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Lipid analysis

Week 3: GAS LIQUID CHROMATOGRAPHY

During this week, you will analyze the fatty acid composition of the individual lipid fractions recovered from the TLC plate. Gas chromatography is a very sensitive method for the separation and quantification of chemicals, and it is perfect for the analysis of fatty acid components. Like in any other chromatographic technique, separation of compounds depends on their partition between a stationary and a mobile phase. In gas chromatography, the mobile phase is a gas that is moved through the column, while the stationary phase is a liquid film that coats the column filling (in packed columns) or the column wall (in capillary columns). Hence, the correct name for gas chromatography is "Gas Liquid Chromatography", abbreviated GLC. Compounds are injected onto the column and carried through it by the mobile phase; depending on their partition into the stationary phase, they move slower or faster. A sensitive detector is required at the end of the column to detect and quantify the compounds as they leave the column.

While the separation principle is simple, practical considerations necessitate rather a complex experimental set-up. Compounds must be present in the gas phase so that partition between the gaseous mobile phase and the liquid stationary phase is possible. Thus, GLC must be carried out at temperatures above the boiling point of the compounds to be separated. In practice, the boiling point of many compounds, including glycerides and free fatty acids, is too high for GLC analysis. Therefore, compounds are frequently derivatized, i.e. chemically transformed into analogs that are more volatile. In the case of lipids, this is achieved by transforming fatty acids into their methyl esters.

If we want to analyze lipids other than free fatty acids, we could release the free fatty acids by hydrolyzing the glycerides and then transform the free fatty acids into their methyl esters. However, frequently it is more convenient to "methanolyze" the acylglycerides, i.e. to immediately transform the fatty acid glycerol esters into fatty acid methyl esters. This was achieved in the transmethylation reaction with BF₃ and methanol.

The mobile phase in GLC is an inert gas (nitrogen); fatty acid methyl esters are retained more or less by dissolving in the stationary phase. The volatility of fatty acid methyl esters and their interaction with the stationary phase depends on the chain length and degree of desaturation. Increased chain length leads to lower volatility and increased retention; hence, methylesters with short fatty acyl chains come out first, while longer ones have higher retention times. In order to avoid extremely long retention times, one can increase the column temperature, thus increasing the volatility of the longer fatty acid methyl esters. However, short fatty acids will then not separate. If one is interested in achieving good separation of both, short and long fatty acid methyl esters, is is advantageous to use a temperature program: For the first part of the run, the column temperature is low; after the short fatty acids have passed the column, the temperature is gradually increased until all components have left.

The interaction of the mobile phase depends on its chemical nature. Non-polar phases (as SP-1) just separate according to chain length, while polar phases bind polar components more strongly than non-polar ones. For fatty acids, the non-polar phase used here separates all fatty acids only according to their chain length. Unsaturated fatty acids are eluted before the saturated molecule with the same number of carbon atoms. A typical elution profile would look like this:

Various possibilities exist for the detection of the eluted compounds. The most commonly used device for fatty acid analysis is a Flame ionization detector. When the eluted compounds enter the FID detector, they are combusted by an intense flame and broken up into ionized fragments, which can be quantitatively detected.
 

The intensity of the signals depends of course on the amount injected onto the column. In capillary GLC, only very small volumes can be carried through the column. Since it is impractical to inject 1/20 of a microliter, for example, our instrument uses a split injection mode; with a split ratio of 1:20, only 5 % of the volume injected actually enters the column, while 95 % are diverted.

For the exact quantitation with the GLC profile and separate standards, it would be necessary to known what proportion of your total lipid fraction was injected onto the column. This, however, is impossible, given the small injection volume and the various steps involved in the sample preparation. Therefore, we added prior to the lipid extraction step a mixture of internal standards; these were equal amounts of phospholipids, mono- di- and triacylglycerol and free fatty acids which only contained odd-numbered fatty acid chains; these do not naturally occur in animals, and thus any peak in the GLC profile belonging to an odd-numbered fatty acid must come from the internal standard. Since losses in handling etc. are identical for natural lipids and internal standards, we can directly determine how much of each lipid class was present in our lipid extract by simply comparing the sum of all even numbered fatty acid peaks with the peak of the internal standard.

Experimental protocol

1. Run a blank with 1 µl of hexane.

2. Run 1 µl of fatty acid methyl ester standard to calibrate the column.

Name       %    Rt(example)      Rt observed
            
14:0ME     1      4.9 min           
16:0ME     4      7.5 min          
18:3ME     5     10.2 min           
18:2ME    12     10.2 min           
18:1ME    60     10.4 min           
18:0ME     3     10.8 min          
20:0ME     3     14.7 min              
22:1ME     5     17.9 min           
22:0ME     1     18.5 min           
24:0ME     3     22.3 min 

3. Run your samples: PL, MAG, DAG, FFA, TAG. You may have to vary the amount depending on the amount of lipid you have. Start with dissolving the PL fraction in 50 µl of hexane, and inject 1 µl. If response is too weak, concentrate down to 10 µl.

Note that PL and DAG contain the most lipid; dissolve MAG, FFA, and TAG in less hexane. You can vary the amount injected between 0.5 and 2 µl.

Experimental Details:

A: Computer setup

1. Turn on computer if it is off.
2. Double click on GPC alias to launch the Labview data collection program, this must be done each time a sample is run through the GC.
3. After a short period of time a grid will appear with the following settings, Ymax should read "100" and Mode should read "Acquire".
4. Press the white arrow "È", the run button, located in the upper left. A grid should appear over the plot and the scale should change.
5. A dialogue box will come up with the desktop file/folder hierarchy, select "New". A new
   dialogue box with "Untitled Folder" highlighted in black will appear.
6. For your first run you must create a folder for your group that will contain the files of all the GLC runs carried out by your group. Type a name representative of your group over the highlighted "Untitled Folder". Select "Folder" after the name is entered.
7. Another dialogue box will appear with your folder name in the header. Select "New". A dialogue box will now appear asking you to name the file for your GLC run. Type over the "Untitled Folder" with your selected name. Make the file name descriptive for the run you are carrying out so it will be easy to discern from the rest later. Select "File" after naming the run.
8. The program will now return to the grid to allow you to begin the run. It is probably best to keep your group folder at the desktop level of the hierarchy so it is easy to find. Just make sure that all subsequent files for your group are placed into this folder.
   
   

For each subsequent run you must quit the current run. To do this click the small box in the upper left of the grid and the grid will disappear. You must now launch the GPC alias on the desktop and go through the above procedure again except for creating your group folder. Just move about the folder/file hierarchy to find your group folder, open it, type in a name for the new run to be carried out and select "New". The program will move you to the grid to begin your next run.

B: GLC procedure

The GLC will be prepared for your use. Please do not attempt to turn on the GLC on your own if it is off. Do not change any of the setup values as they must remain consistent for all runs.

Check out the panel of the upper right side of the GLC. Numerous buttons for controlling functions are located here. Check the following setup values by depressing the button once and acknowledging the readout.

1. Init Temp - should read 180 °C, this is the starting oven temperature.
2. Inj A Temp - should read 260 °C, this is the temperature of the injector port (where the sample is injected into and vaporized).
3. Det B Temp - should read 280 °C, this is the temperature of the detector at the end of the column. It quantifies materials passing out of the column as voltage through flame ionization.
4. Final Temp - should read 260 °C, this is the upper oven temperature.
5. Init Time - should read 1.0, this is the amount of time in minutes the oven runs at the initial temperature. In this case the oven runs for 1 minute at 180 °C and then starts increasing in temperature.
6. Rate - should read 4 °C, this is the rate of increase per minute in oven temperature.
7. Final Time - should read 8 minutes, this is the time the oven will hold at the final temperature. In this case after 21 minutes the oven will reach 260 °C and run for 8 minutes at that temperature.
8. Purge - should be set to on.
9. Time - (located beside purge), should be at 0.0 min (purge begins immediately).
10. Sig 1 - output signal for detector B (FID) detector we are using). Once the run has started leave this on to follow the signal output at the detector. The output value here and on the computer grid may be slightly offset, this is not a problem. A value around 10.0 or less will be your baseline, the reading on the computer grid will likely be slightly higher.

If all the setup parameters have been reached the green "run" light, located above the buttons, should be lit up. If the settings have not as yet been reached the red "not ready" light will be lit up. During a sample run the three yellow "oven" lights will be lit up in turn as the trial progresses.

Air and hydrogen are flowing over the detector and have been ignited with an electric coil. Helium is flowing through the heated column acting as a carrier gas for your sample to be injected.

Do not adjust any of the valves or controls to the left side of the GLC. These valves control the flow of gases and dangerous conditions may occur if the flame goes out and the gases keep flowing. If the signal drops well below the baseline for a period of time while the trial is running bring it to the attention of the instructor or technician.

C: Sample injection

1. 1. Use the 10.0 µl syringe provided and labeled for the GLC only, do not use any other syringe for your samples.
2. Rinse the syringe out with hexane provided. A minimum of 10 times immediately after injecting a sample and before collecting one if the first run. To do this place the needle tip into the hexane and fill the barrel as full as possible with hexane. Eject the hexane into a waste beaker provided to evaporate. Repeat this over and over. Be careful not to bend the needle plunger as it is delicate.
3. Place the syringe into the sample to be run (hexane for your first run). Remove air from the syringe by running the sample in and out of the syringe until bubbles no longer appear in the needle barrel. Load the syringe to the 1.0 µl mark.
4. Sandwich the sample. After loading the sample to the 1.0 µl mark remove the syringe from the sample vial and draw some air into the needle. You should now be able to see the total volume sandwiched in the needle barrel with air on either side of it. The sample volume will be 2.0µl as the needle itself holds 1.0µl of sample.
5. When ready to run the sample (do not let it sit in the needle for long as it is volatile), computer is set to go and the GLC is in "run" mode, place the needle into the injector port. Port A located on top of the machine to the front left of center. The needle can be difficult to inject through the rubber septum as there is pressure on the other side. Drive it down as straight as possible holding the glass barrel. Push it in until the barrel bottoms out (gently though, be careful not the bend the needle as they are expensive. The technique will be demonstrated to you.
6. As soon as the needle is fully inserted you must inject the entire contents in one quick movement, careful not to bend the plunger. Immediately hit the run button on the GLC and use the mouse to click the start button on the computer grid.
7. Remove the needle carefully straight up out of the injector port, if left in it can alter gas flow patterns.
   

The trial has now started and will run for 30 minutes in total. The hexane solvent spike will appear in approximately 3 minutes. The output at from the FID (Sig 1) will jump off scale as the solvent front moves across the detector.
At the end of a run the computer will display the entire 30 minutes of data output, but only 20 minutes during data acquisition. The oven will begin resetting itself for the next run, the "not ready" light will be on during this time.

D: Calibrating peak areas

You may immediately calculate the areas under the peaks of your chromatogram at the end of a run or you can wait and call up the run for analysis later, (see the next section). Once a completed chromatogram is displayed follow this procedure to calculate the area under each peak.

1. Change the "mode" from "acquire" to "measure", (mode is located to lower right).
2. To improve peak area measurement resolution you can select a specific area of the graph and magnify it. You will need to do this for each peak. To do this click the cursor on the magnifying glass (lower left) and hold it, move to the right and select the first of the three graph options given, (dotted box around peaks). You can now encircle any portion of the chromatogram by clicking your positioned cursor and holding the button while moving it around the area to be magnified. Try this a few times to get the hang of it. Make the area to be measured as easy to discern as possible.
3. To return to the full chromatogram click the "X" and "Y" axis boxes located to the lower left.
4. When you have a peak isolated and magnified for area integration locate the two red cursor cross hairs in the lower center area. Click and hold on cursor 1 and select bring to center. Do the same for cursor 2.
5. Now you must change the cursor mode by clicking the plus sign "+" located to the lower left.
6. a. Move cursor #1 to the lower and most left side of the selected peak and b. move cursor #2 to the upper and most right side of the selected peak. If the baseline changes slightly over the width of the peak simply average it by placing the cursor base in the middle region.
7. When you have satisfactorily boxed in the entire peak with the 2 cursors hit the "run" button (white arrow to the top left). The area under the peak will be given to you in the box beside the mode selection.
8. Clicking on the "X" and "Y" axis boxes to the lower left will return you to full chromatogram display and you can start over with another peak area. It takes a little time to display the entire plot. You must start at step 2 in this list for the next area to be measured.

E: Recalling a chromatogram that has been saved

1. If a chromatogram is already open close it, click the box located to the upper left.
2. Launch GPC alias from the desktop by double clicking it.
3. Under the mode box (lower right) change to "reload".
4. Press the run button (white arrow to top left). When the dialogue box comes up move through the hierarchy to the file you wish to launch and select it.
5. It will take a while to load the file (30sec to 1 minute). Adjust the "X" and "Y" axis (lower left corner) after it loads to get the full graph in view.
6. You can now work with the chromatogram as described in the previous section.

    

   (Should the data file be unusuable, and you only have a hard copy of the chromatogram, you could also analyze the peak areas in the NIH Image software)