 # BCEM2201 TLC Data analysis

Points: /10

Formatting:
KEEP THE ORIGINAL FORMATTING only use the tables available to report your results, insert text, graphs, and calculations where you are directed.
Check with your lab instructor about how you need to submit your worksheet. You will be asked to either hand in a paper copy or submit it electronically. Electronic submissions are best in a PDF to preserve formatting!
Failing the above WILL RESULT IN LOST OF MARKS (up to 1 pt.))

1. TLC Data analysis
TLC data 2 pts.

Include a figure representing a picture of your TLC plate. Label your figure to clearly show the Mono- Di- and Trioline bands in the Standard and Fish oil lanes. Follow the figure-making guide for the accepted formatting.
Insert figure here

Amount of neutral lipid in the fish oil tested 3 pts.

1. Pick one type of neutral lipid to quantify in the fish oil (i.e. Mono- or Di- or Trioline), indicate the identity of the lipid in table
2. Calculate the µg of neutral lipid loaded in each standard lane.
3. Using Image J, measure the intensity of the chosen lipid’s band in the standard mixture and fish oil lanes.

Neutral lipid identity (i.e. Mono- Di- or Trioline):
Standard 1
Standard 2
Standard 3
Fish Oil 1
Fish Oil 2

4. Make the lipid Standard curve by plotting intensity/area of peak versus the µg of lipid loaded per lane in the three standard lanes, (report the standard plot in the supplementary info (do not need to have a caption just a well-labelled figure).
5. Use the slope of the curve to calculate the amount of neutral lipid in the original fish oil sample.
Show calculations here

2. MS Data Analysis
Answer specific questions regarding an MS dataset collected from fish oil samples. The MS Data is presented in the following figures. 5 pts.

Figure 1, Figure 2, Figure 3
For your calculations, assume the mass of C, O and H atoms are 12, 16 and 1, respectively. Show all your calculations!
1) What would the m/Z be for the neutral lipid you chose to quantify in the TLC experiment with 1 and 3 ammonium ions bound to it? Can you find a peak on any of the figures? (1 pt)
2) Figure 2 shows an enlargement of the region which can be interpreted to contain long-chain triacylglycerols (~800-1000 m/Z). One distinct feature is that the more intense peaks (i.e., higher relative abundance) have m/Z values separated by ±2 m/Z from neighbouring intense peaks. Suggest what this may be a result of. Also, explain the source of the “grouping” of these peaks into clusters separated by ±~28 from the neighbouring clusters (particularly visible in canola and halibut oil spectra). (Hint: Z = +1 for these species). (1 pt)
3) Figure 3 shows an enlargement of the region containing the medium-chain triacylglycerols. Consider the peak at m/Z 516 again.
a) Assuming that that peak has a +1 charge and that the charge comes from an ammonium ion, what is this lipid’s “real” mass? (1 pt)
b) Assuming it is a triacylglycerol with fully saturated fatty acids, draw a suggested structure of this molecule Show all your calculations! (1 pt)
c) Is your suggested structure the ONLY possible structure consistent with the mass, and why? (1 pt)
Supplementary information

Insert the standard curve here
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BCEM2201

TLC Data analysis
Figure 1: TLC plate with Mono-, Di-, and Trioline bands in the Standard and Fish oil lanes.
TLC plate

Neutral lipid identity: Dioline

Standard 1 40 1569
Standard 2 60 2291
Standard 3 80 3135
Fish Oil 1 N/A 1642
Fish Oil 2 N/A 1593
Calculations:

µg lipid loaded per lane = (amount of lipid (mg)/volume of solution (µL)) x volume spotted (µL) x 1000
µg Dioline loaded per lane in Standards 1-3 = (10/100) x 1 x 1000 = 40, (15/100) x 1 x 1000 = 60, (20/100) x 1 x 1000 = 80
Amount of Dioline in Fish Oil = (Intensity of Fish Oil / Slope of Standard Curve) x µg Dioline loaded per lane in Standards 2 = (1642 / 0.0456) x 60 = 2196 µg
MS Data Analysis
The m/Z for Dioline with 1 and 3 ammonium ions bound to it would be:
Dioline with 1 ammonium ion: [(2 x 12) + (36) + (1 x 22) + (1 x 15)] / 1 = 87
Dioline with 3 ammonium ions: [(2 x 12) + (36) + (1 x 22) + (3 x 15)] / 3 = 103
There is a peak at m/Z 87 in Figure 1 (Fish Oil) that could correspond to Dioline with 1 ammonium ion bound to it.
The grouping of peaks separated by ±2 m/Z from neighbouring intense peaks in Figure 2 may be a result of the presence of different isotopes of the same molecule, each with a slightly different mass. The peaks are grouped into clusters separated by ±~28 from the neighbouring clusters due to the presence of different types of fatty acids, which have different masses and contribute differently to the overall mass of the molecule.

a) The “real” mass of the peak at m/Z 516 in Figure 3, assuming a +1 charge and an ammonium ion, would be [(3 x 12) + (3 x 16) + (3 x 1) – (1 x 15)] / 1 = 165. Therefore, the lipid at m/Z 516 would have a “real” mass of 165 Da.
b) A suggested structure for this molecule would be triolein, which is a triacylglycerol composed of three oleic acid molecules. Oleic acid has a chain length of 18 carbons, which contributes a mass of [(18 x 12) + (32 x 1) + (1 x 16)] = 282 to the molecule. Therefore, the suggested structure for the lipid at m/Z 516 would be:

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O
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O-C-C-C-O-C-C-C-O-C-C-C-O
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H-H-H-H-H-H-H-H-H-H-H-H
c) No, the suggested structure