charged sulfonate groups.
In the isopropyl alcohol solution, all three dye molecules would experience ion–dipole interactions due to the charged
functional groups and polar alcohol group in isopropyl alcohol. In addition to their changed side chains, all of the food
dyes are large organic molecules with significant nonpolar rings and groups. These nonpolar regions interact with relatively nonpolar isopropyl alcohol molecules and thus have a greater affinity for this solvent than for either the NaCl solution or the hydrophilic paper substrate.
4. Chromatography paper, and paper in general, is highly hydrophilic. Paper is made from a natural polymer called cellulose,
which is a long chain of glucose molecules. Glucose is a cyclic structure with a number of —OH groups around the ring.
a. Predict and explain the types of intermolecular forces that would occur between paper and water. How do these interactions account for the hydrophilic nature of paper?
The —OH groups around the glucose rings are sites for ion–dipole interactions and hydrogen bonding with water. The
hydrogen bonding interaction between the paper and water will be strong because the —OH groups will be able to
interact strongly with the hydrogens and oxygen in water due to the large dipole moments. Paper has a strong affinity
for water and draws water up by capillary action.
b. Explain the types of intermolecular interactions that would occur between the FD&C Red No. 40, Blue No. 1 and
Yellow No. 5 food dyes and the paper.
The —OH groups around the glucose rings are sites for hydrogen bonding with the charged functional groups on the
dye molecules. Additionally, Red No. 40 would experience hydrogen bonding at the —OH group and —OCH3 group.
Yellow No. 5 would have hydrogen bonding at the —OH group, as well. In both Red No. 40 and Yellow No. 5, hydrogen
bonding would occur with the double-bonded nitrogen atoms and —OH groups around the glucose rings.
Sample Data for Guided-Inquiry Activity
Comparison of Solvent Concentration on Dye Mixture Separation
Based on the data and observations of the Introductory Activity, the sodium chloride solvent was chosen for further investigation. The concentration of the solvent was increased by a factor of four to 8% and decreased by a factor of four to 0.5%. The data
table below summarizes the findings.
Solvent
Red 40 Rf Value*
Blue 1 Rf Value*
Yellow 5 Rf Value*
2%†
0.093
0.73
0.26
8%
0.10
0.73
0.21
0.50%
0.26
0.86
0.49
*Values are averaged from two trials each.
†Rf values are copied from Introductory Activity here for comparison.
In the 8% sodium chloride solvent, total time for chromatograms to develop ranged from 20–30 minutes. All three dyes were
visible: blue on top, yellow in the middle, and red on the bottom. Blue No. 1 was the largest band. The Yellow No. 5 band overlapped with roughly half of the Red No. 40 band. These two bands were much closer than in the 2% sodium chloride solvent. All
three dyes traveled in the same direction in a straight line (not curved).
– 12 –
© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
Teacher’s Notes continued
In the 0.50% sodium chloride solvent, the total time for chromatograms to develop ranged from 20–30 minutes. The three
dyes were visible: blue on top, yellow in the middle, and red on the bottom. Red No. 40 was the largest band. The Yellow No. 5
band overlapped with the very top of the Red No. 40 band. These two bands were much closer than in the Introductory Activity.
All three dyes traveled in the same direction in a straight line (not curved).
Based on the data collected for the 8% and 0.50% sodium chloride solutions, the more dilute solvent separated the mixture of
dyes better. The three dyes were more distinguishable from one another in the 0.50% solution than in the 8% solution. This was
evident with the overlapping of the Yellow 5 and Red 40 dyes with the 8% solvent. The 0.50% sodium chloride solution was the
more optimal solvent, compared to the 2% and 8% solutions, because the dyes traveled farther on the paper and there were greater
distances separating the dye bands.
Effect of Solvent Concentration on FD&C Food Dyes
The chart below is a comparison of the seven FD&C food dyes at two different concentrations of sodium chloride solutions:
2% and 0.10%.** The dyes were run simultaneously on the same piece of chromatography paper. Green No. 3 and Blue No. 1
have very similar Rf values in both 2% and 0.10% sodium chloride solutions. Similarly, Red No. 40 and Blue No. 2 have similar
Rf values in the two sodium chloride solutions. These two pairs of dyes will be the most difficult to separate.
n No
G re e
.3
N o. 1
.5
w No
B lue
R ed
N o. 2
N o. 4
0
Y ellow No. 5
Y ellow No. 6
G reen No. 3
B lue No. 1
B lue No. 2
N o. 3
R ed
N o. 2
B lue
R ed
N o. 3
0.2
R ed
N o. 4
0
0.3
.6
Y e llo
0.4
R ed No. 40
w No
w No
.5
0.5
0.1
R ed No. 3
Y e llo
0.6
Y e llo
RRf f Value
V alue
0.7
.6
Y e llo
w No
n No
0.8
B lue
G re e
0.9
B lue
1
N o. 1
.3
R f V alues
Rf Values
0
2%
0.10%
ent S odium CChloride
hloride S olutionSolution
PercentP erc
Sodium
** The 0.10% concentration was chosen based on the work of Peter Markow (see the References section).
Answers to AP Chemistry Review Questions
1. Hydrocarbons are nonpolar compounds containing carbon and hydrogen atoms. The properties of three hydrocarbons are
summarized below.
Methane
Octane
Eicosane
CH4
C8H18
CH3(CH2)18CH3
Natural gas
Gasoline
Lubricant (grease)
Gas, bp –161 °C
Liquid, bp 126 °C
Solid, mp 37 °C
a. How do the attractive forces between molecules change in the transition from the gas to the liquid to the solid state?
Attractive forces between molecules increase in the order gas << liquid < solid. Molecules in the gas state are very far
apart—there are almost no attractive forces between the molecules. As gases condense into liquids and then solidify,
the molecules get closer together and the strength of attractive forces between molecules increases. Attractive forces
between molecules are strongest in the solid state, because the molecules are locked into fixed positions.
– 13 –
© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
CH3
H
Methyl Orange
Teacher’sFigure
Notes 2.
continued
b.Based on its properties, which compound has the strongest attractive forces? The weakest attractive forces?
Eicosane, a hydrocarbon with 20 carbon atoms, has stronger intermolecular attractive forces than octane or methane,
which contain eight carbon atoms and one carbon atom, respectively. Methane has the weakest attractive forces.
c. Write a general statement describing how the size of a molecule influences the strength of London dispersion forces
between molecules.
The strength of London dispersion forces between molecules increases as the size of the molecules increases (all other
factors being equal).
2. Dyes are organic compounds that can be used to impart bright, permanent colors to fabrics. The affinity of a dye for a fabric depends on the chemical structures of the dye and fabric molecules and also on the interactions between them. Three
common fabrics are wool, cotton and nylon. Wool is a protein, a naturally occurring polymer made up of amino acids with
ionized (charged) side chains. Cotton is a naturally occurring polymer made up of glucose units with hydrophilic groups
surrounding each glucose unit.
Nylon is afor
synthetic
polymer made
of hydrocarbon repeating chains joined together by
Structure
TEACHER
VERSION
highly polar amide (–CONH–) functional groups.
a. The chemical structure of methyl orange is drawn below. Identify the groups in the dye that will bind to ionic and polar
sites in a fabric.
CH3
.. ..
¯O 3S
N
N
N
CH3
H
Binds
Bindstotopolar
polar
and ionic sites
and ionic sites
Binds
toionic
ionicsites
sites
Binds to
Bindstotopolar
polarsites
sites
Binds
Figure 2. Methyl Orange
b. Complete the following “If/then” hypothesis to explain how the structure of a fabric will influence the relative color
intensity produced by methyl orange.
“If a fabric contains more ionic and polar groups in its structure, then the intensity of the dye color due to methyl
orange should increase, because there would be more sites on the fabric for the dye molecules to bind.”
c. Using this hypothesis, predict the relative color intensity that would be produced by methyl orange on cotton, nylon and
wool. Rank the fabrics from 1 = lightest color to 3 = darkest color.
1 = nylon, 2 = cotton, 3 = wool
References
AP* Chemistry Guided-Inquiry Experiments: Applying the Science Practices; The College Board: New York, NY, 2013.
Markow, P. G. The Ideal Solvent for Paper Chromatography of Food Dyes. J. Chem. Ed. 1988, 65, 10, pp 899–900.
Separation of a Dye Mixture Using Chromatography—Advanced Inquiry Laboratory Kit is available
from Flinn Scientific, Inc.
Catalog No.
AP7661
Description
Separation of a Dye Mixture Using Chromatography—
Advanced Inquiry Laboratory Kit
Consult your Flinn Scientific Catalog/Reference Manual for current prices.
– 14 –
© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
Chemical
Structures ofInformation
FD&C Food Dyes
Supplementary
SO3Na
Cl¯
SO3Na
+
H
O
N (C2H5)CH2
NaO 3 S
N
N
SO 3 Na
O
H
SO3Na
Figure 2. FD&C Blue No. 2
Figure 2. FD&C Blue No. 2
N(C2H5)CH2
Figure 1. FD&C Blue No. 1
Figure 1. FD&C Blue No. 1
SO 3Na
I
Cl¯
NaO
+
SO 3Na
HO
I
N (C 2H5)CH 2
O
O
I
I
CO 2Na
SO 3Na
Figure 4. FD&C Red No. 3
N(C 2H5)CH 2
Figure 4. FD&C Red No. 3
Figure 3. FD&C Green No. 3
Figure 3. FD&C Green No. 3
OCH 3
NaO 3S
N
SO3Na
HO
HO
N
NaO3S
N
H3C
N
N
N
NaO2C
Figure 6. FD&C Yellow No. 5
SO 3Na
Figure 5. FD&C Red No. 40
Figure 5. FD&C Red No. 40
Figure 6. FD&C Yellow No. 5
HO
NaO3S
N
N
SO3Na
Figure 7.
7. FD&C
FD&C Yellow
No.No.
6 6
Figure
Yellow
– 15 –
© 2013 Flinn Scientific, Inc. All Rights Reserved.
IN7661
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Separation of a Dye Mixture Using Chromatography