EXTRACTION AND CHARACTERIZATION OF LIPIDS FROM FOOD SAMPLES

Student Manual

LAB OBJECTIVES

The major goals include:

1. Extraction of lipids from egg yolk and avocado (you are also welcome to bring your own
food; run your food choice by your instructor and stay away from highly processed food);
2. Separation of the different lipid classes using chromatographic methods;
3. Identification of the various lipid classes using thin-layer chromatography and chemical
tests;
4. Characterization of fatty acids methyl esters using gas chromatography;
5. Characterization of lipids using matrix-assisted laser desorption/ionization mass
spectrometry.

INTRODUCTION

Lipids constitute one of the major classes of biological molecules besides carbohydrates,
nucleic acids, and proteins and are involved in a vast array of intra- and extra-cellular functions.
The term 'lipid' is an umbrella term which covers many sub-classes of bio-molecules including
fatty acids, triacylglycerols, glycerophospholipids, sphingolipids, and steroids.
Triacylglycerols, fatty acids and phospholipids are all related in structure. Triacylglycerols serve
- among other functions - as energy reservoirs and contain three fatty acid side chains connected
by ester bonds to a glycerol backbone. Glycerophospholipids have the same structure as
triacylglycerols except that one fatty acid side chain is replaced by a polar phosphate-containing
head group. These molecules are important for cellular membrane structures and make up for the
majority of the cellular bilayer. Fatty acids consist of long typically even-numbered carbon
chains of linear structure connected to a carboxylic acid. They often have chain lengths of 16
(C16) or 18 (C18) carbon atoms and can be either completely saturated or contain one or more
double bonds. Some fatty acids are also known to be components of various oils and waxes.
Lastly, steroids are a class of lipids with a characteristic cycloalkane ring system structure.
Typical endogenous compounds of steroid structure are sex hormones and cholesterol, which
makes up to about 30 - 40% of the plasma membrane. Excessive cholesterol - or, more
specifically, high LDL (a lipoprotein which enables the transport of nonpolar cholesterol within
the polar bloodstream) levels - has been linked to cardiovascular diseases.
It is evident that lipids function in many areas of the cell and the body, however, many of
their roles have yet to be discovered. It is, therefore, of great interest within the scientific
community to be able to extract and categorize them. This laboratory experiment will lead to a
better understanding of the lipid compositions of different food sources and the techniques used
to analyze them.
In order to analyze the lipids, a total lipid extract must first be isolated from the food
sources with the help of a modified Bligh, Dyer, and Folch liquid-liquid extraction. This
technique enriches lipids into the organic phase of a dual phase extraction solution by means of
their hydrophobic nature. Typically, a solution containing organic nonpolar solvents and water
are added to the food source and mortared with sea sand. Chloroform as the originally used
organic solvent causes liver and kidney impairment and is possibly cancerogenic and should be
replaced by dichloromethane (DCM). The Folch salt solution was modified and includes 0.04%
CaCl2, 0.034% MgCl2 and 0.58% NaCl (all v/v) for raising the polarity of the aqueous phase to
improve the separation into the organic phase. Silicic acid column chromatography is used for
dividing the extracted lipids into a fraction of lower polarity by elution with DCM and a second
fraction of higher polarity by using methanol as mobile phase.
A pre-column and both post-column lipid fractions are subsequently analyzed by thin-
layer chromatography (TLC), transesterification followed by gas chromatography (GC) or gas
chromatography coupled to mass spectrometry (GC/MS), and matrix-assisted laser
desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS).
TLC is a traditional chromatographic method, which uses a stationary phase and a solvent
system to separate analyte mixtures based on the specific polarities of the contained components.
The plate consists of an aluminum or plastic sheet coated with a thin-layer of an adsorbent, such
as silica gel. A small amount of the sample mixture, a standard, and a co-spot are placed onto the
plate at the same height. The bottom of the plate is submerged in the solvent, which travels up
the plate through capillary forces. Separation of the applied mixture results from different
affinities of each compound to the mobile (solvent) and stationary (silica plate) phase. TLC will
be performed on both lipid fractions of higher and lower polarity using two different solvent
systems. A solution of high hydrophobicity consisting of hexane/diethyl ether/acetic acid
(49:29:5 v/v/v) will be used for the less polar lipid fraction and a more polar mobile phase
containing DCM/methanol/acetic acid/water (100:23:5:1 all v/v) for the higher polar one. Once
completed, the separated spots can be visualized by different staining methods. The most simple
method uses ultraviolet (UV) light which causes a green fluorescence of fluorescence-dye-
modified TLC plates. Aromatic compounds/compounds with sufficient amounts of conjugated
double bonds absorb the UV light preventing fluorescence of the stationary phase that leads to
dark spots. With the iodine tank method, many organic compounds can be detected by subjecting
the TLC plate to iodine crystal vapors. Lipids that contain primary or secondary amine groups
can be stained by spraying ninhydrin on the plate and lastly, cholesterol and its derivatives can
be visualized by iron (III) chloride solutions followed by heating. Spot analysis starts with
determination of the retardation factor (Rf) of each visualized compound by calculating the ratio
of the distance traveled by the spot above the origin to the distance traveled by the solvent front
above the origin. By comparing the Rf values from the sample with that of lipid standards, the
components of the sample can be determined. The co-spot is traditionally used to confirm
compound identification. A specific compound must exhibit identical Rf values in both (spot and
co-spot) lanes, since its interaction with the mobile and stationary phase is the same.
A technique specific to cholesterol is the Liebermann-Burchard test. This reaction is used
for detection of steroids such as cholesterol by oxidative dehydratization followed by generation
of a colored polyene cation. After dissolving the sample in DCM, acetic anhydride and sulfuric
acid are added. In the presence of steroid derivatives, the color of the solution changes from
purple/pink to light green and finally to dark green. This test will be performed on the total lipid
extract and the more and less polar lipid fractions in addition to a positive control containing
pure cholesterol and a blank (negative control).
Transesterification with subsequent GC separation can be used on the total lipid extract
fraction to determine the percentage of each fatty acyl residue present in the sample. The
transesterification reaction is shown in Figure 1. The triacylglycerols are subjected to heat with
sulfuric acid as the catalyst in the presence of methanol to yield volatile fatty acid methyl esters
(FAMEs) suitable for GC analysis.

Figure 1. Acid catalyzed transesterification of triglycerols.

FAME’s are then injected into the GC instrument and carried through a long and narrow column
filled with a stationary adsorbent using helium or hydrogen gas. After separation by polarity, the
intensity of each specific compound is detected and plotted versus the corresponding retention
time (RT). The specific RT values can be used for peak correlation by comparison with the
retention times of commercially available FAMEs. The determined compound peak areas can be
analyzed for determination of the respective FAME percentages. The two most widely used
detectors are the flame ionization detector (FID) and thermal conductivity detector (TCD). The
FID is an extremely sensitive detector with a wide dynamic range, however, this method
pyrolyzes the separated compounds. The TCD on the other hand exhibits a lower sensitivity but
does not destroy the sample. Another detection system with high sensitivity used in GC is mass
spectrometric (MS) detection. In a GC-MS experiment, the compounds are first separated in a
column by polarity and vapor pressure. The separated species are successively transferred into
the mass spectrometer where the compounds are ionized, separated by their mass-to-charge (m/z)
ratio and detected by multi-channel plates or time-to-digital converters.
Matrix-assisted laser ablation/ionization time-of-flight mass spectrometry (MALDI-TOF
MS) will be used to determine the analyte masses and to identify the components of the higher
and lower polarity lipid fractions. The samples are diluted in a matrix solution and spotted on a
steel target plate for analysis. The matrix serves numerous purposes, e.g. it promotes sample
crystallization with subsequent analyte ablation and ionization and prevents excessive
fragmentation. 2,5-Dihydroxybenzoic acid (DHB) is a well-known matrix for lipid analysis and
will be used for the lipid fractions of higher polarity. The lower polar lipid fractions will be
investigated by using 4-chloro-α-cyanocinnamic acid (ClCCA).

SAFETY
Precautions should be taken for the following listed materials. Dichloromethane [75-09-2],
methanol [67-56-1], and toluene [108-88-3] are flammable and toxic. Hexane [110-54-3] is
flammable, neurotoxin, and irritant. Sulfuric acid [7664-93-9] is very corrosive and toxic. Acetic
acid [64-19-7] and acetic anhydride [108-24-7] are corrosive and toxic. Ninhydrin [485-47-2]
spray is toxic. Iodine [7553-56-2] is toxic and volatile. Iron (III) chloride [7705-08-0] spray is
 very corrosive. Acetonitrile [75-05-8 ] is toxic upon ingestion in significant amounts. The
following solids are used in relatively small amounts or in solutions and do not pose significant
risks: 2,5-dihydroxybenzoic acid [490-79-9], 4-chloro-α-cyanocinnamic acid [69727-07-7],
silicic acid [1343-98-2], calcium chloride [10043-52-4], magnesium chloride [7786-30-3],
sodium chloride [7647-14-5], sodium sulfate [7757-82-6], and sodium bicarbonate [144-55-8].
All food samples should be handled with gloves or, alternatively, hands should be carefully
washed subsequently. All volatile solvents should be handled in a hood.

PRE-LAB ASSIGNMENTS AND QUESTIONS

1. Lipids are one of the major constituents of the cell membrane. What property makes them
perfect for this job?
2. Which of the two solvents listed below would be used to separate phosphatidylcholine
from phosphatidylethanolamine in thin-layer chromatography? Why?
 DCM/methanol/acetic acid/water (100:23:5:1, all v/v)
 hexane/diethyl ether/acetic acid (49:29:5, all v/v)
3. What is the structure of cholesterol? What are its functions in your body? You may need
to do some additional reading, besides the introduction, to answer questions 3 and 6.
4. Which part of this laboratory is directly related to the process of production of biodiesel?
5. Using the National Nutrient Database for Standard Reference (website is listed below),
look up either Hass avocado or egg yolk AND a food of your choice. Record the total
lipid percentage and any other useful lipid information. For example, record the percent
of monounsaturated lipids, cholesterol, etc. This website will be useful later when
analyzing and discussing your results: http://ndb.nal.usda.gov/ndb/search/list. Consider
bringing your own food sample for lipid analysis. Run your food choice by your
instructor and stay away from highly processed food.
6. It is generally believed that unsaturated fats are healthier than saturated fats. Is this belief
accurate? Explain.

PROCEDURE

Outline
100 µL MALDI-TOF
Liebermann-Burchard Test MALDI-TOF
sample preparation
MS

Lipid Extraction
Volume: 1200 µL

900 µL Polar and TLC

Column chromatography
nonpolar fractions

GC sample
100 µL Transesterification GC
preparation

Day 1 Day 2 Day 3

Day 1: Lipid extraction and transesterification
Part A: Extraction
1. Place 1 g of wet weight food sample, 1 g of sea sand, and 20 mL of DCM: methanol (2:1,
v/v) solution into a mortar and grind thoroughly.
2. Set up a glass filtering funnel containing a fluted filter paper over a 50-mL centrifuge
tube. Moisten the filter paper with DCM: methanol (2:1, v/v).
3. Pour the ground mixture onto the filter. Wash the mixture with 2 mL of DCM: methanol
(2:1, v/v) solution. Collect both the flow through and wash in the centrifuge tube as the
filtrate.
4. Assuming you have 20 mL of filtrate, add 4 mL of the Folch salt solution (0.04% CaCl2,
0.034% MgCl2, 0.58% NaCl as aqueous solution).
5. Cap the centrifuge tube and invert a few times gently. Carefully release any build up
pressure.
6. Centrifuge at 5,000 x g at room temperature for 2 minutes. This will break any existing
emulsions and separate the mixture into organic and aqueous layers.
7. Use a Pasteur pipette to remove and discard the top aqueous layer.
8. Add 9 mL of DCM: methanol: water (3:48:47, all v/v) to the lower phase. Repeat the
extraction and centrifugation (steps 5-7). If organic layer becomes less than 5 mL, add
DCM.
9. Clean, dry, and pre-weigh a round bottom flask. Please keep in mind that all round
bottom flasks should also be pre-weighted prior their use in other steps.
10. Use anhydrous sodium sulfate (Na2SO4) to dry the lower DCM layer. The test for dryness
is the "snowflake effect".
11. Create a plug in a Pasteur pipette using cotton and use it to filter out the solid drying
agent. Let the liquid drain into the round bottom flask from step 9.
12. Use a rotary evaporator to remove all solvent. Make sure that the extract is dry. The
separation using column chromatography (Day 2, Part B) will fail or not work efficiently
if water or methanol is present. Use anhydrous Na2SO4 and the rotary evaporator again, if
needed.
13. Weigh the flask again. Determine the weight of your total lipid extract and record it in
your notebook.

Part B: Transesterification
1. Dissolve the total lipid extract in 1.2 mL DCM.
2. Transfer 100 µL to a 5 mL labeled capped vial. Place the remaining 1,100 µL into a
labeled capped vial. Mark the level of solvent with a permanent marker. Cover the top
with parafilm to minimize evaporation and store it in the refrigerator until the next lab
period.
3. Put on the gloves. Carefully add 1 mL of methanol: sulfuric acid (50:50, v/v) and 1 drop
of toluene to the 100 µL sample.
4. Label and tightly close the vial. Minimize evaporation by placing parafilm over the top.
5. Place the vial in a 60o C incubator until the next lab period.
Day 2: GC sample preparation of transesterified lipids and separation of the more and less
polar lipid fractions using silicic acid column chromatography
Part A (may be done after part B): Transesterification work-up/ GC sample preparation
1. Put on the gloves. Remove the transesterified total lipid extract from the 60o C incubator
and cool the mixture to room temperature.
2. Use a glass Pasteur pipet to transfer all organic fractions/solvents. Place 1 mL of water
into a 15 mL tube. Slowly add the acidic transesterified mixture and add 2 mL of hexane.
Be careful when adding the acidic mixture to the water. The black residue that is left in
the vial can be discarded.
3. Gently shake the tube making sure to periodically release any pressure buildup by
loosening the cap.
4. Use a Pasteur pipette to remove and save the hexane layer.
5. Repeat the extraction (steps 2-4) twice.
6. Combine the hexane layers into a 15 mL tube.
7. Perform a 1:1 (v/v) wash with saturated sodium bicarbonate (NaHCO3) solution and
discard the aqueous wash layer.
8. Using pH paper, check the pH of the water you are about to add, it should be close to
neutral. Add 2 mL of water to wash the hexane layers.
9. Remove the water layer and check its pH again. Repeat the water extractions until the pH
of the aqueous layer is about neutral. Discard all water fractions.
10. Add 1 g anhydrous sodium sulfate to the hexane phase to remove any remaining water.
11. Clean, dry, and pre-weigh a round bottom flask.
12. Create a cotton plug in a Pasteur pipette and use it to filter out the solid drying agent. Let
the liquid collect in the round bottom flask.
13. Use a rotary evaporator to remove all solvent.
14. Weigh the flask again. Determine the weight of FAMEs in the flask and record it in your
notebook.
15. Dissolve the residue in 1 mL of DCM and transfer it to a capped vial for GC analysis.
Cover the top with parafilm to minimize evaporation. Store the properly labeled vial in
the refrigerator for the next lab period.

Part B: Silicic acid column chromatography

1. Remove the 1,100 µL of total lipid extract (day 1) from the refrigerator.
2. Check the volume of your solution. If evaporation has occurred, adjust the volume to the
1,100 µL mark using DCM.
3. Clean, dry, and pre-weigh two round bottom flasks. Label one "polar lipids" and one
"nonpolar lipids". Add your name or initials to both labels.
4. Vertically fasten a polyethylene or glass column to, e.g., a ring. Make sure the bottom is
closed.
5. Add 10 mL of DCM to 1 g of silicic acid in a beaker.
6. Gently swirl the mixture and pour the slurry into the column. To ensure to get all silicic
acid, rinse the beaker with 2 mL of DCM and add it to the column.
7. Wait until the silicic acid settles to the bottom of the column. Place the non-polar labeled
pre-weighted round-bottomed flask below the column.
8. Open the column tap and allow the fluid to drip out just until the liquid reaches the top of
the silicic acid bed.
 9. Add 900 µL of the total lipid extract to the column and let the liquid flow through the
column until the liquid level reaches the top of the silicic acid bed.
10. Place the remaining 200 µL of total lipid extract back into the refrigerator for the next
laboratory period.
11. Add 1 mL of DCM to the column and let the liquid flow until the liquid level reaches the
top of the silicic acid bed.
12. To elute the lipids of lower polarity from the column, add 10 mL of DCM and collect the
flask.
13. When the liquid level reaches the top of the silicic acid bed, add 10 mL of methanol.
Keep collecting the nonpolar fraction. Watch the clearly visible methanol front and
switch to the polar pre-weighted round bottom flask only when the front reaches the
bottom of the column and the DCM has eluted out of the column. Collect the polar
fraction.
14. Use a rotary evaporator to remove all DCM from the flask containing nonpolar lipids and
all methanol from the flask with polar lipids. Weigh both flasks and determine the weight
of the two post-column fractions. Record these values in your notebook.
15. Dissolve both residues in 1 mL of DCM.
16. Transfer both solutions into separately labeled vials and store them in the refrigerator.

Day 3: TLC, L-B, GC, and MALDI-TOF MS analysis of the prepared lipid fractions
Part A: TLC
More polar lipid fraction
1. Gently draw a line with a pencil across a silica gel TLC plate 1 cm from the bottom.
Label the plate: polar lipids.
2. Get the more polar lipid fraction from the refrigerator.
3. Spot three evenly spaced lanes on the TLC plate: the lipid dilution of higher polarity, the
phosphatidylethanolamines and phosphatidylcholines standard mixture, and a co-spot
containing sample and standards. The sample should be applied to the TLC plate in small
aliquots using a capillary tube. Do not scratch the surface of the plate and keep the spot
under 2 mm in diameter.
4. Fill a TLC tank with a small volume of a DCM/methanol/acetic acid/water (100:23:5:1,
all v/v) solvent mixture, place the TLC plate vertically in the solvent and close the tank.
The solvent must not reach above the pencil line.
5. Allow the solvent to travel about 80% to the top of the plate and mark the final solvent
front position.
6. Remove the plate from the TLC tank and dry it in the fume hood.
7. Place the plate in a TLC tank with iodine crystals. When the spots become visible,
remove the plate and circle the spots with a pencil.
8. Evenly spray ninhydrin on the plate and wait for the plate to dry. Heat for 5 minutes at
100o C or until a color change appears. Note that spraying too much reagent or heating for
too long might cause the plate to disintegrate. Keep an eye on the plate and remove it
when red spots become visible. Circle these amine-containing lipid spots and record the
color change. Do this quickly because over time these spots will fade.
9. Photograph the TLC plate for the written report.
Less polar lipid fraction
1. Draw a line with a pencil across a silica gel TLC plate 1 cm from the bottom. Label the
plate: nonpolar lipids.
2. Get the less polar lipid fraction from the refrigerator.
3. Spot three evenly spaced lanes on the TLC plate: the less polar lipid dilution, the
cholesterol/ squalene/ triolene standard mixture, and a co-spot containing sample and
standards. The sample should be applied to the TLC plate in small aliquots using a
capillary tube. Do not scratch the surface of the plate and keep the spot under 2 mm in
diameter.
4. Fill a TLC tank with a small volume of the hexane/diethyl ether/acetic acid (49:29:5, all
v/v) solvent mixture, place the TLC plate vertically in the solvent and close the tank. The
solvent must not reach above the pencil line.
5. Allow the solvent to travel close to the top of the plate and mark the final solvent front
position.
6. Remove the plate from the TLC tank and dry it in the fume hood.
7. Shine a UV lamp set at 254 nm on the TLC plate to see which spots contain unsaturated
lipids and circle them.
8. Place the plate in a TLC tank with iodine crystals. When the spots become visible,
remove the plate and circle the spots with a pencil.
9. Evenly spray iron (III) chloride on the plate and wait for it to dry. Heat it for 5 minutes at
100o C or until a color change appears. Note that spraying too much reagent or heating for
too long might cause the plate to disintegrate. Keep an eye on the plate and remove it
when dark spots become visible. Circle these squalene and cholesterol/ cholesterol
derivative containing spots and record the color change. Do this quickly because over
time these spots will fade.
10. Photograph the TLC plate for the written report.

Part B: Liebermann-Burchard test

1. Place 100 µL of the total lipid extract and the more and less polar lipid fraction into three
separate test glass tubes.
2. Add 0.9 mL of DCM to all three tubes.
3. Set up your positive and negative controls. Place 1 mL of DCM into two separate glass
test tubes. Add a microspatula tip full of cholesterol to one of the test tubes to create your
positive control. The second tube will be a blank and serve as negative control.
4. To each of the five tubes, add 2 mL DCM and 1 mL acetic anhydride.
5. Add 1 drop of concentrated sulfuric acid to each tube.
6. Mix the contents and record any color changes in your notebook.
7. Wait 5-10 minutes and record any color changes in your notebook again.

Part C: GC analysis
1. Remove the 1000 µL of transesterified fatty acid methyl ester mixture previously
dissolved in DCM from the refrigerator (day 2, part A).
2. Take your flask to the 2400 Varian GC and wait until the word READY is shown on the
display, indicating the completion of the previous run.
3. Use a Hamilton syringe to draw up 4 µL of your mixture.
 4. Inject the solution into the GC injector connected to FID detector with slight downward
pressure. It is important that the top and bottom metal pieces surrounding the slot make
contact. Immediately press RUN on the GC and RUN on the integrator.
5. Remove the syringe from the injection slot and wash it with DCM.
6. Wait about 12 minutes for your run to finish and use this time to record the retention
times from the chromatogram containing the standard FAMEs. Once your run is done,
tear off the chromatogram from the printer slot. If the run does not stop automatically,
press RESET to manually stop it.
7. GC-MS data analysis will require the remaining FAME sample to be transferred to a
specialized vial for an automatic procedure for all runs. Sign your name on the sign–in
list on the instructor’s table.
8. Place your vial on the tray of GC-MS autosampler for run. Your instructor will put the
standard sample for run as well. The autosampler will take 1 μL of each sample and inject
it to the GC-MS. The total run for each sample is 25 min. Your instructor will send you
the chromatograms for your sample and for the standard as well as mass spectrum for
each peak. If this instrument is not available, your instructor will provide you with all
appropriate data.

Part D: MALDI-TOF MS
Sample Preparation
1. Calculate the initial concentration of your more and less polar lipid fractions and dilute
both fractions to a final concentration of 1 mg/mL using DCM.
2. Perform a 1:10 (v/v) dilution of the more polar lipid fraction with the DHB matrix
solution. Keep in mind that you will only need 2 µL of the dilution for the analysis.
3. Perform a 1:1 (v/v) dilution of the less polar lipid fraction with the ClCCA matrix
solution. Keep in mind that you will only need 2 µL of the dilution for the analysis.
4. Vortex your final solutions.
5. Spot 2 µL of each dilution on a steel target plate. Do not let the pipette tip touch the plate.
Record the alphanumeric ID of your spots and submit this information to your instructor.
6. Place the plate at the edge of the fume hood for 10 minutes to allow the solutions to dry
and crystallize.
7. Load the target plate into the instrument.

Operate the MALDI-TOF mass spectrometer using automated features. This section may be
performed by your instructor. General parameter settings:
- Accumulate at least 500 shots per spectrum;
- Use laser fluences optimized for highest analyte signal-to-noise ratios;
- Use positive polarity and reflector operation mode;
- Select the mass range to detect to about m/z 600-2000;
- Use a low mass gate set to about 100 Da below the lower mass limit;
- Activate the mass peak filter function and select only monoisotopic peaks, if available;
- Calibrate the mass spectrometer externally by means of a standard with known analyte
masses;
- In addition, calibrate all spectra internally using spectra provided.
The directions below pertain specifically to methods and settings regarding to a Bruker Autoflex
and files saved on the instrument.
1. Open the program Flex Control and when prompted, select the lipids_RP.par method
and press Open. The range on the x-axis should be about m/z600-2000.
2. Click on the autoXecute tab and click Select. This will open a window that will allow
you to choose the test sequence. Choose the autotest.txt sequence and click open.
3. This sequence has the option of being edited. To do so, select edit and the autoXecute
sequence window will open.
4. To adjust the plate position, go to the sample position section of the window and click on
the drop down menus next to the word 'sample' to select your fraction coordinates.
Next to the word chip, make sure that MTP 384 steel is chosen from the drop down
menu. Under the section named spectrum location open the browse directory by clicking
browse. From the drop down menu, select the data folder. Click on the folder named
Lipids lab and then click on the Lipids folder. Click the choose button. Press the new
folder icon and name the file with your name. If an error window pops up, just hit okay.
You will now see your named file in the Directory. Click on your file name and hit
choose. Click choose again. This will take you back to the autoXecute sequence window.
Under spectrum location you will now be able to name your spectrum (Example:
PolarEggK2).
5. Go to the autoXecute method section of the window and select Lipid method from the
drop down menu.
6. Once these configurations are finished go to the new entry section of the window and
click add. Your file will appear in the white top part of the window. Adjustments can be
made to the entry by selecting your file from the table, making necessary adjustments
within the parameters below and then clicking replace under the new entry section.
7. Repeat this set of directions until all the necessary spots on the plate have been added to
the sequence. Then select save. This will bring you back to the original Flex Control
screen.
8. In top left corner select Start Automatic Run. Clicking this button the second time will
abort the run.
9. If the run is not completed by the end of the lab period, the instructor will email the
graphs.

Part E: Share data with your partner who had a different food source. Your report should
contain information from at least two different food sources. Graphs that supplement and
confirm GC and/or TLC data will be provided by the instructor if MALDI-TOF MS and/or GC-
MS instruments are not available.

REPORT INFORMATION
Result section
1. All results should be shown in your report in neatly organized tables with proper labeling.
All appropriate graphs from each analysis should be presented in your report.
2. During the laboratory period, you were asked to determine the weight of the total lipid
extract as well as of the more and less polar lipid fractions. Use this to determine the
percent composition of the total lipid extract in the starting food sample and the percent
 of polar and nonpolar lipids in the total lipid extract. Keep in mind that you used only a
fraction of total lipids sample for this task.
3. During the laboratory period, you were asked to determine the weight of the FAMEs after
transesterification. Use this to determine the percent yield of FAMEs in the total lipid
extract.
4. Use the TLC plates labeled “polar” and “nonpolar” to calculate the Rf value of each
visualized spot. Compare the standards' Rf values to the samples' Rf values to identify the
contents of your samples. Use the co-spot lane to confirm identification. Arrange your
results in a table. Relative standard Rf values are shown below.

Polar Standards Nonpolar Standards

Phosphatidylcholines Phosphatidylethanolamines Cholesterol Squalene Triolein
0.15 0.95 0.5 0.88 0.94

5. Use your sample GC chromatograms and the supplied standard GC chromatogram.

Compare the standards' RT values to the samples' RT values to identify the contents of
your samples. Note that RT values should be adjusted based on solvent peak elution
times. Then use peak areas to determine the percent composition of each FAME that you
identified.
6. Provide a table showing your Leiberman-Burchard test results.
7. Provide a table highlighting significant results from the MALDI-TOF MS analysis.
Please note that the matrices may also contribute to the peaks seen on the spectrum.
Common peaks from DHB are m/z 554.38, 573.75, 668.94, and 694.19 and peaks from
ClCCA are m/z 553.98, 568.96, 570.79, 572.88, 581.19, 676.01, and 678.03.

Discussion and post-laboratory questions

1. For your discussion, refer to the website from the pre-lab questions to compare
experimental results with the reference value lists. Discuss all your results from each
analysis and note all unexpected data. For all analyses, make comparisons between
food samples: are the results similar or different between the two? Use questions 2-4 as
guides when comparing data between methods.
2. Discuss the accuracy of the percentages you calculated. Are they close to theoretical
values or values obtained from the reference website?
3. Use the Leiberman-Burchard test and TLC results to discuss the efficiency of separating
your total lipid fraction into lipid fractions of different polarity on the silicic acid column.
Refer to the tables made in steps 7 and 8 of the results information section.
4. Compare and contrast your TLC and your MALDI-TOF MS data. Do they show similar
results? Refer to the tables made in steps 8 and 9 of the results information section. The
table provided by your instructor will show previous lipid MALDI-TOF MS research
from various sources, identifying common egg yolk peaks.
5. Compare the actual and reference percentages of fatty acids from the FAMEs analyses.
6. You have already identified and calculated the relative abundances for the FAMEs. Use
the provided GC/MS chromatograms to discuss if they confirm your lipid identification.
7. Compare any given GC-MS chromatogram to an online database or a peer-reviewed
paper of your choice and discuss your findings and the accuracy of the peaks.
8. Why is it necessary to do a transesterification on the total lipid fraction?
9. Draw the products formed in a transesterification reaction performed on glycerol
tripalmitate.
10. What effect would the following conditions have on the retention time of a compound
during GC?
 Increase of the flow rate of helium through the column;
 Increase of the temperature of the column;
 Increase of the length of the column.
11. Why is it necessary to use a co-spot containing both standards and sample when running
TLC?
 EXTRACTION AND CHARACTERIZATION OF LIPIDS FROM FOOD SAMPLES

Instructors’ Laboratory Notes

All amounts and volumes listed are exact amounts needed for one student using one food source.
Note that there should always be excess of each solution.
This is a comprehensive and, thus, somewhat lengthy experiment. It takes three 3-hour periods to
complete it. Use it for junior/senior and graduate level laboratory biochemistry classes or project
laboratories. Please provide GC-MS and MALDI data (available below) to your students if you
do not have access to these instruments.

Day 1: Lipid extraction and transesterification

 Today, students will be using a modified Folch extraction to isolate lipids from one of
two food samples. Once the lipids are extracted, the students will carry out a
transesterification reaction on a portion of the total extract and let it incubate until the
next lab period.
 The two food samples being studied are egg yolk (from chicken or duck egg) and Hass
avocado (typical avocado) pulp. If students want to analyze their own food sample, they
should do it in parallel with one of the typical samples. Note that highly processed food
or ones with low fat content rarely yield any interesting results.
 It is recommended that students work in pairs where one student studies egg yolk and the
other studies avocado. At the end, they should share and compare the data. These two
food sources have very different types of lipids in abundance.
 Techniques being studied today include: mortaring, liquid-liquid extraction,
centrifugation, rotary evaporation, and transesterification.
For extraction (Part A)
 Materials/Instruments:
o Food samples, 1 gram/student
o Sea sand, 1 gram/student
o Mortar and pestle, 1 set/student
o Filter funnel, filter paper, 50 mL test tubes, 1 set/student
o Centrifuge for 50 mL tubes, 1/class
o Pasteur pipette, 2/student
o Round bottom flask, 1/student
o anhydrous Na2SO4, 1 bottle/class
o cotton, 1 bag/class
o rotary evaporator, 2 or more/class
o balance, 2/class
 Solutions needed for today:
o DCM: methanol (2:1 v/v) solution, ~26 mL/student
o Folch salt solution (0.04% CaCl2, 0.034% MgCl2, 0.58% NaCl in DI water), 4
mL/student
o Modified stock solution of DCM: methanol: water (3:48:47 v/v/v). 10 mL/student.
Acetone for washing the round bottom flasks
For transesterification (Part B)
 Materials/Instruments:
 o Screw cap tube, if possible, with Teflon lining, 2/student
o Parafilm, 1 role/class
o 60o C incubator for the class to share. The incubator should be reserved for
enough time so that students can leave their reactions in it until the next lab
period.
 Solutions:
o Stock DCM, 1.2 mL/student
o methanol: sulfuric acid (50:50 v/v), 1 mL/student
o stock toluene, 1 mL total (1 drop/student)

Day 2: Transesterification work up and separation of polar and nonpolar samples

 Today, students will isolate the FAMEs.
 They will separate their total liquid extract into samples of different polarity using silicic
acid column chromatography.
 For time management, we recommend to have students start with column
chromatography and finish with the transesterification work-up.
 Techniques being used today include: liquid-liquid extraction work up, rotary
evaporation, and silicic acid column chromatography.
For transesterification work up/GC sample prep (Part A)
 Materials/Instruments:
o 15 mL tube, 2/student
o Pasteur pipette, ~4/student
o pH strips, 1 pack/class
o sodium sulfate, 1 gram/student
o cotton, 1 pack/class
o round bottom flask, 1 /student
o rotary evaporator, 2 or more/class
o balance, 2/class
o small glass vial, 3/student
 Solutions:
o Access to DI water
o Hexane, 2 mL/student
o DCM stock, 4-8 mL/student
o Saturated NaHCO3, 6 mL/student
o Acetone for washing the round bottom flasks
For silicic acid column chromatography (Part B)
 Materials/Instruments:
o round bottom flask, 2/student
o polyethylene column and stand, 1/student
o silicic acid, 1 g/student
o rotary evaporator, 2 or more/class
o balance, 2/class
 Solutions:
o DCM, ~30 mL/student
o methanol, 10 mL/student
Day 3: TLC, Liebermann-Burchard, GC, MALDI-TOF MS analysis of prepared lipid
fractions
 Today, students will use TLC to analyze their more polar and less polar lipid fractions.
Below is a table showing the standards' compositions followed by the expected results
from each food source. Note that squalene is not present in egg yolk or Hass avocado and
is just used to emphasize the color change of cholesterol derivatives during the ferric
chloride test. Students should photograph their TLC plates and then discard them. It is
our recommendation that you experiment with several concentrations of standards a few
days prior to running experiments with a class to ensure that TLC standards work well.

Polar Standards Egg yolk Hass Nonpolar Egg yolk Hass

avocado standards avocado
Phosphatidylethanolamines Yes No Triolene No Yes
Phophatidylcholines Yes No Squalene No No
Cholesterol Yes No
 GC will be used to analyze fatty acid methyl esters and to compare them to standards ran
on 2400 Varian GC as well as a GC-MS. The GC should be prepared early that morning
to ensure that enough time for temperature conditions are met. A standard method with
specific instrument parameters is attached. Each student’s run should take approximately
12 minutes. It is recommended to set up time schedule for students at the beginning of the
laboratory period for efficient time management.
o Students should be given a chromatogram of standards (ran immediately before
the class period) for the GC instrument containing the following FAMEs
(Standard mixture FAME GLC-20 from Sigma-Aldrich): methyl arichidate 20 wt.
%, methyl linolenate 20 wt. %, methyl oleate 20 wt. %, methyl palmitoleate 20
wt. %, methyl stearate 20 wt. %)
o Students will also use GC-MS data collected at another institution to compare to
their results if GC-MS is not available. If GC-MS is available, run it instead of
GC.
o A list of all FAMEs and lipid molecules as well as both GC protocols are
attached.
 A quantitative Liebermann-Burchard cholesterol test will be performed on the total lipid,
polar lipid, and nonpolar lipid fractions.
 Finally, MALDI-TOF MS will be used to analyze polar and nonpolar lipids using an
automated feature of the spectrophotometer. MALDI-TOF MS will be somewhat
technical because it applies a new automated procedure. It is expected that students will
need your assistance for this step. Both matrixes should be prepared fresh before each
class. All students can spot duplicates of their diluted polar and nonpolar lipid fractions
on the same plate. All spots can be added into the automated configuration and the
program can be run without assistance for all the students at the same time. If the
program is not finished by the end of the laboratory period, students will have to stop by
at a later time to retrieve their data and take a screen shot.
 IMPORTANT: Use matt steel for the MADLI-TOF MS target plate. Ground steel or
aluminum plates cause samples to spread during spotting.
  Techniques being studied today include: TLC, chemical tests via ninhydrin spray, ferric
chloride spray, and Liebermann-Burchard cholesterol test, gas chromatography, and
MALDI-TOF MS preparation and analysis.
For TLC (Part A)
 Materials/Instruments:
o silica gel plates, 2/student
o capillary tubes, at least 6/student
o TLC tank, 2/student
o TLC tank containing iodine (I2) crystals, 2/class
o UV lamp set at 254 nm, 1/class
o hot plate, 2/class
o sprayer, 2/class
o a box in a hood for spraying plates, 1/class
 Solutions:
o DCM stock, 1 mL/class (~36 µL/student for dilutions)
o standard phosphatidylethanolamine and phosphatidylcholine mixture in DCM, 1
mL total (2 µL/student)
o polar DCM/methanol/acetic acid/water (100:23:5:1 v/v) solvent (~800 mL stock)
o standard cholesterol/squalene/triolene mixture, 1 mL total (2 µL/student)
o nonpolar hexane/diethyl ether/acetic acid (49:29:5 v/v) solvent (~800 mL stock)
o ferric chloride spray reagent, 1 bottle full/class
o ninhydrin spray reagent, 1 bottle full/class

For Liebermann-Burchard test (Part B)

 Materials/Instruments:
o Glass test tubes, 5/student
o cholesterol standard, 1 bottle/class (spatula tip full/student)
 Solutions:
o DCM, 15 mL/student
o acetic anhydride, 5 mL/student
o concentrated sulfuric acid, 1 mL total (1 drop/student)

For GC-FID and GC-MS (Part C)

 Materials/Instruments:
o Hamilton syringe, 1/class
o 2400 Varian GC (or equivalent) with FID port, 1/class
o Thermo Fisher GC-MS: ISQ TRACE 13010 with an autosampler (or equivalent),
1/class
 Solutions:
o DCM for washing the syringe, ~10 mL/class
o Standards: 1892-1AMP SUPELCO GLC-20 FAME mix from Sigma-Aldrich
For MALDI-TOF MS (Part D)
 Materials/Instruments:
o matt steel target plate, 1/class
o external MALDI standards for calibration in positive ion mode
 o MALDI-TOF MS reserved for the class period plus additional time in case the
analysis runs past class time: Bruker Daltonics Autoflex
 Solutions:
o ACN, 1 bottle/class (enough for dilutions)
o 0.5 M or ~77 mg/mL DHB (2,5-dihydroxybenzoic acid, C7H6O4, MW:154 g/mol)
solution in 90% methanol, 10% water, 1 mL total (~18 µL/student)
o 30 mM or 6.21 mg/mL ClCCA (4-chloro-α-cyanocinnamic acid, C10H6ClNO2,
MW: 207.61 g/mol) solution in 70/30 ACN/ 1.5% TFA (v/v), 1 mL total (~10
µL/student)
o Peptide &/ lipid standards for external calibration; use internal calibration for
samples run using numerical data provided in tables and graphs.

For report: All goals of the day should be touched upon and results should be analyzed.
Questions from report in regards to gas chromatography should also be answered. Lab partners
should compare data and make general conclusions about their own data and partner’s data. For
example: types of fatty acids in egg yolk vs. avocado, general lipid structure (phospholipids vs.
triglycerides). What type of food is healthier (unsaturated vs. saturated comparison, and
cholesterol comparison)? Have them refer to the website from the National Nutrient Database for
reference values.

HAZARDS:
Precautions should be taken for the following listed materials. Dichloromethane [75-09-2],
methanol [67-56-1], and toluene [108-88-3] are flammable and toxic. Hexane [110-54-3] is
flammable, neurotoxin, and irritant. Sulfuric acid [7664-93-9] is very corrosive and toxic. Acetic
acid [64-19-7] and acetic anhydride [108-24-7] are corrosive and toxic. Ninhydrin [485-47-2]
spray is toxic. Iodine [7553-56-2] is toxic and volatile. Ferric (III) chloride [7705-08-0] spray is
very corrosive. Acetonitrile [75-05-8 ] is toxic upon ingestion in significant amounts. The
following solids are used in relatively small amounts or in solutions and do not pose significant
risks to participants: 2,5-dihydroxybenzoic acid [490-79-9], 4-chloro-α-cyanocinnamic acid
[69727-07-7], silicic acid [1343-98-2], calcium chloride [10043-52-4], magnesium chloride
[7786-30-3], sodium chloride [7647-14-5], sodium sulfate [7757-82-6], and sodium bicarbonate
[144-55-8]. All food samples should be handled with gloves or, alternatively, hands should be
carefully washed subsequently. All volatile solvents should be handled in a hood.
Gas chromatography protocol:

Gas chromatography-mass spectrometry protocol:

 1 µL undiluted sample injected in split mode (Split/ column flow 16.8:1)
 Hydrogen carrier gas 0.8 mL/minute
 Injector: 225o C
 Oven: 50o C for 1 minute, increase to 250o C over 15 minutes. (10o C/min) then hold for 4
minutes
 25 minute total run
 Interface tube 280o C
 For MS: EI, 70 eV, ion source temperature 200o C with solvent delay of 15 minutes
Table 1S. Percent lipid composition (w/w) for the both analyzed food sources. From all trials, the
averaged values ± SD were calculated.
Egg yolk Avocado
Class Average Data (N=18) 26 ± 12% 17.6 ± 6.6%
Student Research Data (N=6) 20.1 ± 1.4% 12.2 ± 0.4%
Reference Value6 26.6% 14.7%

Table 2S. Silicic column lipid separation data.

Egg yolk Hass avocado
More Polar Lipid Less-Polar Lipid More Polar Lipid Less-Polar Lipid
Composition Composition Composition Composition
Class average 11±8% 34±12% 21±23% 65±45%
data (N=18)
Student research
data (N=6) 53±5% 13±2% 17±1% 29±3%

Table 3S. Fatty acyl residues identified using GC. Relative abundances (w/w) were calculated
using instrument generated peak area data.
Experimental Reference6 Retention time
Fatty acyl residue
relative abundance (%) relative abundance (%) (minutes)
Myristoyl (14:0) 0.7 1.0 4.6
Palmitoleyl (16:1) 1.9 3.2 6.4
Egg yolk

Palmitoyl (16:0) 36.8 26.1 6.6

Oleoyl/Linoleoyl (18:1/18:2) 45.6 58.8 8.2
Stearoyl (18:0) 10.4 8.9 8.5
Arachidonyl (20:0) 4.6 2.0 9.6
Palmitoleyl (16:1) 4.2 6.2 6.4
Avocado

Palmitoyl (16:0) 28.4 23.3 6.6

Oleoyl/Linoleoyl (18:1/18:2) 63.8 69.6 8.2
Arachidonyl (20:0) 3.6 0.3 9.6

Table 4S. TLC analysis of the egg yolk and avocado lipid extract fractions of higher polarity.
The Rf values and ninhydrin spray results (as a probe for primary and secondary amines)
correspond to the standards. Dichloromethane/methanol/acetic acid/water (100:23:5:1 (all v/v))
was used as mobile phase.
Primary or Detected in
Standard Rf
secondary amine sample
Avocado Egg yolk

Phosphatidylcholines 0.15 No Yes

Phosphatidylethanolamines 0.90 Yes Yes

Phosphatidylcholines 0.15 No No

Phosphatidylethanolamines 0.90 Yes No

Table 5S. TLC analysis of the egg yolk and avocado lipid extract fractions of lower polarity. The
Rf values, UV lamp results (detection of unsaturated lipids), and iron(III) chloride spray results
(detection of cholesterol as a phenolic structure and detection of squalene via charring)
correspond to the standards. Hexane/diethyl ether/acetic acid (49:29:5 (all v/v)) was used as
mobile phase.
Detected in
Standard Rf UV active at 254 nm Reaction with FeCl3
sample
Cholesterol 0.51 Yes Yes Yes
yolk
Egg

Squalene 0.94 Yes Yes No

Triolein 0.88 Yes No No
Avocado

Cholesterol 0.51 Yes Yes No

Squalene 0.94 Yes Yes No
Triolein 0.88 Yes No Yes

Table 6S. Results of the Liebermann-Burchard tests for unsaturated steroids

Unsaturated
Sample Color
steroids
Positive control Blue/Green Yes
Negative control Colorless No
More polar egg yolk fraction Yellow No
Less polar egg yolk fraction Dark blue/green Yes
Total lipids egg yolk fraction Green Yes
More polar avocado fraction Very light green No
Less polar avocado fraction Light green/purple No
Total lipids avocado fraction Light green/purple No
From the literature and the web
(data may significantly vary depending on the timing and specific source of food):

Egg yolk lipid composition

 Phospholipids: phosphatidylcholines (PC, 80.5%), phosphatidylethanolamines (PE,
11.7%), phosphatidylinositols (PI), lysophosphatidylcholine (LPC), sphingomyelin (SM)

 cholesterol

Egg yolk fatty acyl residue composition

Unsaturated (62% total):
 Oleic acid (18:1) 42.6%
 Palmitoleic acid (16:1) 3.2%
 Linoleic acid (18:2) 16.2%

Saturated (38% total):

 Palmitic acid (16:0) 26.1%
 Stearic acid (18:0) 8.9%
 Arachidic acid (20:0) 2%
 Myristic (14:0) 1%

Avocado (Hass) lipid composition

 (~96%) Di/Triacylglycerols: dioleoyl palmitin, triolein, dipalmitoyl olein, linoleoyloleoyl
palmitin, and linoleoyl diolein

Avocado fatty acyl residue composition

Unsaturated (75.9% total):
 Palmitoleic acid (16:1) 6.2%
 Oleic acid (18:1) 59.3%
 Linoleic acid (18:2) 10.3%
 Linolenic acid (18:3) 0.2%

Saturated (23.95% total):

 Palmitic acid (16:0) 23.3%
 Arachidic acid (20:0) 0.3%
 Stearic Acid (18:0) 0.3%
Table 7S. Identified and assumed phosphatidylethanolamine (PE) and phophatidylcholine (PC)
lipids of the more polar egg yolk lipid fraction using MALDI-TOF MS with a mass accuracy of
±0.04 Da.
Detected mono- Calculated monoisotopic peak Chemical composition of Assumed lipid
isotopic peak (m/z) of protonated species (m/z) protonated/sodiated species composition
718.53 718.54 C39H77NO8P PE 18:0/16:1
PE 16:0/20:4
740.51 740.52 C41H75NO8P
PE 18:2/18:2
PE 18:1/18:1
744.54 744.55 C41H79NO8P
PE 18:0/18:2
746.57 746.57 C41H81NO8P PE 18:0/18:1
PC 16:0/18:2
758.58 758.57 C42H81NO8P
PC 16:1/18:1
760.61 760.59 C42H83NO8P PC 16:0/18:1
768.57 768.55 C43H79NO8P PE 18:0/20:4
C42H82NO8PNa PC 16:0/18:1
782.59 782.57
C44H81NO8P PC 18:2/18:2
PC 16:0/20:2
786.63 786.60 C44H85NO8P
PC 18:0/18:2
PC 16:0/20:1
788.66 788.62 C44H87NO8P
PC 18:0/18:1
790.61 790.63 C44H89NO8P PC 18:0/18:0
PC 16:0/22:6
806.56 806.57 C46H81NO8P
PC 18:2/20:4
PC 16:1/22:4
808.60 808.59 C46H83NO8P
PC 18:1/20:4
PC 16:0/22:4
810.64 810.60 C46H85NO8P
PC 18:0/20:4
PE 22:2/20:4
820.55 820.59 C47H83NO8P
PE 20:2/22:4
PE 20:1/22:4
822.59 822.60 C47H85NO8P
PE 22:1/20:4
PE 20:0/22:4
824.60 824.62 C47H87NO8P
PE 20:2/22:2
Table 8S. Identified and assumed cholesteryl esters (CE) of the more polar egg yolk lipid
fraction using MALDI-TOF MS with a mass accuracy of ±0.02 Da.
Chemical
Detected
Calculated composition of Assumed lipid
monoisotopic
monoisotopic peak (m/z) sodiated cholesteryl composition
peak (m/z)
ester species
637.49 637.50 (sodiated) C43H66O2Na CE(16:5)
647.59 647.57 (sodiated) C43H76O2Na CE(16:0)
663.56 663.57 (sodiated) C43H76O3Na CE(16:0(OH))
685.56 685.55 (sodiated) C45H74O3Na CE(18:3(OH))
693.56 693.56 (sodiated) C47H74O2Na CE(20:5)
Table 9S. Cholesteryl esters (CEs) assumed to be additionally present in the less polar egg yolk
lipid fraction in addition to those listed in the Table 5S with a mass accuracy of ±0.2 Da due to
peak overlapping at m/z 665.72 and 687.41.
Chemical
Detected
Calculated composition of Assumed lipid
monoisotopic
monoisotopic peak (m/z) sodiated cholesteryl composition
peak (m/z)
ester species
639.51 639.51 (sodiated) C43H68O2Na CE(16:4)
665.72 665.53 (sodiated) C45H70O2Na CE(18:5)
687.41 687.57 (sodiated) C45H76O3Na CE(18:2(OH))

Figure 1S. Part of positive ion mode mass spectrum of the more polar egg yolk fraction from
MALDI-TOF MS analysis.
Figure 2S. Part of a mass spectrum of triolein standard from positive ion mode MALDI-TOF
MS analysis.
Figure 3S. Part of a mass spectrum of the less polar Hass avocado lipid fraction from positive
ion mode MALDI-TOF MS analysis.
Figure 4S. Part of a mass spectrum of a cholesterol standard from positive ion mode MALDI-
TOF MS analysis.
Figure 5S. Part of a mass spectrum of the less polar egg yolk lipid fraction from positive ion
mode MALDI-TOF MS analysis.
GC-MS runs and FAME spectra (listed alphabetically;note the retention times):

Figure
6S. Standard GC-MS total ion trace.

Figure 7S. Avocado GC-MS total ion trace.

Figure 8S. Egg yolk GC-MS total ion trace.

Figure 9S. Arachidic acid methyl ester.

Figure 10S. Oleic acid methyl ester.

Figure 11S. Palmitic acid methyl ester.

Figure 12S. Stearic acid methyl ester.

 Table 10S. Student evaluations
Average SD Questions
As a consequence of performing this laboratory experiment, how well
4.17 0.72 do you understand the classification of lipids?
4.33 0.78 How well do you understand the principles of liquid chromatography?
4.25 0.75 How well do you understand the concept of GC?
How well do you understand the principles of MALDI-TOF
4.00 0.74 spectroscopy?
4.42 0.67 How well do you understand the concept of TLC?
3.92 0.90 How well do you understand the process of transesterification?
4.25 0.62 Were the lab directions presented in a clear manner?
Did you find this lab to be more interesting than a typical laboratory
3.96 0.81 experiment?
Would you recommend continuing this lab as part of this course in the
4.08 0.90 future?
4.15 0.77 Average for all

Numerous lower abundant lipids, including entire groups of lipids found in eggs and avocado are
listed in the text above. Please note that lipid dimers were not noted in our spectra. Sodium
adducts of PC’s and PE’s were not abundant due to the hydrophobic nature of extraction used. It
is expected to have difficulties when differentiating between different lipid isomers and sodium
adducts without MS/MS fragmentation analysis. The use of negative-ion mode MALDI MS
analysis is possible and could be explored by students willing to do research or earn extra credit.

Additional literature sources:

1. Christie, W. W. The AOCS Lipid Library. http://lipidlibrary.aocs.org/lipids/tag1/index.htm
(accessed April 17, 2013).
2. Voet, D; Voet, J.G.; Pratt, C.W. Fundamentals of Biochemistry, Life at the Molecular Level, 4th
ed.; John Wiley & Sons: New York, 2013
3. Farooqui, A. A. Glycerophospholipids. In: eLS. John Wiley & Sons Ltd, Chichester. 2009;
http://www.els.net/WileyCDA/ElsArticle/refId-a0000726.html (accessed April 17, 2013).
4. Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 7th ed.; W. H. Freeman: New York, 2010.
5. Christie, W. W. The AOCS Lipid Library. http://lipidlibrary.aocs.org/Lipids/cholest/index.htm
(accessed April 17, 2013).
6. Garrett, R. H.; Grisham, C. M. Biochemistry, 5th ed.; Brooks/Cole, Cengage Learning, Boston,
MA, 2012
7. Levine, G. N., Keaney, J. F., Vita, J. A. Cholesterol Reduction in Cardiovascular Disease -
Clinical Benefits and Possible Mechanisms. The New England Journal of Medicine. 1995, 332,
512-521.
8. Xiong, Q.; Wilson, W. K.; Pang, J. The Libermann-Burchard Reaction: Sulfonation,
Desaturation, and Rearrangement of Cholesterol in Acid. Lipids, 2007, 42, 87-96.
9. Detectors In Gas Chromatography. Ševĉík , J., Eds; Journal of Chromatography Library , Vol. 4;
Elsevier Scientific Publishing Company: New York, 1975
10. McMaster, M. C. GC/MS: A Practical User's Guide, 2nd ed.; Wiley-Interscience: Hoboken, NJ,
2008
11. National Nutrient Database for Standard Reference; U.S. Department of Agriculture (Online)
http://ndb.nal.usda.gov/ndb/search/list (accessed June 26, 2013)
12. Garrett, R. H.; Grisham, C. M. Biochemistry, 4th ed.; Thomson Brooks/Cole: Belmont, CA, 2010
13. Ayyagari, A.; Nigam, A. Lab Manual in Biochemistry, Immunology and Biotechnology, 2nd ed.;
Tata McGraw Hill: New Delhi, India, 2008
14. Juaneda, P.; Rocquelin, G. Rapid and Convenient Separation of Phospholipids and Non
Phosphorus Lipids from Rat Heart Using Silica Cartridges. Lipids 1985, 20, 40–41.
15. Moran, L.A.; Scrimgeour, K.G.; Horton H.R.; Ochs, R.S.; Rawn J.D. Principles of Biochemistry,
2nd ed. Prentice-Hall Inc.: Upper Saddle River, NJ, 1996
16. Moran, L.A.; Scrimgeour K.G. Biochemistry Resource Book. Neil Patterson Publishers/ Prentice-
Hall, Inc., Upper Saddle River, NJ, 1994
17. Ozdemir, F.; Topuz, A. Changes in Dry matter, Oil Content and Fatty Acids Composition of
Avocado During Harvesting Time and Post-Harvesting Ripening Period. Food Chemistry, 2004,
86, 79–83.
18. Plummer, D.T. An Introduction to Practical Biochemistry. McGraw-Hill Book Co. (UK), Ltd.,
Maidenhead, Berkshire, UK, 1971
19. Schiller, J.; Süß, R.; Fuchs, B.; Müller, M.; Petković, M.; Zschörnig, O.; Waschipky, H. The
Suitability of Different DHB Isomers as Matrices for the MALDI-TOF MS Analysis of
Phospholipids: Which Isomer for What Purpose. Eur. Biophys. J. 2007, 36, 517–527.
20. Teuber, K.; Schiller, J.; Fuchs, B.; Karas, M.; Jaskolla, T. W. Significant Sensitivity
Improvements by Matrix Optimization: a MALDI-TOF Mass Spectrometric Study of Lipids
from Hen Yolk. Chem. Phys. Lipids 2010, 163, 552–560.