An Inquiry-based Teaching Module Using the Separation
Techniques of the Chemical Engineer
Donald McQuarrie
and
Mari Knutson
Lynden Public High School
1201 Bradley Rd.
Lynden, WA 98264
Summer, 2008
WSU Mentors: Dr. Neil Ivory, Jeff Burke, Dr. Richard Zollars
Department of Chemical Engineering
Washington State University
Pullman, WA 99164-2710
National Science Foundation Grant No. EEC-0808716 supports this project: Dr. Richard L.
Zollars, Principal Investigator and Dr. Donald C. Orlich, co-PI. The module was developed by
the authors and does not necessarily represent an official endorsement by the National Science
Foundation.
TABLE OF CONTENTS
INTRODUCTION .......................................................................................................................... 3
BSCS FORMAT
PROBLEM STATEMENT
THE ESSENTIAL ACADEMIC LEARNING REQUIREMENTS
4
5
5
CHEMICAL ENGINEERING ....................................................................................................... 6
MATERIALS.................................................................................................................................. 9
PREREQUISITE KNOWLEDGE AND SAFETY CONSIDERATIONS
9
DAILY ACTIVITIES ................................................................................................................... 10
EVALUATION.. .......................................................................................................................... 10
EXTENSION.. .............................................................................................................................. 10
REFERENCES. ............................................................................................................................ 11
ACKNOWLEDGEMENTS .......................................................................................................... 11
APPENDIX A: PRE-ASSESSMENT ......................................................................................... 12
APPENDIX B: SCIENCE NOTEBOOKING ............................................................................. 13
APPENDIX C: "I'M SEEING SPOTS" ACTIVITY ................................................................... 14
TEACHER NOTES
Seminar Example
15
16
APPENDIX D: SPINACH CHROMATOGRAPHY .................................................................. 17
TEACHER NOTES
20
APPENDIX E: INQUIRY ACTIVITY-CSI ................................................................................ 21
TEACHER PAGE
22
APPENDIX F: NOTEBOOK ASSESSMENT ............................................................................ 23
APPENDIX G: LAB REPORT ASSESSMENT ......................................................................... 25
APPENDIX H: EXTENSION ACTIVITY PART I .................................................................... 26
TEACHER NOTES
32
EXTENSION PART II: STEP GRADIENT SEPARATION ..................................................... 35
TEACHER NOTES
37
2
INTRODUCTION
Overview. This year, the Lynden School District is implementing the 2nd year of the BSCS
integrated, inquiry based science program. The first chapter of the text is meant to reinforce
science process skills and set the stage for further inquiry-based learning. However, the initial
activities involve growing bacteria from unknown places. With the increasing abundance of
methicillin-resistant Staphylococcus aureus (MRSA), however, the National Science Teacher‟s
Association (NSTA) has published a warning against the indiscriminate use of random bacteria
in the high school, even the college classroom.
Searching for an alternative, these chromatography activities were developed and will be used in
place of the suggested curriculum but this module is designed to follow the “5E” model from
BSCS Science: An Inquiry Approach, Level 2. We have organized the “5Es” into 3 parts. Part 1
is an “aha” moment, short and to the point as the first day of school is always somewhat chaotic.
Part 2 is more guided, having the student separate the pigments of a plant (spinach) and analyze
what they are seeing, both qualitatively and quantitatively. Part 3 has the students trying to solve
a „murder‟ based on a handwritten word found in the victim‟s hand. They will have to determine
which of several pens wrote the word, and either convict or exonerate the suspects.
Please inquire:
Inquiry, notebooking, and coffee filter and spinach activites…
Mari Knutson
[email protected] or [email protected]
Technical and experimentation including mystery and extension activites…
Don McQuarrie
[email protected] or [email protected]
3
BSCS format followed (more or less):
“E”
Engagement
Definition
The learners‟ prior knowledge and
helps them become engaged in a new concept
through the use of a short activity
that promotes curiosity and elicits prior
knowledge.
Action
Students review basic
notebooking and science
process skills. A short survey
and pre-activity assessment is
given.
Exploration
Exploration experiences provide students
with a common base within which current
concepts (i.e., misconceptions), processes,
and skills are identified and conceptual
change is facilitated. Learners may complete
lab activities that help them use prior
knowledge to generate new ideas, explore
questions and possibilities, and design and
conduct a preliminary investigation.
The explanation phase focuses students‟
attention on a particular aspect of their
engagement and exploration experiences and
provides opportunities to demonstrate their
conceptual understanding, process skills, or
behaviors. This phase also provides
opportunities for teachers to directly
introduce a concept, process, or skill.
Learners explain their understanding of the
concept. An explanation from the teacher or
the curriculum may guide them toward a
deeper understanding, which is a critical part
of this phase.
Teachers challenge and extend students‟
conceptual understanding and skills.
Through new experiences, the students
develop deeper and broader understanding,
more information, and adequate skills.
Students apply their understanding of the
concept by conducting additional activities.
The evaluation phase encourages students to
assess their understanding and abilities and
provides opportunities for teachers to
evaluate student progress toward achieving
the educational objectives.
Students are given 2 coffee
filters each with a black spot
in the middle. Water is used
as the solvent and students
make observations and
inferences about the nature of
the black spots. Students share
observations in larger group
setting (seminar).
Students prepare and separate
pigments in spinach using
paper chromatography.
Students learn about stationary
and mobile phases, solvents,
and retention factor (Rf).
Explanation
Elaboration
Evaluation
4
Students share observations
and results in larger group
setting (seminar).
Students solve a “who-doneit” by designing and testing
their own paper
chromatography experiments.
Students share results in group
setting (seminar) and then
present evidence for their
findings to the entire class.
All work done in notebook is
also assessed by teacher. Preassessment is retaken.
Problem Statement. An understanding of the actual principles and technology used to identify
the components that allow for the engineering of pharmaceuticals, foods, and other new products
will give students the tools to evaluate information more critically. Students will be asked to
determine the components in various mixtures by using chromatography.
In addition, students need to practice science process skills and argue their findings based on
evidence from their experimentation. "Linking Questions” from the BSCS Science: An Inquiry
Approach, Level 2 text guide the activity selection and sequencing for this module. They are:

"How can I use what I have learned to design a scientific investigation of my own?

"How does my design reflect the process of scientific inquiry?

"How can the process of scientific inquiry help me to evaluate scientific claims in the
media?"

"How can I demonstrate what I have learned about the process of scientific inquiry?"
The Essential Academic Learning Requirements. When students finish this module they will
have achieved many of the items specified by the Washington State “Essential Academic
Learning Requirements” (EALRs) and by national standards.
1. The student understands and uses scientific concepts and principles.
2. The student conducts scientific investigations to expand understanding of the natural
world.
3. The student applies science knowledge and skills to solve problems or meet challenges.
4. The student uses effective communication skills and tools to build and demonstrate
understanding of science.
5. The student understands how science knowledge and skills are connected to other subject
areas and real-life situations
Scope. Activities may be mixed and matched according to time constraints and laboratory
accessibility. Activities are designed to be cost-effective and not to require extensive preparation
on the part of the student or teacher. If all components are included, the module may be
accomplished over seven days or less. Extension/elaboration activities are included in the
appendices.
*Teacher and Student worksheets are included in the appendices. Student worksheets are
designed to be used in a „notebooking‟ context.
5
Chemical Engineering. Chemical engineers utilize chemistry to solve problems for industry.
“Chemical engineers concern themselves with the chemical processes that turn raw materials into
valuable products. The necessary skills encompass all aspects of design, testing, scale-up,
operation, control, and optimization, and require a detailed understanding of the various “unit
operations”, such as distillation, mixing, and biological processes, which make these conversions
possible. Chemical engineering science utilizes mass, momentum, and energy transfer along with
thermodynamics and chemical kinetics to analyze and improve on these “unit operations.”
(Pafko, 2000)
This module focuses primarily on separation techniques used by chemical engineers. As science
and technology seem to be producing information at astonishing speed, the „making it work‟ is
the job of the chemical engineer.
The American Institute of Chemical Engineers (AIChE) has compiled a list of the “10 Greatest
Achievements of Chemical Engineering”. The two on the list that pertain to this module on
separation of substances by chromatography are:
1. Chemical engineers have helped develop processes like catalytic cracking to break
down the complex organic molecules found in crude oil into much simpler species.
These building blocks are then separated and recombined to form many useful
products.
2. Chemical engineers have long studied complex chemical processes by breaking them
up into smaller “unit operations.” Such operations might consist of heat exchangers,
filters, chemical reactors and the like. Fortunately this concept has also been applied
to the human body. The results of such analysis have helped improve clinical care,
suggested improvements in diagnostic and therapeutic devices, and led to mechanical
wonders such as artificial organs. Medical doctors and chemical engineers continue
to work hand in hand to help us live longer fuller lives.
To see a summary of each of the ten achievements and to learn more about the field of Chemical
Engineering visit: http://www.cems.umn.edu/~aiche_ug/history/h_whatis.html
Chemical Engineering Application and Inspiration. Chemical engineers at Washington State
University (WSU) are working on lowering costs in the identification, separation and mass
production of desirable proteins. This work seeks to produce an analytical tool for commercial
use in determining which particular protein is being expressed out of many possibilities. Control
6
of the removal of particular fractions for further chromatography analysis would allow minute
impurities to be identified and potentially removed from drug compounds. Designing the
apparatus is the task of Jeff Burke, a PhD candidate in Dr. Neil Ivory's laboratory.
Jeff Burke’s Work: Dynamic Field Gradient
Focusing of Proteins
 Electrophoretic focusing technique
 Analytical scale: 0.5-10 µg
 Separation of R-phycoerythrin,
Allo-phycocyanin, and myoglobin
(This apparatus is only 2 inches long!)
Isotachophoresis of Dyes
 Electrophoretic apparatus
 Preparative scale: 10-100mg
This apparatus was designed by
people working in Dr. Ivory’s
lab…You can’t buy this!
7
Background Information on Chromatography. “Chromatography is used in many different
industries and labs. The police and other investigators use chromatography to identify clues at a
crime scene like blood, ink, or drugs. More accurate chromatography in combination with
expensive equipment is used to make sure a food company‟s processes are working correctly and
they are creating the right product. This type of chromatography works the same way as regular
chromatography, but a scanner system in conjunction with a computer can be used to identify the
different chemicals and their amounts.
Chemists use chromatography in labs to track the progress of a reaction. By looking at the
sample spots on the chromatography plate, they can easily find out when the products start to
form and when the reactants have been used up (i.e., when the reaction is complete). Chemists
and biologists also use chromatography to identify the compounds present in a sample, such as
plants.” (Hess, 2008)
Type of
chromatography
Paper chromatography
Thin-layer
chromatography
Column
chromatography
Gas chromatography
"Physical Methods of Separating Mixtures
Mobile Phase
Stationary phase
Solvent such as methanol
Solvent such as ethyl
acetate
Solvent such as 2-propanol
Filter paper
A thin layer of an adsorbent solid such
as alumina or silica gel on the surface of
a flat sheet of plastic, glass, etc.
An adsorbent solid such as alumina or
silica gel, in a glass or plastic column
An adsorbent packing
An inert carrier gas such as
N2 or He
The type of mixture to be separated will determine the type of chromatography to be used."
(Rasheed, 2008)
8
Materials to Obtain Before Starting the Basic Module.
coffee filters. (We used “Melita, Junior Basket Coffee Filters")
Whatman qualitative filter paper in sheets or strips
chromatography solvents (water, acetone, methanol)
Mr. Sketch, Vis a Vis, and Flair pens in black, purple, and blue.
beakers or jars (400ml size)
large test tubes or jars with lids
Additional Materials Needed for Extension Activities.
C18 Sep-Pac® cartridge (available from Flinn Scientific)
10 mL syringe with male Luer® tip
1 mL syringe with male Luer® tip
Grape Kool-Aid® drink mix, unsweetened (1 pack)
2-propanol (2-propanol)
Vernier Spectrometer
Logger Pro 3.5
Prerequisite Knowledge and Skills. Students should have experience with science process
skills and perhaps notebooking. (See Appendix B for example.) Students should be able to use
glassware and follow safety instructions. No chemistry background is necessary for the basic
module activities. Experience with polarity and basic chemistry laboratory is useful for the
extension activities.
Safety precautions around solvents such as acetone and methanol should be observed. If these
solvents are provided, students should be aware not to breathe in the vapors and to keep
containers capped at all times or use a fume hood. Eye protection is always a good idea when
working with solvents.
Use of glassware may involve handling beakers, stirring rods and test tubes.
9
Daily Activities. Each day‟s activities are designed for 85 minutes periods although the module
could be divided into shorter periods.
Day One – First day of school! Classroom expectations are covered. Students review basic
notebooking and science process skills. A short survey and pre-activity assessment is given.
Initial chromatography experiment is done. See Appendices A, B and C.
Day Two – Students seminar (groups of 5-6) and discuss results with others (20 minutes). See
end of appendix C for example to use during seminar. Discussion of chromatography basics and
students are asked to pre-lab “Separation of Spinach Pigments” (Appendix D).
Day Three –Students separate spinach pigments. See appendix D addendum for teacher notes.
Day Four – Students seminar and discuss spinach chromatography results. Several seminar
groups will be asked to present their findings to the entire class. Discussion of what 'good'
notebooking and seminar discussion looks like will be included today. Include teacher-led
discussion of how chromatography works and possible applications as time permits.
Day Five –Students are presented with the inquiry activity in the form of a forensic challenge.
See Appendix E.
Day Six – Students finish any further experimentation and prepare findings to share in seminar.
Scoring guide for notebook evaluation and formal report is handed out (see Appendix F and G).
Day Seven-Seminar representatives present findings to the class. Wrap up with pre-assessment
retake. Notebooks and formal report due.
Evaluation. Students will have a short pre-assessment (see Appendix A) designed to give the
teacher an idea of student's knowledge about chemical engineering, chromatography and science
process skills. This will also be given at the end of the unit. In addition, students will be given a
rubric (see Appendix F) before turning in notebooks (see Appendix F for notebook example) so
they may check their own work before the teacher uses that same rubric to assign a score.
Examples of student notebook work will be shown using a document camera so that students
receive formative assessment and notebooking gets off to a good start. The formal lab report
(last activity) will require students to use their notebooks to clearly communicate their findings
(see Appendix G).
Extension. Column chromatography using C18 Sep-Pac® cartridges and grape-flavored KoolAid® allow advanced work in chromatography. If both activities are done, advanced math skills
are required. Students also use spectrometers (see Appendix H).
10
References.
Advanced Chemistry with Vernier, Vernier Software and Technology, Beaverton,
Oregon.
BSCS Science: An Inquiry Approach, Level 2, Kendall/Hunt Publishing Co., Dubuque,
IA, 2008
Hess, A., Paper Chromatography: Basic Version, http://www.sciencebuddies.org/sciencefair-projects/project_ideas/Chem_p008.shtml?from=Home
Pafko, W., The History of Chemical Engineering, September 2000,
http://www.pafko.com/history/h_whatis.html
Quach, H. T.; Steeper, R L.; Griffin, G. W., Separation of Plant Pigments by Thin Layer
Chromatography, J. Chem. Educ. 2004, 81, 385-7
Rasheed, L., Paper Chromatography, Spring 2008, http://www1.brcc.edu/turner/Chm101/
101%20Paper%20Chromatography.htm
Acknowledgements. Our thanks go to:
Dr. Neil Ivory and Jeff Burke for the opportunity to work with chromatography in their lab at
Washington State University.
Dr. Richard Zollars for the opportunity to work with chemical engineers and colleagues during
project SWEET at Washington State University and for his support and encouragement.
Dr. Donald C. Orlich of Washington State University for his advice and encouragement in the
writing of this module.
Dr. Ken Wareham of Lewis and Clark State College for his expertise in developing assessment
tools.
The National Science Foundation for the opportunity to perform unique research and for their
support in development of this curriculum.
11
Appendix A
Chromatography: pre-assessment
Name _____________________Per____
This assessment is given to check your understanding about the nature of science and certain
basic science concepts. It will NOT affect your grade. Please answer all items honestly.
1. Please finish the sentence.
A chemical engineer's job is to…
2. Please finish the sentence. Chromatography is a scientific technique designed to…
T = true, or I agree, F = not true, or I disagree, ? = I don‟t know, or, I am undecided
_____1.
Science is primarily a method for inventing new devices.
_____2.
Science can prove anything, solve any problem, or answer any question.
_____3.
Science is primarily concerned with understanding how the natural world works.
_____4.
Science involves dealing with many uncertainties.
_____5.
Science requires a lot of creative activity.
_____6.
Science always provides tentative (temporary) answers to questions.
_____7.
A "hypothesis" is just an "educated guess" about anything.
_____8. Science is most concerned with collecting facts.
_____9. Most engineers and medical doctors are practicing scientists.
_____10. Something that is "proven scientifically" is considered by scientists as being a fact,
and therefore no longer subject to change.
_____11. Science can be done poorly.
_____12. Science can study things and events from the past.
_____13. Knowledge of what science is, what it can and cannot do, and how it works, is
important for all educated people.
_____14. Different scientists may get different solutions to the same problem.
_____15. Disagreement between scientists is one of the weaknesses of science.
12
Appendix B
SCIENCE NOTEBOOKING
Leave 2 pages (1 front/back) for “Table of Contents”. Label this now.
3rd page: page #1, in upper right corner with this page (and then the outer, upper corner
thereafter). Trim and Tape the following onto page 3:
SCIENCE PROCESS SKILLS
1.
2.
3.
4.
5.
6.
Define the problem (ask the question)
Collect background information
Formulate a hypothesis: “If…then…because…” or “This happens because…
Design an experiment which will test the hypothesis (must be able to disprove it)
Keep accurate/complete records of data and observations
Interpret data and draw conclusions
LAB FORMATTING
1. Title (each activity/lab starts a new page) and Date (month/day/year) at the top of the page.
Use blue or black ink, no erasures. Instead of erasing or scribbles, use strikethrough only.
2. Objective/Focus/Purpose (usually a brief statement describing the how and why of the
activity. May be required to include background information for some labs).
3. Methods/Procedure (I did....) Write down what you are doing as you perform tasks and
make observations.
Pictures/diagrams are good!
Example:
Bubbles forming in the vinegar
solution and these bubbles seemed to
be coming out of the vinegar. Rapid
fizzing, eventually stopped bubbling
after 4 minutes.
1g of baking soda added
to 25ml of vinegar in a
250ml beaker
4. Data organized into table or chart (use ruler, include a title and units)
5. Analysis in the form of graphs and/or calculations (title and label graphs, label calculations so
it is obvious what they are for and always includes units)
6. Conclusion relates objective to data, discussion of precision/error, impact/importance (what
can we infer?). Conclusion is always written in paragraph form.
13
Appendix C
“I’M SEEING SPOTS!”
Scientists must be very observant and be able to describe their actions and results clearly. Here
is your opportunity to review and practice these skills…and find out something about
chromatography!
Follow the “LAB FORMATTING” handout. (You need a title, purpose….).
 At your station you should find:
o 3 beakers (2 large and 1 small)
o a dropper pipette
o 2 coffee filters, each with a black dot in the center (labeled A and B)
 Get about 25 mL of tap water in the small beaker.
 Place the coffee filters loosely on top of the large beakers (same position as if making
coffee).
 Draw a diagram of each set-up. Use this to explain your actions.
 Make a table to record what happens each time you add water, and final observations for
each dot.
 Using the pipette, place 1 drop of water from the small beaker on the center of each dot.
 Record your observations in your lab book.
 In order to continue the movement of the color, place 1-2 drops of water in several
places, about 1 cm behind the front of the ink line.
 When most of the ink reaches the pleated area of the filter, stop adding water and record
your final observations.
 Allow filter papers to dry. Please dry beakers, and return pipette and beakers where you
found them
 Trim and tape a section of your filter papers so that each person in your team has a piece.
Cut this fluted part off to
leave the circle at the
bottom. Then cut circle in
half or in thirds so that
each person has a piece
showing the results. Tape
into notebook.
Analysis/Conclusion:
In your lab book, copy the following sentence starters and complete the statements. (This should
make a paragraph!)
1. The ink on filter A …
2. The ink on filter B …
3. I think that the reason that the solvent (water) moved is…
4. I think that the similarity between the two is because…
5. I think that the difference between the two is because…
Be prepared to share your observation and conclusions with a larger group.
14
Teacher Notes: I'm Seeing Spots



Use 2 coffee filters per team and label A and B with a pencil.
Mark one filter with a spot in the center, using a 'Mr. Sketch' or 'Vis a Vis' transparency,
black marker. *These show more definite color if not made more than a day ahead.
Mark other filter with a spot using a 'Flair' black marker.
Spot should be about the size of a
pencil eraser.

'Flair' marker will not separate in water.
Probable Results

'Mr. Sketch‟ or “Vis a Vis‟ will separate in water.
15
SEMINAR AGENDA: “I’M SEEING SPOTS”
1. Introduce yourself to tablemates.
2. Compare data (filter paper slices) with the others at your table. Note similarities and
differences in your notebook under “SEMINAR” after your analysis/conclusion section.
3. Compare your „because‟ statements (from your analysis/conclusion) statements with
those at your table. Make any adjustments to your analysis/conclusion at this time and. . .
4. Explain why you changed or added to your analysis conclusion.
Teacher Notes: Seminars


students should be in groups of 5-6 and not with their lab team members. This requires
each lab team member to know what happened and be able to relay that to an 'outside'
group.
this should take 20 minutes the first time but after students get the hang of seminars, they
are more efficient at sharing and comparing results. If they are to present a consensus to
the class a bit more time should be allotted.
16
Appendix D
Paper Chromatography Using Spinach Leaves
Paper chromatography is a separation technique that can be used to separate all sorts of
molecules such as metals or pigments in solution. The substance to be separated is usually
dissolved in a solvent and then placed on chromatography paper. This paper is porous (feels
rough and fibrous) and allows a solvent to travel through it. As the solvent moves, it carries the
separated pigments or metals along with it. Some molecules travel farther and others are left
behind.
Pigments are large protein molecules and are what cause coloration in cells. You will be
looking at pigments found in spinach leaves. What color pigment do you expect to see on your
paper strip? Let‟s see what is really there.
In your notebook: Title, purpose (summarize introduction in one sentence), predict pigment
color. As you proceed, make a diagram and description using „label format‟ to indicate your
preparations.
Part A. Preparing Leaf Pigments and Making the Chromatogram
1. Put a small amount (just enough to cover the bottom) of methanol in the bottom of the
test tube and push the cork in lightly. Seal the tube but don‟t push so hard the cork gets
stuck! *METHANOL warning. Vapors may be toxic…take care not to breathe them.
*Set the tube aside (in
the test tube rack).
2. Tear up ½ of a spinach leaf into small pieces and place it in the mortar (grinding dish).
3. Add a small pinch of sand (like
adding salt to your soup!).
4. Add some acetone (fingernail polish remover). A
tablespoon or so. It should be soupy. Acetone is the
solvent used to dissolve components of the cell and
release the pigments.
5. Grind with the pestle until the liquid is very green.
Grind some more! This breaks open the spinach
cell walls to release the contents (including the
pigments). Grind until you have a smooth solution
without any visible pieces.
17
*Handle the paper strip so that you do not get finger oils
on the paper, as though you‟re handling a Compact Disc.
6.
7.
Prepare the paper strip by drawing a line, in pencil, about 2.5cm up from one end.
Use a glass stirring rod to add 4-5 drops of the spinach solution to the paper, across the line
(by touching the line). Stir each time you get a drop but only put liquid on your paper strip.
Let dry 20 seconds (or until it looks dry) and add another line of drops. Continue adding
drops in this way until you have 10 layers and a dark, green line. *Do not let the green
solution run down below the line very far.
8. Remove the stopper from the test tube and carefully, but quickly, slide
your paper down inside the tube so that the bottom edge, (but not the
green line) is in the methanol (solvent) at the bottom.
*If you slop solvent on your spinach solution you will have to make another
paper strip. Replace the stopper. It will hold your paper strip in place and
keep a saturated environment in the tube. Place in the rack and don't disturb
until it is time to collect data.
**Remember to diagram your apparatus and label it to indicate what
you did and what it looks like at the start of your experiment. See the
example on page 1 of your notebook if you need help.
9. While you are waiting for the solvent to move up the paper, wipe out your mortar and pestle,
with a paper, into the trash and then wash and dry it. Throw away trash. Be careful not to
disturb your chromatography chamber (test tube). While you are waiting for the solvent to
move up the paper, prepare a diagram and table in order to collect and analyze your data (see
ANALYSIS).
10. Remove the paper from the test tube when the solvent leaves the pigment(s) behind. This
18
should take about 25 minutes. Mark with a pencil where the solvent line stops. Lay the
paper on your table to air dry.
ANALYSIS:
 How many pigments do you see on your paper strip? What colors?
 Diagram and label a representation of your own paper strip.
Common plant pigments: Orange = carotene, Yellow = xanthophyll
Bright green = chlorophyll a, Khaki green = chlorophyll b, Anthocyanin = blue

Determine the retention factor for each pigment. Show all of your calculations (with
units) and organize your work into a table.
The retention factor, Rf, is a quantitative indication of pigment movement. It is defined as the
distance the solute (D1) moves divided by the distance traveled by the solvent front (D2)
Rf = D1 / D2
* In this experiment, the solutes are the pigments (what was dissolved)
and the solvent was the methanol.
**Here is a sample chromatogram to show you how to mark your paper strip.
1. Draw another pencil line down the area where you can
see the pigments most clearly and the bands/spots look
consistent.
Appendix F
2. Mark each segment of color and determine the midpoint
of the segment. How will you be most accurate in this?
Use this midpoint as D1 for each pigment.

After measuring your chromatogram (paper strip),
Cut it into thin strips so each lab team member can
tape a portion into their notebook alongside their
labeled diagram.
CONCLUSION: In your lab book, copy the following
sentence starters and complete the statements. (This should
make a paragraph!)
I expected spinach pigments to be _[color(s)]__ because…
Spinach actually contains __[number of]_ different pigments. My evidence is….
Pigments can be identified by their retention factor. I am confident/not confident in my numbers
because (describe any steps you took to be accurate and any difficulties you had)…
I think these pigments are on different places on my paper strip because…
Plants use pigment(s) to…
I think plants have more than one pigment because…
Chromatography is useful because…
Be Prepared to seminar and some seminar groups will be asked to present to the class.
19
Spinach Chromatography: Teacher Notes
These make a difference:
 students set up test tube with methanol first, stopper on. Methanol works best out of
various inexpensive solvents tried. (Flinn 'chromatography solvent' works well although
results will be different...see below.)
 grind spinach thoroughly, let solids settle, use stirring rod to transfer only liquid.
Likely results using methanol. Carotene is not
soluble and stays behind. Chlorophyll b is
khaki colored and is just above the carotene,
chlorophyll a is above that and xanthophyll is at
the top.
Markings for
determining
retention factors.
Pigments will fade so it is
important for students to
make a diagram or take a
picture as soon as possible.
Results using 'chromatography solvent' from Flinn
Scientific. (80-90% petroleum ether, 10-20% acetone)
The green in spinach is mainly due to chlorophyll a and chlorophyll b.
beta carotene
The yellow dyes in spinach include beta carotene and xanthophylls.
Quach, et al., 2004
20
Appendix E
CRIME SCENE INVESTIGATION
J.B. Quick was just one of “those” lab partners. When lab day came around, he always had an
excuse as to why he wasn‟t prepared. The excuses were fairly transparent, but he stuck to them.
When working in the lab, he always left a mess, with unknown liquids in puddles and in beakers.
Broken glass was often found at his lab station.
One day, the entire science building had to be evacuated, due to an experiment with
ammonium sulfide (the stuff that‟s in stink bombs) gone awry. It seemed like this might have
been the last straw for his teacher Dr. L.B. Blue, and lab partner L.M. Muffit, as months of their
research on gradient chromatography was destroyed when they were forced to leave the lab at an
inopportune time.
The next morning, Quick‟s lifeless body was found at the bottom of the stairs leading to
the second floor labs. He was clutching a piece of paper with some scribbled writing in his hand.
The police immediately suspected Blue and Muffit, as they were the people with the most
obvious motive for murder.
Recognizing that Blue always used a 'Vis a Vis' pen for work, while Muffit was addicted
to the sweet smell of 'Mr. Sketch' markers, it is up to you to determine the identity of the person
writing the note, and assumedly, the identity of the murderer. Perhaps, however, you can
exonerate both of them, leaving the authorities baffled.
Do you have all the cool toys we see on CSI Miami/Las Vegas/New York? Not a chance.
You‟re using paper chromatography. A bit rudimentary, perhaps, but it works.
In your somewhat rudimentary lab, you will have access to the following materials:
Solvents
Writing implements
 Alcohol (methanol)
 'Vis a Vis' pens in blue, black and
 Distilled water
purple
Chromatography materials
 10cm by 10 cm Whatman paper
 Strips of Whatman chromatography
paper
 Filter paper
 Coffee Filters


'Flair' pens in blue, black and purple
'Mr. Sketch' pens in blue black and
purple.
Labware
 beakers, test tubes, stoppers
 stirring rods, mortar/pestle
*Follow proper formatting in your lab book as you proceed. Keep detailed records.
Report (Evaluation). In addition to your lab book, you will compile a one page, word
processed report to the prosecution summarizing your laboratory techniques and results. Include:
 Purpose
 Briefly describe procedure. (What did you do? This includes everything, not just what
you consider to be your successful trial.)
 Why did you choose to proceed in a particular fashion (reasons/background)?
 Your results (include a visual for clarity). Any calculations made.
 Your conclusion. (Who is guilty and evidence to support your choice)
21
Teacher Page for Determination of the Murder of J.B. Quick
This activity is designed to be done as an inquiry. Students will have to rely on experiences from the first
2 activities in order to be successful. The less direction given, the more they will have to experiment in
order to find the correct method.
Ideas that might be brought out include:
 Students need to determine the original color of the pen used.
 Students will need to build standard chromatograms from the known pens for comparison
 Using vertical samples of the unknown – not the entire sheet at once.
o It‟s the teacher‟s decision as to what to do if the entire sheet us used. We recommend that
another sheet be given, but that it might or might not be the same pen, thus they‟ll have to
start over.
 Seminar groups will need to be determined. Generally these are made up of representatives of
different lab groups. Size generally should not be greater than 6 unless that‟s unavoidable.
 „Seminaring‟ on this project might be done more than once. First after the first day of the project,
students might want to see what others are doing to solve the problem. After the activity is done
or almost done, students should compare their results and findings. Note that groups will
generally not have the same suspect, but the techniques of solution should be similar.
Samples of the 'Knowns':
Blue pens. Left to right:
Vis a Vis
Flair
Mr. Sketch
Black pens. Left to right:
Vis aVis
Flair
Mr. Sketch
Purple pens. Left to right:
Vis a Vis
Flair
Mr. Sketch
We made a pencil line and then wrote
along it in even, small letters. We
recommend not writing the note more
than 3-4 days before use.
Students will obtain slightly different results when comparing the „murder note‟ with standards they make
with fresh ink. This may be a variable they will have to account for.
22
Appendix F
Lab Journal Check: Chromatography Unit
Name ________________________Per_____
Seeing Spots
"C.S.I" Pen Analysis
_____title and date (1)
_____title and date (1)
_____Purpose is clear (1)
_____ purpose is clear (1)
_____procedure(s) clearly diagrammed and
labeled; someone else could recreate your
work (5)
_____ procedure(s) clearly diagrammed and
labeled; someone else could recreate your
work (6)
_____observations clear, every three minutes,
informative; and organized in a titled,
labeled table(5)
_____ results are diagrammed, titled, labeled and
complete for any and all trials (6)
sample of actual materials included (2),
organized and easy to follow (2)
_____ Analysis/Conclusion clearly compares A
and B filters; answers to 'I think'
_____ results are analyzed using mathematical
questions show thoughtful consideration (5)
calculations (shown and explained) (10)
_____ seminar discussion/revisions clear (3)
_____ conclusion includes discussion of
evidence supporting your findings (7)
_____ Total (20)
Paper Chromatography (Spinach Leaf)
_____title and date (1)
_____purpose is clear and prediction made (2)
_____procedure(s) clearly diagrammed and
labeled; someone else could recreate
your work (5)
_____data is diagram and section of actual
paper strip - both labeled with pigment
colors/molecule names(6)
______analysis includes Rf for each pigment
organized into a table, with all
calculations, units/labels shown (10)
_____ Total (35)
Lab Journal Format
_____ table of contents updated (2)
_____ pages numbered properly(2)
_____ no erasures, strikethrough only (2)
_____ no doodles (2)
_____writing is readable (2)
_____ Total (10)
_____conclusion includes completion of all 8
sentence starters organized into a
paragraph. (8)
________ TOTAL
(100)
_____seminar discussion notes include
similarities and differences between team
results (3)
______Total (35 pts)
23
Notebook (first 2 pages) Example of "Seeing Spots" Activity.
24
Appendix G
Assessment of Formal CSI Report
Research Process
Needs Rewrite
Name_______________________Per____
Developing
Proficient
*The research
*The research purpose is
Research
described but some detail is
Purpose/Obj. purpose is not
What do I want described.
missing.
to find out?
Points (10)
*A description of the *A description of the methods
Procedure:
How will I find methods of data
of data collection is incomplete.
out?
collection is absent *Diagrams not labeled
or seriously flawed. completely or difficult to
follow.
And:
Why am I
*Limited
choosing a
background
*Background information is
particular
information is
provided and it accurate.
method?
provided or has
.
obvious mistakes
Points (25)
*The research purpose is
described clearly, in great detail.
*The information
Results of
collected is
Study:
What
incompletely
information did displayed or
I collect from described.
my experiment? *Limited or no
visuals.
Points (25)
*The information collected
adequately reflects the stated
procedure.
*Visuals included.
.
*The information collected is
highly detailed and accurate and
is clearly displayed and
explained.
*The conclusion
Conclusion:
What did I find does not
out?
communicate the
meaning of the
results.
*The conclusion adequately
communicates the meaning of
the results and makes use of
inferences or deductions.
* Some evidence cited.
*The conclusion clearly
communicates the meaning of
the results and makes
comparisons, interpretations,
inferences or deductions from the
data or information.
*Claims supported by evidence.
*Most components and
formatting appropriate.
*Title, Name, Date, Class Per.
*Word processed
*Single page
*Work summarized clearly and
concisely
*There is a highly detailed
description of the methods of
data collection. Someone could
recreate the work.
*Background information is
accurate and comprehensive.
Points (25)
*Components
Format:
Did I follow
missing.
proper
formatting for a
formal report?
Points (15)
Total Points (100)
25
Appendix H
COLUMN CHROMATOGRAPHY
This procedure is a modification of experiment 18 “Liquid Chromatography” as found in
Advanced Chemistry with Vernier, published by Vernier Software and Technology, Beaverton
Oregon. Used with permission.
Background.
Chromatography is a process used to separate the components of a mixture. Like paper
chromatography, a soluble mixture is placed on a solid substrate. The degree to which it is
adsorbed onto the substrate determines how far and how fast it travels.
In column chromatography, a mixture is injected into a chromatography column, where it lands
on a substrate, also known as the stationary phase. The stationary phase may be polar, attracting
polar substances, or non-polar, attracting non-polar substances. Next, a solvent is injected into
the column. The solvent is called the mobile phase. As the solvent moves along the stationary
phase, it will carry the components with it. When and how quickly the substances are carried out
of the column by the solvent depends on properties such as the molecule size or polarity of the
substances and their solubility in the solvent. If the solubility's and/or polarities of the individual
parts of the mixture are significantly different, the substances in the mixture will separate from
each other as the mixture travels along the substrate. The substance that is the most strongly
attracted to the solvent will be the first to move through the chromatography column.
In this experiment, you will use column chromatography to separate the dyes, FD&C Blue and
FD&C Red that are found in grape-flavored Kool-Aid®, from the other ingredients in the dry
drink product. You will use a special column, called a C18 Sep-Pac® for the experiment. This
column contains a silica solid with a C18 hydrocarbon bonded to it, which renders the solid nonpolar.
Vocabulary.
 Stationary phase; the solid material in the column.
 Mobile phase; the solvent used for the column, also called eluate.
 Eluent/Eluting; the sample that comes off a column/the process of the sample coming off
a column
 Wash/Washing; solvent used to clean and moisten the column prior to loading the
sample.
Summary. There are two parts to this experiment.
 Part I is an isocratic separation, in which one solvent passes through the column at a
specified rate. This process allows you to separate the two food dyes from the other
ingredients in the mixture. Eluents will be placed in a spectrometer to obtain
absorbance spectra for analysis.
 Part II is a step gradient separation. In this process, three solvents are used (each of a
different polarity and concentration) to separate the substances in the mixture. Eluents
will be placed in a spectrometer to obtain absorbance spectra for analysis.
26
MATERIALS
C18 Sep-Pac® cartridge
10 mL syringe with male Luer® tip
1 mL syringe with male Luer® tip
two 10 mL graduated cylinders
two 25 mL graduated cylinders
2-propanol (2-propanol)
Grape Kool-Aid® drink mix, unsweetened
dissolved in 2L distilled water
four 50 mL beakers
three 100 mL beakers
cuvettes
PROCEDURE
Part I: Isocratic Separation
1. Obtain a 10 mL sample of grape Kool-Aid in a small beaker.
2. Prepare the solvent (mobile phase) in a 100 mL beaker by mixing 9.3 mL of
2-propanol with 40.7 mL of distilled water to make an 18% (v/v) 2-propanol solution.
3. Wash the C18 Sep-Pac column chromatography cartridge as follows:

Fill a 10 mL syringe with undiluted 2-propanol. Attach the tip of the syringe to
the long end of the Sep-Pac cartridge and inject the 2-propanol into the column at
a rate of 1 drop/sec. Collect the eluate into a small beaker. Once you are confident
of your ability to control the rate of drops you may push the eluent through faster.

Wash (in the same way) the Sep-Pac cartridge with 10 mL of distilled water.
4. Load the cartridge:
 Use a 1 mL syringe to draw up 1 mL of your
sample of grape Kool-Aid.
 Slowly inject the 1 mL of Kool-Aid into the SepPac cartridge.
 Collect and discard the effluent that washes out of
the column as you inject the sample. (It is just
wash water left in column.)
27
5. Elute the components of the grape Kool-Aid sample.

Fill the larger syringe with exactly 10 mL 18% 2-propanol solution (the solvent).
Note your starting volume. (Should be 10ml on the syringe.)

Set up a small beaker to collect the dyes as they leave the column.

Slowly inject the 18% 2-propanol solution into the column at the steady rate of 1
drop/sec until the red dye has been eluted and the next drop is blue-ish.
Note the volume on the syringe.

Set up a second beaker for collection of the blue dye.

Continue to inject the18% 2-propanol solution into the column at the steady rate
of 1 drop/sec until the blue dye has been eluted and the next drop is clear.
Note the volume on the syringe.
NOTE: If there is not a perfect separation of the red and blue bands you will see purple.
Record the data (syringe volumes) for the beginning and end of the purple band. Use the
mid-point of the purple volume, as the end of the red band and the beginning of the blue
band.

Perform two more trials by washing the Sep-Pac cartridge and loading a new
sample of the Kool-Aid. Proceed with Step 4. Make a data table for each trial.
See a sample table below. Remember to add a column for purple if you need to.
6. Wash the Sep-Pak column with 5ml undiluted 2-propanol and then 5ml distilled water.
DO NOT THROW AWAY...THESE ARE REUSABLE
*Please put all waste solutions (except water) in the container labeled 'CHROMATOGRAPHY
WASTE'. You will find this in the fume hood.
28
Isocratic Separation Data
Red Dye
Blue Dye
Sep-Pak
specifications
VR (starting volume)
VR (ending volume)
W (VR ending – VR starting)
Vavg (VR starting + 0.5W
L (length of Sep-Pak)
1.25cm
r (radius of Sep-Pak)
0.5cm
VM (see note below)
k' (see note below)
α (see note below)
N (see note below)
R (see note below)
* VM is the mobile phase volume, determined by the following equation: VM = 0.5 πr2L. This
factor represents about half of the total empty column volume. The unit for VM will be cm3
(or mL) if the values of r and L are measured in cm.
* k ′ is the capacity factor, which is a unit-less measure of the retention for each of the dyes and
is determined by solving the following equation: k ′ = (VRavg – VM)/ VM. In this experiment,
you will calculate k′ values for each dye. (Optimum values of k ′ are commonly between 1
and 10.)
* α is the selectivity, or separation, factor and it is the ratio of the separation of the k′ values. In
this experiment, you will calculate one selectivity factor, because you separated only two
substances (the two food dyes). The equation for the selectivity factor is: α = k ′2 / k ′1. The
value of α is always larger than 1, therefore you will use the larger of your k′ values as k ′2.
* N represents the number of theoretical plates in the column. Think of N as the number of times
a dye molecule is exchanged back and forth between the stationary phase (the silica in the
column) and the mobile phase (the isopropanol solution). The equation for N is: N = 16
29
(VR/W)2. The value of N is generally based on the dye which is eluted last. A large value of N
means that the column is more efficient. The range of N values is normally between 20 and
200.
*R is the resolution, which is the major objective of a chromatographic separation. R measures
how well the two dyes were separated by the Sep-Pac cartridge. The equation for R is:
R = (VR1 – VR2) / 0.5 (W1 + W2). The numerator is the volume between the bands made by the
two dyes when they were in the column, which is related to the selectivity factor (α). The
denominator is the average band width, which is proportional to the efficiency of the column.
As the value of R increases above a value of 1, there is much greater total separation of the
dyes.
Finding the Absorbance Spectrum.
1. Take your blue and red dye samples (and purple if you have it) and obtain absorbance spectra
of each using the following protocol:
 Pour each dye into a separate cuvette, supplied by the teacher. (2/3 full).
 Take the cuvettes to the spectrometer (spec), which will be on and calibrated.
 Establish a “new” document. A spectrum will appear by default.
 Place your red dye into the spec. Make sure you do not touch the clear sides and
that the cuvette is placed into the (spec) properly.

1. Click start (on tool bar).
2. Observe the spectrum
3. Click stop
4. Press ctrl-L to save the run.
Replace the red dye cuvette with the blue dye cuvette and repeat steps 4 a-d.
2. Save the document on a memory stick or on the I:/ drive if available, and take it to your
computer to work with it. Save it on your own H:/ drive. Make sure your partner also has a
copy on their drive. Import into a word document and 'smallify' so that you can print, trim
and tape into your notebook.
ANALYSIS (Be prepared to discuss in seminar)
 Complete all calculations. Show your work so someone else can follow your thinking.
 Compare your calculated values to the normal range of values as given in the data
descriptions for k', α, N, and R. If you are not within acceptable range, discuss sources of
error.
 The most important factor is R. What do your results indicate?
 Describe your spectra (number and location of peaks).
 What does your spectra tell you about each dye?
 Use the 'analyze/examine' function on Logger Pro to collect accurate numbers. Describe
how you decided which number to use.
CONCLUSION (Be prepared to discuss in seminar)
 Isocratic separation is/isn't efficient because...
(Hint: use R values and absorbance spectra to support your statement)
30
Teacher Notes:
Sample Data...
Isocratic Separation Data
Red Dye
Blue Dye
VR (starting volume)
0.0mL
0.8 mL
VR (ending volume)
0.8 mL
3.3 mL
W (VR ending – VR starting)
0.8 mL
2.5 mL
Vavg (VR starting + 0.5W
1.4 mL
3.1 mL
Sep-Pak
specifications
L (length of Sep-Pak)
1.25cm
r (radius of Sep-Pak)
0.5cm
VM (see note below)
0.49 mL
k' (see note below)
1.8
5.2
α (see note below)
2.9
N (see note below)
25
R (see note below)
1.0
* VM is the mobile phase volume, determined by the following equation: VM = 0.5 πr2L. This
factor represents about half of the total empty column volume. The unit for VM will be cm3
(or mL) if the values of r and L are measured in cm.
VM = .5π r2L = .5 π (0.50 cm)2 1.25 cm = 0.49cm3
* k ′ is the capacity factor, which is a unit-less measure of the retention for each of the dyes and
is determined by solving the following equation: k ′ = (VRavg – VM)/ VM. In this experiment,
you will calculate k′ values for each dye. (Optimum values of k ′ are commonly between 1
and 10.)
k’ = (VRavg- VM) / VM
k’red = (1.4 mL – 0.49 mL)/0.49 mL = 1.86
k’blue = ( 3.1 mL -0.49 mL) / 0.49 mL = 5.3
31
* α is the selectivity, or separation, factor and it is the ratio of the separation of the k′ values. In
this experiment, you will calculate one selectivity factor, because you separated only two
substances (the two food dyes). The equation for the selectivity factor is: α = k ′2 / k ′1. The
value of α is always larger than 1, therefore you will use the larger of your k′ values as k ′2.
α = k’2/k’1 = 5.3/1.9 = 2.8
* N represents the number of theoretical plates in the column. Think of N as the number of times
a dye molecule is exchanged back and forth between the stationary phase (the silica in the
column) and the mobile phase (the isopropanol solution). The equation for N is: N = 16
(VR/W)2. The value of N is generally based on the dye which is eluted last. A large value of N
means that the column is more efficient. The range of N values is normally between 20 and
200.
N = 16 (VR/W)2 = 16(3.1mL/2.5mL)2 = 24.6 ≈ 25
*R is the resolution, which is the major objective of a chromatographic separation. R measures
how well the two dyes were separated by the Sep-Pac cartridge. The equation for R is:
R = (VR1 – VR2) / 0.5 (W1 + W2). The numerator is the volume between the bands made by the
two dyes when they were in the column, which is related to the selectivity factor (α). The
denominator is the average band width, which is proportional to the efficiency of the column.
As the value of R increases above a value of 1, there is much greater total separation of the
dyes.
R =(VR1 – VR2)/0.5(W1 + W2)= (3.1mL-1.4mL)/(.5(0.8mL + 2.5 mL) =1.03 ≈ 1.0
Students should be able to discern
these eluents.
Waste disposal – place an open container in a fume hood or really well ventilated area. Solvents
will evaporate.
32
Part II: Step Gradient Separation
1. Prepare the solvents (mobile phase).

Mix 2.45 mL of 70% 2-propanol with 47.55 mL of distilled water into a 100 mL
beaker to make a 5% 2-propanol solution.
Mix 14 mL of 70% 2-propanol with 36 mL of distilled water into a 100 mL beaker to
make a 28% 2-propanol solution.
Use distilled water as the third solvent for the step gradient separation.


2. Wash the C18 Sep-Pac column chromatography cartridge as follows:
 Fill a 10 mL syringe with undiluted 2-propanol. Attach the tip of the syringe to the long
end of the Sep-Pac cartridge and inject the 2-propanol into the column at a rate of 1
drop/sec. Collect the eluate into a small beaker. Once you are confident of your ability
to control the rate of drops you may push the eluent through faster.

Wash (in the same way) the Sep-Pac cartridge with 10 mL of distilled water.
3. Load the cartridge:
 Use a 1 mL syringe to draw up 1 mL of your sample of
grape Kool-Aid.
 Slowly inject the 1 mL of Kool-Aid into the Sep-Pac
cartridge.
 Collect and discard the effluent that washes out of the
column as you inject the sample. (It is just wash water
left in column.)
4.
4. Elute the components of the grape Kool-Aid sample and separate by the step gradient
process. You will need 4 small beakers.

Beaker 1: Use the larger syringe, inject 5 mL of distilled water through the column to
elute the polar components. Rate should be 1 drop/sec.

Beaker 2: Inject 5 to 10 mL of 5% 2-propanol solution through the column to elute the
red dye. Stop as soon as the red dye is out. Rate should be 1 drop/sec.

Beaker 3: Inject 5 to 10 mL of 28% 2-propanol solution through the column to elute the
blue dye. Again, stop when the blue dye is out. Rate should be 1 drop/sec.

Beaker 4: Inject 8 mL of 70% 2-propanol solution through the column to elute flavor
oils and other non-polar ingredients. Rate should be 1 drop/sec.
33
Finding the Absorbance Spectrum for the Step Gradient Separation.
5. Determine and save the absorbance spectrum for the red and blue dyes only.
 Pour each dye into a separate cuvette, supplied by the teacher. (2/3 full).
 Take the two cuvettes to the spectrometer (spec), which will be on and calibrated.
 Establish a “new” document. A spectrum will appear by default.
 Place your red dye into the spec. Make sure you do not touch the clear sides and
that the cuvette is placed into the (spec) properly.
Click start (on tool bar).
Observe the spectrum
Click stop
Press ctrl-L to save the run.

ribbed
sides may
be
touched,
clear
should
face light
beam
Replace the red dye with the blue dye and repeat steps 4 a-d.
6. Save the document on a memory stick or on the I:/ drive if available, and take it to your
computer to work with it. Save it on your own H:/ drive. Make sure your partner also has a
copy on their drive. Import into a word document and 'smallify' so that you can print, trim
and tape into your notebook.
Additional Observations on the Four Beakers.
7. Have one team from your lab bench place the four beakers of eluents in a hood (labeled) and
allow the solvents to evaporate. When the beakers are dry, observe the contents of each
beaker and record your observations in your lab book.
8. Wash the Sep-Pak column with 5ml undiluted 2-propanol and then 5ml distilled water.
DO NOT THROW AWAY...THESE ARE REUSABLE
*Please put all waste solutions (except water) in the container labeled
'CHROMATOGRAPHY WASTE'. You will find the container in the fume hood.
ANALYSIS (Be prepared to discuss in seminar)

Describe the contents of the four 50 mL beakers in which you collected the
various ingredients of the grape Kool-Aid mix during your step gradient
separation.

Look at the step gradient spectra. How many peaks do you see for each dye?
Describe your spectra and discuss the number and location of peaks. Use the
'analyze/examine' function on Logger Pro to collect accurate numbers. Describe
how you decided which number to use.
34

After solvent has evaporated (from beakers in fume hood), observe by noting
color, texture (DO NOT TOUCH with skin), and odor.
CONCLUSION (Be prepared to discuss in seminar)
 Your solvent solutions were made from highly polar water and highly non-polar
2-propanol. The reason the polarity of the solvents change even though the chemicals do
not is because…

The difference between isocratic and step gradient separations is...

Similarities between isocratic and step gradient separations are...

My spectra results and direct observations indicate __(method)__ is more efficient at
completely separating the dyes. The data to support this is....
35
Teacher page. Step-gradient chromatography.
Like paper chromatography, liquid chromatography relies on differential adhesion of a solute to
a stationary substrate in the presence of a moving solvent. The stationary phase may be polar,
attracting polar substances or non polar, attracting non polar substances. In the case of the work
done here, the substrate is a column of silica solid, a polar substance to which are attached a
myriad of C 18 chains, which renders the substance non-polar. (Vernier, p 18-1) Specifically,
this product will be contained in a small cartridge, called a SEP-PAK cartridge.
Initially the substance to be separated is grape Kool-Aid ®, a purple
substance, having two different dyes, FD&C red and blue (find numbers).
The solvent you will use will be 2-propanol, see left.
The protocol for the actual separation is included by permission of Vernier
Software and Technology of Beaverton, Oregon.
Summarizing the protocol; for the first test of separation, the cartridge is loaded with 1 mL of
grape Kool Aid. A 17% (V/v) solution of 2-propanol is then run through the loaded cartridge.
The red dye separates nicely, followed by the blue dye. There is an overlap between the two,
allowing a purple color to be eluted for a short time. In this protocol, measurements for the
amount of each color in the compound are made and a retention factor for each is determined.
In the second test of separation, two very different concentrations of 2-propanol are run through.
Distilled water will first elute the most polar substances. The 5% solution will elute the red dye,
but not the blue. A much more concentrated solution, 28% (v/v) of alcohol will elute the blue
dye. A more fully concentrated (70%) solution will elute the flavors and any other materials. In
the protocol, the student is told to allow the 4 beakers to evaporate in a fume hood, and then
observe the results. The purpose for this is to look at the uncolored substances to show that
something was actually separated during the first and fourth phases.
It is not clear in the student documentation how increasing the amount of alcohol will change the
polarity of the solvent. The reason for the increasing ability to carry compounds from the column
has to do with the decreasing amount of highly polar water in the solution, thus actually
decreasing the polarity of the solvent.
The final separated samples are then placed in a Vernier (Ocean Optics) spectrometer for
analysis of purity. The isocratic elution showed a marked overlap of colors. The step gradient
showed very little overlap, indicating a much more complete separation.
If the eluent samples are allowed to
evaporate in a fume hood, one beaker should
have a distinct odor (safe to sniff). You may
want to have hand lenses or a microscope
available for further observations but
students should not touch the residue.
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Absorption spectra of the two mixtures
resulting from an isocratic separation of
grape Kool Aid. Note that both samples
have some degree of each dye as a
component.
Absorption spectra of the two mixtures
resulting from a step gradient separation of
the grape Kool Aid. Note that there is very
little if any absorption of more than one dye.
This is how the spectra appear on the Logger Pro screens.
Teacher notes
A spectrometer should be on and calibrated at a central point in the room. Students can take their
measurements quickly if each team has 2 cuvettes.
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