Phospholipids, Dietary Supplements, and Chicken Eggs: An Inquiry-Based Exercise Using

Thin Layer Chromatography

Sara E. Potteiger, Julie M. Belanger*

Supplemental Information Contents

Instructor Notes: Pages 2-11, as follows:

Table of examples of supplements (with ingredients) used…………….. Page 2

Chemical and equipment list…………………………………………….. Pages 3

Methods and related references ……………………………….……....... Pages 4 – 5

Answer key to the optional pre-lab activity …………………………..... Page 6

Answer key to the student handout ..……………………………..…….. Pages 7 – 11

Student handouts:

Student optional pre-lab activity………………………………………… Page 12

Student in-lab handout………………………………………………..…. Pages 13 – 19

Assessment:

Sample student data………………………………………………………. Page 20

Summary of assessment data…………………………………………….. Page 21– 22

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 Instructor Notes

The supplementary information is separated into two sections: one for the instructor’s reference
and one for the student handout. It is highly recommended that to preserve the guided-inquiry
nature of the exercise that the instructor notes are not provided to the students, but instead as a
resource for the instructor to help guide the students’ understanding during the laboratory
experiment.

Dietary supplements that were chosen are shown in Table 1S. Some contained various lipids
(especially lecithin, which is mostly phosphatidylcholine), whereas fish oil was chosen as a
negative control. The students were not told ahead of time that this sample should not give them
a positive result and care was taken to remove the ingredient list from the labels of the bottles.

Before the students ran the experiment, samples were tested via TLC for the instructor’s
reference with the chosen samples, and results were obtained for each one of the stains. Ideal
TLC plate results are shown in the student handout answer key (page 8) and sample student data
is shown on page 20. Pure lipid samples (obtained from Avanti Polar Lipids) were included for
reference in the instructor’s TLC data, however, students participating in the guided-inquiry
experiment did not run the standard lipids, since they were given the relative Rf values instead.
Actual Rf values for the lipids (as reference to the instructor) are also given in the student
handout answer key.

Table 1S: Suggested dietary supplements purchased from a local pharmacy. Supplements should
be selected that contain phospholipids (krill oil, lecithin), as well as some that do not (fish oil).
Substance Source Ingredients
Omega-3 Fatty Acids (300 mg) (EPA
Fish Oil Anchovy, mackerel, (eicosapentaenoic acid), DHA (docosahexaenoic
(1000 mg) sardine acid), Other Fatty acids), Gelatin, Glycerin,
Mixed Natural Tocopherols
Krill Oil (300 mg) (Omega-3 Fatty Acids (90
100% Pure
mg), EPA (eicosapentaenoic acid) (50 mg),
(300 mg)
Crustacean shellfish (krill) DHA (docosahexaenoic acid) (24 mg),
Omega-3 Krill
Phospholipids (130 mg)), gelatin, glycerin,
Oil
sorbitol, purified water, natural flavor
Soy Lecithin (8000 mg) (Linoleic acid (2160
Vegetarian
mg), Phosphatidylcholine (1680 mg),
Lecithin Soy
Phosphatidylinositol (960 mg), Linolenic acid
Granules
(240 mg))
Soy Lecithin (1200 mg) (Phosphatidylcholine
Lecithin
Soy (168 mg), Phosphatidylinositol (96 mg)), soy
(1200 mg)
lecithin, gelatin (bovine), glycerin

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Chemical Information

The natural materials needed for extraction are vegetarian lecithin, soy lecithin, fish oil,
krill oil, and raw chicken eggs. The chemicals (solvents and spray ingredients) needed are
bismuth nitrate (CAS#[10035-06-0], ACS reagent grade, MP Biomedicals), ninhydrin
(CAS#[485-47-2], Aldrich Chemical Co.), glacial acetic acid (CAS#[64-19-7], 99.7%,
Spectrum), sodium nitrite (CAS#[7632-00-0], 98.7%, Fisher Scientific Co.), potassium iodide
(CAS#[7681-11-0], 99%, Aldrich), 1-butanol (CAS#[71-36-3], 99.9%, Sigma Aldrich),
ammonium molybdate (CAS#[12054-85-2], 82.3%, Fisher Scientific Co.), copper wire or tinsel
(CAS#[7440-50-8], 99.9%, J.T. Baker), concentrated sulfuric acid (CAS#[7664-93-9], 95-98%,
Fisher Scientific Co.), chloroform (CAS#[67-66-3], 99.8%, Sigma Aldrich), and methanol
(CAS#[67-56-1], 99.8%, Sigma Aldrich), iodine (CAS#[7553-56-2], 99.8%, Aldrich Chemical
Co.). The lipid standards used for the authors’ “ideal data” TLC were obtained from Avanti Polar
Lipids Inc.

Equipment List (per group)

• Beakers • Glass Pipettes with Bulbs

o 250 mL • Spatulas
o 400 mL (3) • Scoopulas
o 600 mL • Forceps
• Graduated Cylinders: • Aluminum Backed TLC Plates (20 x
o 100 mL (3) 20 cm, Aldrich catalog number
o 10 mL (2) 56524)
• 25 mL Vials (with lids) (5) or 5 large • Aluminum Foil
glass test tubes with a test tube • Ruler
holder • Scissors
• 250 mL Flask (preferred: with • Pencils
stopper) • Paper Cup
• Buchner Funnel • Paper Towels
• Watch Glass(s) • Weight Boats
• Hot Plate • Rubber tubing
• Filter Paper (4) • Colored Pencils
• Sprayer (50 mL tube-type, Aldrich • Safety Glasses/Gloves
catalog number Z126292 or a similar
sprayer, although a dip method may
be used for some stains)
• Rubber Policemen
• Capillary Tubes
• 10 mL Pipettes with Pipette Bulb

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Methods

A detailed procedure is outlined in the student handout. The below comments provide
information on how to prepare the sprays and dip and some helpful hints on the set-up for the
lab.
Each spray and dip made was adapted from previously published material. The
molybdenum sprayS2 contained 0.08 g Cu, 0.25 g of ammonium molybdate and 1 mL of de-
ionized water that were added to a glass scintillation vial. The vial was then chilled in an ice bath
for approximately 20 minutes. A milliliter of concentrated sulfuric acid was added slowly to the
vial. The solution was left to sit overnight (or up to 5 days). 40 mL of de-ionized water was
added while the residual copper was removed and 3.2 mL of concentrated sulfuric acid was
added and the solution was mixed. The molybdenum spray was used as is and could be stored for
several weeks in the refrigerator, in the dark. Spots were visualized as dark blue spots on a
white/light blue background upon heating of the freshly sprayed plate.
When making the molybdenum spray, caution is recommended when adding the sulfuric
acid to the copper metal. In our experience, using copper turnings or wire, and allowing the
solution to sit overnight, or up to 5 days in advance, yielded very reproducible results with
minimal exotherm. If copper tinsel or powder (not advised) were used to shorten the incubation
time the sulfuric acid should be added very slowly on ice since the reaction is highly exothermic.
Dragendorff dipS3 with a nitrite “enhancing” co-dipS4 were made as follows: 0.85 g
bismuth nitrate, 40 mL de-ionized water, and 10 mL of glacial acetic acid were combined
(solution A). 20 g KI and 50 mL of de-ionized water were mixed together (Solution B).
Immediately before using the spray, 20 mL of solution A was mixed with 1.25 mL of solution B
then diluted with 17.5 mL de-ionized water to create the working Dragendorff reagent. The TLC
plate was dipped into the Dragendorff reagent after the spots were eluted. The nitrite spray was
made using 0.25 g of NaNO2 and 25 mL of de-ionized water added together, and the TLC plate
was dipped into the aqueous sodium nitrite solution immediately following the Dragendorff dip.
Spots were made visible as light orange spots on a white background by touching the freshly
dipped TLC plate to a warmed hot plate in a chemical fume hood.
The ninhydrin dipS5 was made by combining 3 mL acetic acid, 100 mL n-butanol, and 0.3
g ninhydrin. The resulting dark green solution was made fresh at the beginning of the experiment
as it quickly degraded upon storage.
Caution is also advised when using the plastic-backed TLC plates, as they melt very
quickly when placed onto a hot plate. A way to avoid this is to use the aluminum TLC plates, or
to put aluminum foil under the TLC while on the hot place and then remove the aluminum foil
after the completion of the experiment. A heat gun or hair-dryer set on high setting can also be
used to develop the spots, if available.
It is also advised that the instructor set-up one separate “heating” station that has 1 or 2
hot plates, as well as the needed TLC developing agents, and colored pencils, that students then
rotate through as needed to develop the spots on their TLC plates. This allows for the instructor
to be nearby when this step is completed and minimizes the amount of concentrated acid that is
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being sprayed or dipped to one location. The plates can be set on paper towel during spraying
and can be blotted on paper towel to remove excess acid before heating. In our experience this
worked well, as each group reached this step in their procedure at a slightly different time since
some took longer with the initial extraction step than others.

Literature Cited

S1) Solvent Systems - Lipid Migration.

http://avantilipids.com/index.php?option=com_content&id=1690&Itemid=409 (accessed
October 15, 2013), Avanti Polar Lipids, Inc.

S2) Goswami, S. K.; Frey, C. F. J. Lipid Res. 12. 1971, 509-510.

S3) Wagner, H.; Horhammer, L.; Wolff, P. Biochemische Zeitschrift, 1961, 334, 175-184 (in
German).

S4) Rubia, L. B. and Gomez, R. J. Pharm Sciences. 66 (11), 1977. 1656-1657.

S5) Gordon, A. J.; Ford, R. A. The Chemist’s Companion: A Handbook of Practical Data,
Techniques, and References; John Wiley & Sons: New York, 1972; p 378.

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 Answer Key for Optional Pre-Activity Questions

Note that these questions were given one week prior to the start of the lab as a homework assignment. As
an alternate option to this handout, these questions can be covered in a brief pre-lab lecture before the
start of lab. The students in the organic laboratory section that performed the experiment already had
previous experience with TLC and Rf calculations so those questions were a review.

1. What is thin layer chromatography?

In this answer, the students should describe the basics of separation based on polarity, description
of the stationary phase (the coating on the plate itself), the mobile phase (the solvent used), and
that each spot represents either one compound or a group of compounds that have similar
polarity. Also, that TLC is used to determine the purity of a sample by the number of spots
present.

2. What is a retention factor and how is it calculated?

In this answer, students needed to define Retention Factor (Rf) as the distance the spot traveled
divided by the distance that the solvent front traveled. The retention factor gives a value for a
particular spot on a plate that relates to the compound’s polarity in a particular mobile phase.
Retention factors are useful since they can be compared between plates (provided the same
mobile and stationary phases were used) to identify similar compounds. Additionally, retention
factors will change if either the stationary or mobile phase is changed.

3. What is a lipid and where are lipids found in nature?

In this answer, the students needed to define that lipids had a polar and nonpolar component that
were covalently bonded together. Lipids are found in biological membranes.

4. Research and draw the structures for the following lipids (phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,
cholesterol, glucosylceramide, phosphatidic acid, and phosphatidylglycerol) and comment on any
similarities or differences that you see. (Attach additional paper if necessary)

Please refer to student handout for the structures. Students should comment on the polar and
nonpolar components, as well as the differences in heteroatoms and/or functional groups (for
example, some have phosphorus/phosphates, some don’t) between the lipids.

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 Answer Key for Student Handout

1. Why do some spots react with the molybdenum stain and not ninhydrin? Why do some
spots react with ninhydrin and not molybdenum?

The ones that react with molybdenum contain phosphorus. If they don’t also react with
ninhydrin, then they only have phosphorus and not a free amine. If one only reacts with
ninhydrin and not molybdenum, then the spot contains a compound with a free amine and
no phosphorus.

2. Why are not all the positive iodine spots also positive with the molybdenum spray?

Iodine is less specific and reacts with any unsaturation. Some of these spots do not
contain phosphorus, so therefore they are not stained with the molybdenum spray.

3. What can you say about the number of compounds in each pill/egg?

There are many spots so there are many different compounds present.

4. In general terms, what can you say about the polarity of each of these spots?

The less polar spots move farther on the TLC plate, therefore the spots that are lower on
the plate (smaller Rf) are more polar than those closer to the solvent front (higher Rf).

5. Why do you think that these tests are all performed instead of just one?

Some of the visualization agents don’t stain all the spots. Each stain is dependent on the
functional group present. In order to visualize all the spots (so nothing is missed), more
than one stain is used. In addition, the different stains reveal which functional groups are
present in each spot.

6. In general terms, what does each spot mean on the TLC plate? (More detail will be
provided for questions #8 and #9)

Each spot represents a compound or group of compounds with similar polarity, that has
been separated from a mixture.

7. When looking just at the two different egg extracts (D and E) why are there more spots in
the chloroform:methanol extract than in the acetone extract?

The two washing steps extract compounds with different polarities. There were more
compounds in the egg that had a similar polarity to the chloroform:methanol and were
therefore soluble in the solvent, and ultimately appear on the TLC plate.

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 8. What functional groups can you definitively say are present in each spot for each
pill/egg? Redraw all of your TLC’s below and write the functional groups that you think
correspond to each spot.

The TLC plates (Iodine (top left), Molybdenum (top right), Ninhydrin (bottom left), and
Dragendorff(bottom right)) run against standards where the samples were A=Lecithin,
B=Fish Oil, C=Krill Oil, D=Vegetarian Lecithin, E= Egg Crude, F= Fruit Pectin,
G=Gelatin, and the pure lipids (phosphatidylcholine, phosphatidylethanolamine, and
phosphatidylserine, from Avanti Polar Lipids). These plates were run by the authors and
represent “ideal” data. Note that the students did not run the aforementioned standards
on their plates. These are here for the instructor’s reference only. Sample student data
(answer) for this question is found on page 20.

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 9. Given the ranks of literature Rf values below (1=largest Rf, 7=smallest Rf), fill in which
Rf values you believe correspond to each structure (calculate them from your TLC plates)
and circle which sample you think contains each lipid (note that you will not be able to
assign all the spots on the TLC plates):

A=Lecithin, B=Fish Oil, C=Krill Oil, D=Egg (acetone), E=Egg (Chloroform:Methanol)

Your
Relative Circle which sample Contains
Structure/Name Positive Tests calculated
Rf Value Each Lipid
Rf Value
R

Iodine
Molybdenum 0.34 6 A B C D E
Dragendorff
R

Phosphatidylcholine
R

Iodine 0.79
Molybdenum 2 A B C D E
Ninhydrin
R
Phosphatidylethanolamine
R

Iodine
Molybdenum 7 A B C D E
0.33
Ninhydrin
R

Phosphatidylserine

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 R

Iodine
0.39 5 A B C D E
Molybdenum

Phosphatidylinositol

R
Iodine
(weak
1 A B C D E
R Ninhydrin 0.94
possible)
Glucosylceramide

Iodine
0.55 4 A B C D E
Molybdenum
R

Phosphatidic Acid

Iodine
0.60 3 A B C D E
Molybdenum

Phosphatidylglycerol
*Rf values given are in 65:25:4 v/v/v Chloroform:Methanol:Water on silicaS1.

**For guided inquiry handout: Assign group numbers and have them label their plates for when
collecting them.
2 week lab: keep as is
1 week lab: omit a few of the lipids from problem #8 (phosphatidylserine,
glucosylceramide, and either phosphatic acid, or phosphatidylglycerol). Also consider using two
pill (suggested is the krill oil and fish oil) and one egg.

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 10. Are any of the spots in any of the TLC plates you developed due to cholesterol (structure
shown below)? Can you definitively say that cholesterol is or is not in any of your
samples? Explain.
H3C
CH3
H CH3
CH3 H
H3C
H H

HO

For this answer, students should reflect on a few things:

- Eggs are known to contain cholesterol
- The molecule would not stain with molybdenum (no phosphorus), ninhydrin (no free
amines) and Dragendorff (no choline and not an alkaloid) reagents.
- It could stain with iodine, but then they would also need to have some idea of the
retention factor to make any conclusion

After reflecting on these points, the student should come to the conclusion that, no, you
cannot definitively say that cholesterol is present in any of the samples since more
information is needed. The instructor can also guide the student here with the idea that a
known sample can also be run to positively identify cholesterol’s presence.

11. How could the knowledge gained from this experiment be used to purify/obtain pure
lipid?

This required guidance from the instructor to answer for some. If students get stuck, ask
them:
- Was each sample pure? (No.)
- How do you know? (There were many spots)
- So then what is each spot? (A compound, or possibly a mixture of similar compounds)
- What does it mean if something is pure? (There is only one spot by TLC. Of course
this is idealized)

Usually by this point the students come to the conclusion that the spots can be
scraped off the plate to obtain the purified compound. Additionally, instructors are
encouraged at this point to briefly describe column chromatography as a way to
“scale-up” this process. Anecdotally speaking, the students found this interesting.

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 Using Thin Layer Chromatography to Separate Natural Products:
A closer examination of dietary supplements and chicken eggs

Pre-Activity Questions

Name: _______________________________________

1. What is thin layer chromatography?

2. What is a retention factor and how is it calculated?

3. What is a lipid and where are lipids found in nature?

4. Research and draw the structures for the following lipids (phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,
cholesterol, glucosylceramide, phosphatidic acid, and phosphatidylglycerol) and comment on any
similarities or differences that you see. (Attach additional paper if necessary)

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 Using Thin Layer Chromatography to Separate Natural Products: A closer
examination of dietary supplements and chicken eggs
Main Question
How can we use chemistry to separate and tentatively identify lipids that are in the dietary
supplements and foods that we eat?

Overview of Goals
Thin layer chromatography and selective stains will be used to deduce the probable identity of
lipids that are extracted from dietary supplements and chicken eggs.

Experiment
You will be preparing several samples to analyze using thin layer chromatography. Keep track of
each sample as you prepare them. Be sure to label everything carefully!

Hazards
Use gloves, lab coats and safety glasses. Handling of solvents and all visualization agents
(sprays and dips for developing the TLC plates) should be done in a chemical fume hood. Strong
acids are used in these sprays and can burn bare skin. When handling and heating sprayed TLC
plates in the fume hood, wear gloves and use tweezers.

Egg Extraction
1. Set up the extraction in a hood
2. Take one egg and separate the yolk from the whites. Discard the egg white and shell in
trash can and place the yolk in a 200 mL beaker with 100 mL of acetone in it. Stir with a
spatula to break apart the yolk.
3. Filter the mixture through a Buchner funnel containing filter paper.
4. If the solid is still yellow, place it back into a beaker and add ~100 mL more acetone.
Repeat acetone washes until the solid is no longer yellow.
5. Place ~2.0 mL of the filtrate in a test tube for later and discard the remainder (label this
“D”).
6. Place the extracted solid into a clean beaker
7. Cover the solid with a minimal amount of 2:1 v/v chloroform:methanol and stir
8. Filter mixture and save 2 mL of the extract (filtrate) (label this “E”)
9. Concentrate the solution if necessary using a stream of air or nitrogen

Pill Extraction
1. Obtain one pill of each different dietary supplement available**
**Some lecithin or other supplements may be in granule form. Weigh out granules (0.2
g) and dissolve in minimal amount of chloroform

2. Split each pill open with a spatula and squeeze the insides out into a test tube or small
beaker

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 3. Dissolve in minimal amount of chloroform (~1-2 mL)
4. Label the test tubes A for the soy, B for the Fish Oil, and C for the krill oil

TLC Characterization: Preparing the following reagents to be used for TLC

• Solvent for running the TLC
1. Combine 65 mL chloroform, 25 mL methanol, and 4 mL water into a 250 mL
flask (this is 65:25:4 v/v/v)
2. Stir thoroughly (using a glass stir rod or magnetic stir bar) until mixed

• Running the three TLC Plates (also refer to instructor’s guidance)

1. Cut out three 5x10 cm aluminum backed plates (or obtain pre-cut plates if
available)
2. Using a pencil put a line 1 cm above the bottom of each TLC plate
3. Measure in 0.5 cm from side along this line and measure equal number of spaces
for the amount of samples
4. Label where samples will be placed on the line by gently marking an “x” on the
plate (be careful not to scrape off or scratch the silica)
5. Spot each sample using a capillary tube in its designated area. Keep track of
which “x” is which extracted sample. Spot A-E on each plate.
6. Put 0.5 cm layer of solvent (65:25:4 Chloroform:Methanol:Water) in bottom of
each 400 mL beaker
7. Place filter paper inside the beaker so that it sticks to the beaker (may need to dip
in solvent at bottom of beaker to get it to stick)
8. Place spotted TLC plates in beakers, by carefully lowering them into solvent
without splashing
9. Cover beaker with aluminum foil (or watch glass) to prevent evaporation of
solvent
10. Let sit until solvent line is ¾ of the way up the plate. Do not move or bump the
beaker.
11. Take the TLC plate out of beaker and mark the solvent line with a pencil
12. Lay the TLC plate on paper towel in the hood to dry

• Characterizing the TLC Plates: Always use gloves and safety goggles
o UV (if fluorescent indicator in plates): Set plate #1 under a UV lamp/light. Circle
any spots using a black/gray pencil and record observations
o Ninhydrin: Dump contents of nalgene bottle into a 600 mL beaker. Dip the TLC
plate #3 in the ninhydrin, remove the TLC plate and place the plate on a heated
hot plate. Circle any spots that appear with this stain using a pink colored pencil
and record observations.
o Iodine: Place TLC plate #1 in an iodine chamber and wait about 5-10 minutes.
Circle any spots using a red pencil and record.

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 o Molybdenum: Use the iodine TLC (plate #1) after mark up. Spray the plate with
the sprayer containing the molybdenum spray until thoroughly covered and lightly
touch the plate to a hot plate. Circle any spots using a blue pencil and record
observations.
o Dragendorff: Use TLC plate #2 and dip it into the Dragendorff reagent provided
and then blot the excess Dragendorff reagent off of the back of the TLC plate onto
a paper towel. Then dip the TLC plate into the nitrite solution (which is also
provided). Before the TLC is dried tap it to a hot plate in hood and circle any
spots using an orange pencil and record observations. Be careful not to overheat
because then spots will disappear.

Apply Your Knowledge

Each stain and the functional groups it reveals:

R1
R2 O
+
N

*Structure of Choline: R3 R4

1. Why do some spots react with the molybdenum stain and not ninhydrin? Why do some
spots react with ninhydrin and not molybdenum?

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 2. Why are not all the positive iodine spots also positive with the molybdenum spray?

3. What can you say about the number of compounds in each pill/egg?

4. In general terms, what can you say about the polarity of each of these spots?

5. Why do you think that these tests are all performed instead of just one?

6. In general terms, what does each spot mean on the TLC plate? (More detail will be
provided for questions #8 and #9)

7. When looking just at the two different egg extracts (D and E) why are there more spots in
the chloroform:methanol extract than in the acetone extract?

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 8. What functional groups can you definitively say are present in each spot for each
pill/egg? Redraw all of your TLC’s below and write the functional groups that you think
correspond to each spot.

Ninhydrin Iodine Molybdenum Dragendorff

9. Given the ranks of literature Rf values below (1=largest Rf, 7=smallest Rf), fill in which
Rf values you believe correspond to each structure (calculate them from your TLC plates)
and circle which sample you think contains each lipid (note that you will not be able to
assign all the spots on the TLC plates):

A=Lecithin, B=Fish Oil, C=Krill Oil, D=Egg (acetone), E=Egg (Chloroform:Methanol)

Positive Your
Relative Circle which sample Contains
Structure/Name Tests calculated Rf
Rf Value Each Lipid
Value
R1

6 A B C D E

R2

Phosphatidylcholine
R1

O
O
+
H3 N
–
O O 2 A B C D E
O P O O
O R2

Phosphatidylethanolamine

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 R1

7 A B C D E

R2

Phosphatidylserine
R1

5 A B C D E

R2
Phosphatidylinositol

R1

1 A B C D E
R2

Glucosylceramide
R1

4 A B C D E

R2

Phosphatidic Acid
R1

3 A B C D E

R2

Phosphatidylglycerol

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 10. Are any of the spots in any of the TLC plates you developed due to cholesterol (structure
shown below)? Can you definitively say that cholesterol is or is not in any of your
samples? Explain.
H3C
CH3
H CH3
CH3 H
H3C
H H

HO

11. How could the knowledge gained from this experiment be used to purify/obtain pure
lipid?

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 Sample Student Data

Representative data from one group of two students is shown below. Below are the TLC plates
that the students obtained after staining. Please note that the spots have faded upon storage but
the original location of each spot after using the visualization agent was circled with colored
pencil and is still visible. The students’ representation of the TLC plates (as their answer to #8 in
the student handout) is also shown for reference.

The TLC plates were stained as follows: Ninhydrin (left, plate “8.1”, red colored pencil), Iodine
(plate “8.2”, orange colored pencil), Molybdenum (also “8.2”, blue colored pencil), and
Dragendorff(right, plate “8.3”, red colored pencil) where A=Lecithin, B=Fish Oil, C=Krill Oil,
D=Egg (acetone), E=Egg (Chloroform:Methanol)

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 Assessment of Learning Outcomes

Assessment of the learning outcomes was done using the collected student handouts from
the guided-inquiry experiment as performed by 16 sophomore-level undergraduate students in an
organic chemistry lab in two 3-hour lab periods. Table 2S indicates the majority of students (88
%) were able to collect the needed data without issue (refer to representative student data on
page 20). Phosphatidylcholine is shown as one representative lipid, that 72.9% of students were
able to correctly identify in the krill oil, lecithin and egg (chloroform:methanol) extract.
Additionally, Table 2S shows that just over half the students were able to apply what they
learned to a novel lipid, while 16 of the 16 students were able to describe the purity of their
samples and how to isolate one pure spot.

Table 2S. Results for each learning outcome obtained from graded student worksheets.

Number of
Student Learning Outcomes Percent of students
students able to
At the completion of this exercise, students should able to perform task
perform task
be able to: successfully
successfullya
Successfully perform thin layer chromatography to
14 out of 16 88 %
obtain data
Successfully stain TLC plates using visualization
14 out of 16 88 %
agents
Identify that spots visualized with different stains
16 out of 16 100 %
contain different functional groups
Correctly identify specific phospholipids from their
------ 72.9 %c
datab
Apply knowledge obtained in the experiment to a
new phospholipid 9 out of 16 56.3 %
(Question #10 student handout)d
Apply knowledge gained in the experiment to the
idea of “purity” via TLC and phospholipid 16 out of 16 100 %
purification (Question #11 student handout)d
a
One group (2 students) did not successfully obtain TLC data. Their plate (after staining) indicated that their material
was lost presumably due to having their spots below the solvent. Sample TLC data (a printout of an ideal TLC) was
supplied to them instead.
b
The data represented here indicates the number of students that correctly identified the phosphatidylcholine (PC) spot,
even if their calculated retention value was different than accepted (see Table 3S below). The use of the “relative
retention factor values” helped to correct for minor differences in students’ mobile phase preparation (the
chloroform:methanol:water solution), and still allowed for the correct identification of the spot.
c
Three of the samples contained PC lipid. The percent shown represents the average identification rate for the lipids
based off the students’ data. For example, if a student correctly identified 2 of the 3 samples that contained PC based
on their data, they received a “2/3 = 66.7%” as their score.
d
Asssessment was based on the number of students obtaining a 3 or 4 on the following scale:
0 = Question was left blank
1= answered but incorrect
2= answer contains basic information, with some inaccuracies
3 = answer is correct but lacks depth
4= answer shows exceptional depth and relates several topics together

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More detailed analysis in Table 3S shows the average retention factors that the students
calculated from their TLC data for each lipid based on the staining patterns they observed and
the relative retention factors that were given. The accepted values (reference S1, page 5) for the
retention factors are also shown in Table 3S, along with standard deviation and percent error
from the accepted value. The majority of the student averages were remarkably close to the
accepted values for the retention factors for each lipid. The one notable exception was the
phosphatidylserine (PS) which had a percent error of 42.6% from the accepted value. Looking
back at the students’ data, some of them obtained a ninhydrin positive spot at a very low
retention factor value. The source of this spot is unknown, but led to the false identification of
PS at a lower Rf than expected.

Table 3S. Average of the student calculated retention factor (Rf) values for each lipid
from the student worksheet.
Accepted Student Averages with Percent
Value Standard Deviations Error
Phosphatidylcholine (PC) 0.34 0.40 +/- 0.22 19.0%
Phosphatidylethanolamine (PE) 0.79 0.73 +/- 0.19 7.2%
Phosphatidylserine (PS) 0.33 0.19 +/- 0.10 42.6%
Phosphatidylinostitol (PI) 0.39 0.39 +/- 0.18 0.4%
Glucosylceramide 0.99 0.87 +/- 0.12 12.6%
Phosphatidic acid (PA) 0.55 0.50 +/- 0.09 8.4%
Phosphatidylglycerol (PG) 0.6 0.51 +/- 0.16 14.3%

Based off of these results, it is concluded that the students are able to successfully perform the
experiment. They are able to collect the needed data by performing extractions, running several
TLC plates in a mixed solvent and staining several TLC plates using various visualization
agents. Furthermore, the calculation of the accepted retention factor values from their data was
successful, as the accepted values for the Rf values falls within one standard deviation of the
students’ data, with the sole exception of the phosphatidylserine as previously mentioned.

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