An Inquiry-based Teaching Module Using the Separation Techniques of the Chemical Engineer Donald M c Quarrie 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.
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An Inquiry-based Teaching Module Using the Separation
Techniques of the Chemical Engineer
Donald M
cQuarrie
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
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
21
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
Alcohol (methanol)
Distilled water
Chromatography materials
10cm by 10 cm Whatman paper
Strips of Whatman chromatography
paper
Filter paper
Coffee Filters
*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)
Writing implements
'Vis a Vis' pens in blue, black and
purple
'Flair' pens in blue, black and purple
'Mr. Sketch' pens in blue black and
purple.
Labware
beakers, test tubes, stoppers
stirring rods, mortar/pestle
22
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':
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.
Black pens. Left to right:
Vis aVis
Flair
Mr. Sketch
Blue pens. Left to right:
Vis a Vis
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.
23
Appendix F
Lab Journal Check: Chromatography Unit Name ________________________Per_____
Seeing Spots
_____title and date (1)
_____Purpose is clear (1)
_____procedure(s) clearly diagrammed and
labeled; someone else could recreate your
work (5)
_____observations clear, every three minutes,
informative; and organized in a titled,
labeled table(5)
_____ Analysis/Conclusion clearly compares A
and B filters; answers to 'I think'
questions show thoughtful consideration (5)
_____ seminar discussion/revisions clear (3)
_____ 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)
_____conclusion includes completion of all 8
sentence starters organized into a
paragraph. (8)
_____seminar discussion notes include
similarities and differences between team
results (3) ______Total (35 pts)
"C.S.I" Pen Analysis _____title and date (1)
_____ purpose is clear (1)
_____ procedure(s) clearly diagrammed and
labeled; someone else could recreate your
work (6)
_____ 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)
_____ results are analyzed using mathematical
calculations (shown and explained) (10)
_____ conclusion includes discussion of
evidence supporting your findings (7)
_____ 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)
________ TOTAL
(100)
24
Notebook (first 2 pages) Example of "Seeing Spots" Activity.
25
Appendix G
Assessment of Formal CSI Report Name_______________________Per____
Research Process Needs Rewrite Developing Proficient
Research
Purpose/Obj.
What do I want
to find out?
*The research
purpose is not
described.
*The research purpose is
described but some detail is
missing.
*The research purpose is
described clearly, in great detail.
Points (10)
Procedure:
How will I find
out?
And:
Why am I
choosing a
particular
method?
*A description of the
methods of data
collection is absent
or seriously flawed.
*Limited
background
information is
provided or has
obvious mistakes
*A description of the methods
of data collection is incomplete.
*Diagrams not labeled
completely or difficult to
follow.
*Background information is
provided and it accurate.
.
*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)
Results of
Study:
What
information did
I collect from
my experiment?
*The information
collected is
incompletely
displayed or
described.
*Limited or no
visuals.
*The information collected
adequately reflects the stated
procedure.
*Visuals included.
.
*The information collected is
highly detailed and accurate and
is clearly displayed and
explained.
Points (25)
Conclusion:
What did I find
out?
*The conclusion
does not
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.
Points (25)
Format:
Did I follow
proper
formatting for a
formal report?
*Components
missing.
*Most components and
formatting appropriate.
*Title, Name, Date, Class Per.
*Word processed
*Single page
*Work summarized clearly and
concisely
Points (15)
Total Points (100)
26
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 non-polar.
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.
27
MATERIALS
C18 Sep-Pac® cartridge 2-propanol (2-propanol)
10 mL syringe with male Luer® tip
Grape Kool-Aid
® drink mix, unsweetened
dissolved in 2L distilled water 1 mL syringe with male Luer
® tip four 50 mL beakers
two 10 mL graduated cylinders three 100 mL beakers two 25 mL graduated cylinders 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 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.)
28
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.
29
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
30
(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
(Hint: use R values and absorbance spectra to support your statement)
31
Teacher Notes:
Sample Data...
Isocratic Separation Data
Red Dye Blue Dye Sep-Pak
specifications
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
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 = 0.49cm
3
* 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
32
* 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.
Waste disposal – place an open container in a fume hood or really well ventilated area. Solvents will evaporate.
Students should be able to discern
these eluents.
33
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.
34
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.
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.
ribbed
sides may
be
touched,
clear
should
face light
beam
Click start (on tool bar).
Observe the spectrum
Click stop
Press ctrl-L to save the run.
35
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....
36
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.
37
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