The methods described here concern compounds that can be operationally defined as marine lipids. The basis of this definition is their amenability to liquid-liquid extraction in non-polar organic solvents, which provides a convenient means of separating them from other compounds in an aqueous matrix. Their hydrophobic nature facilitates their isolation from seawater or biological specimens, as well as their enrichment and the removal of salts and proteins.
For seawater samples, the first step normally involves the separation into operationally-defined dissolved and particulate fractions, usually by filtration. The particulate fraction is that retained by filters, and pore size is important in defining the cutoff. Using lipid-clean forceps, place a 47-millimeter glass fiber filter that has been ashed on a lipid-clean filtration system.
Take the sample and gently swirl it to resuspend any particles that may have settled. Measure out a known volume. The volume will depend on the amount of particulate material in the sample.
When the volume is measured out, add suction to the filtration system, and gently wet the filter with filtered seawater. Add the entire sample slowly to the filtration funnel and rinse the graduated cylinder with sea water to ensure that all particles are added to the filter. Rinse the funnel of the apparatus to ensure all particles are rinsed down onto the filter.
Pull all the liquid through the filter, but don't let the filter dry out, as it can rupture cells that are on the filter. Take the lipid-clean forceps, fold the filter in half, then fold it in thirds once again, and then in half lengthwise. The filter is then placed in a lipid-clean vial.
Cover the filter with two mls of chloroform, cap under nitrogen, and seal with Teflon tape. At this point, the sample will be stable in storage at minus 20 Celsius for up to a year. Prepping the sample.
Measure a known volume of the filtrate into a lipid-clean glass graduated cylinder. Place the sample into a lipid-clean separatory funnel, and add approximately 20 mls of chloroform. Shake this mixture for at least two minutes venting frequently.
After first separation, removal of first extract and addition of acid. After shaking the solution for two minutes, wrap the funnel in aluminum foil and wait until separation has occurred, approximately five minutes. After waiting for separation, peel back the bottom of the aluminum foil to see the two layers.
Collect the bottom layer into a lipid-clean round-bottom flask, being careful not to include any of the top layer. Cap the round-bottom flask under nitrogen, and place in a freezer. Recover the separatory funnel and add sulfuric acid to the liquid.
Add 0.25 mls for every liter of seawater present. Once the sulfuric acid has been added, shake the seawater gently, then add another 10 mls of chloroform, and shake vigorously while venting for a further two minutes. Then allow separation.
Second and third separations. Add the bottom layer to the round bottom flask again without including water. Add a third amount of 10 mls of chloroform and shake for another two minutes with venting.
After separation, place the final portion of chloroform in the same flask, and the combined extracts can be evaporated by rotary evaporator and transferred into a two-ml vial.Setup. For the extraction setup, you'll need an insulated container filled with ice. Solvents include chloroform, chloroform-extracted water, and 2:1 chloroform-methanol.
Of this solution, you need approximately one ml per sample, so make it up accordingly. All these solvents should be placed on ice so that they are cold by the time the extractions are started. The samples also go on ice so that everything remains cold.
Grinding and extraction. Samples have been stored frozen in two mls of chloroform, so add one ml of ice-cold methanol or half a pipetful. Be careful when adding the methanol not to touch the vial with the pipet.
Clean and homogenize three times with methanol and three times with chloroform. When grinding, be careful not to cut the extraction vial in your hand in case you break the vial and to avoid warming the sample. Grind quickly.
Once the sample has been completely ground, stand it in ice and wash any remaining particles from the grinder back into the vial using a ml of 2:1 chloroform-methanol. If necessary, use a lipid-clean set of forceps to force particles back into the vial before washing. Be careful not to touch the pipet to the grinder or the grinder to the vial.
Then add 0.5 mls or a quarter pipet of chloroform-extracted water without touching the grinder or the grinder to the vial. When the grinder has been washed, touch the drop hanging from the grinder to the vial. Cap and keep it in ice until ready to sonicate.
Double pipetting. After centrifuging, there will be two layers in the vial:the organic and aqueous layers. To get the organic layer, the double pipetting technique is used.
To do that, a short five-centimeter pipet with the bulb put on loosely is grasped between the index finger and the middle finger. Gently push the pipet down through the two layers while squeezing the bulb causing bubbles to come out to the end. When the bottom of the second layer is reached, use your thumb to pop the bulb off without drawing the organic layer into the pipet.
With a nine-centimeter pipet squeeze the bulb completely. Put the long end of the pipet into the five-centimeter pipet and remove the bottom layer through the five-centimeter pipet. This extract is then placed in a lipid-clean vial.
Continue to take off the bottom layer until it is all removed. All layers of pooled in the lipid-clean vial. Washing pipets.
Remove the bulb from the nine-centimeter pipet. Place it in the vial with the extract in it. Take a pipetful of clean chloroform, and without touching the pipet to the one being washed, squirt the chloroform around the inside of the pipet.
Gently turn that pipet while washing. Take a second pipetful of chloroform and do the same thing to the outside of the pipet. Make sure that all the chloroform runs down the pipet and into the extraction vial.
When finished with the long pipet, take the short one and repeat, but wash this into the vial with the sample. Spotting a sample. Clean a syringe with chloroform.
Take a sample of known volume and rinse the syringe in the sample. Pull the sample past the amount you want to spot. Then bring the plunger down to the amount you want ensuring no air bubbles.
Use the button to dispense 0.5 microliters and touch the drop to the rod. Allow to dry before placing the next drop on the same spot. Spot all samples in a line on rods held over the end of a warm hot plate.
Focusing in acetone. The focusing of the sample is done in 100%acetone. Add approximately 70 mls of acetone to the bottom of the development tank.
Take the two racks and gently lower them into the development tank. Watch the solvent front as it climbs the rod until the bottom of the spot merges with the top of the spot. Remove the rods.
Dry them for around five seconds, then repeat the procedure. If the samples are very concentrated, this focusing can be done a third time. First development system.
The first development system consists of hexane, ethyl ether and formic acid at 98.5 to one to 0.5. Start by rinsing a graduated cylinder three times with hexane and discarding. Fill the graduated cylinder a little beyond 85 mls with hexane.
Then use a pipet and the bottle of hexane to fill the graduated cylinder to 90 mls. Then bring the bottom of the meniscus to the 91-ml line using ethyl ether, which will give one ml of ethyl ether. Add 0.5 mls of formic acid using the Hamilton syringe.
But first rinse the Hamilton syringe three times with the formic acid to ensure that no chloroform that was used to rinse the sample out of the syringe remains. Add two complete syringefuls or 25 microliters at a time for a total of 50 microliters. It is important that the formic acid is rinsed out of the syringe immediately with chloroform to avoid corroding the metal in the syringe.
Once the Hamilton syringe is clean, make the solution up to exactly 100 mls using hexane and a pipet. Then cap and invert three or four times. Pour in approximately 30 mls into the development tank.
Use this 30 mls to wet the paper and rinse the tank. Discard the rinse solution. Add the remaining 70 mls to the development tank.
Adding racks to development system. Adding rods to the development system is the same for every development. Ensure the timer is set for the time the rods are to be developed.
And then take the racks and gently lower them into the tank. Watch until the solvent front reaches the sample spots, then start the timer. Second development system.
The second development system is hexane, ethylene, and formic acid, 79 to 20 to one. So as before, rinse the graduated cylinder three times with hexane. Add exactly 20 mls of ether and then one ml of formic acid, which brings the volume to 21 mls.
Then bring the volume to 100 mls with hexane. Add about 75 mls of hexane using the bottle pumps. And for the portion, use a pipet.
Cap and invert several times. Add approximately 30 ml to the development tank to wet the paper and rinse the tank. Discard the 30 mls.
Add the remaining 70 and it's ready for the set of rods. Third development system. The third development system is a mixture of chloroform, methanol, and chloroform-extracted water, 50, 40 to 10.
Rinse the graduated cylinder three times with chloroform. Add about 45 mls of chloroform. Using a pipet, bring the bottom of the meniscus to the 50-ml mark.
Add about 38 mls of methanol. Bring the bottom of the meniscus to the 90-ml line. Then fill the last 10 mls with chloroform-extracted water.
As with the other development systems, invert this three or four times, and then pour 30 mls into the tank to wet the paper. Discard that 30 mls and add the last 70 mls into the tank. Preparing the methanol.
To make up the Hilditch solution used for derivatization, fill a lipid-clean clean volumetric flask that has also either been dried or rinsed an extra three times with methanol to remove any chloroform. Add methanol until the bottom of the meniscus is at the to contain line. After measuring the methanol, add sodium sulfate that has been dried for at least 24 hours at 60 degrees Celsius.
Add enough sodium sulfate so that it covers the bottom of the volume metric flask. Once covered, invert twice so that any water that's in the methanol is absorbed by the sodium sulfate. After inverting and shaking, let it sit for at least five minutes.
Adding the acid. After the methanol has settled five minutes, slowly decant it into the final glass bottle ensuring all the sodium sulfate stays in the bottom of the volumetric flask. The sodium sulfate usually remains at the bottom, so all the methanol should pour off.
Leave the volumetric with the sodium sulfate in the back of the fume hood to dry. Slowly add sulfuric acid to the methanol using a PIPETMAN. Add a few drops at a time so the methanol doesn't spit.
Once all the acid has been added, cap and gently stir to mix. The solution is now ready to be used for derivatives, but it must be made up on a weekly basis. Making the derivatives.
From an extract vial that has had the volume brought up to a known amount, swirl, then remove a portion of the extract into lipid-clean, marked 15-ml vial. The amount that you remove will be determined by the concentration of the sample from the Iatroscan. Use a lipid-clean Drummond pipet to remove the sample.
Once the sample has been removed, place the 15-ml vial under nitrogen to completely evaporate the chloroform. The original sample can be kept and returned to the freezer. When the sample is dried, add 1.5 mls of dichloromethane or one pipetful and three mls of the Hilditch solution that was made up earlier that day or two pipetfuls.
This solution is then kept under nitrogen, vortexed and placed in the sonicator for four minutes before being placed in an oven at 100 degrees Celsius for one hour. Stopping the reaction. After heating at 100 Celsius for an hour and cooling, slowly add 0.5 mls of saturated sodium bicarbonate solution.
Bubbles will be produced as the acid is neutralized. Then add 1.5 mls of hexane. Recap and shake vigorously.
Let it stand so that it separates into two layers. Process all samples. By the time they're all finished, the first one will be ready for the next step.
Removing the top layer. Once the derivatization has been halted and there is a clear separation in the 15-ml vial, remove the top layer and place it into a lipid-clean two-ml vial. It's better to leave some of the top layer than and get any of the bottom layer into the two-ml vial.
Once the majority of the top layer has been removed, the rest of the solution can be discarded. The extract that is in the two-ml should be evaporated completely under a gentle stream of nitrogen. Once the vial is completely dry, hexane can be readded, and then the sample should be sonicated, kept under nitrogen, and then it is ready to go to the GC.Representative results.
This figure shows raw TLC-FIC chromatograms of our standard, a diet made with rapeseed oil and liver tissue from a salmon fed that diet. Aquaculture, the fastest growing food production sector, has to reduce its dependence on wild-sourced fishmeal and fish oil. Plant oils are being investigated as replacements for fish oil in aquafeeds.
And liver, the primary site for lipid metabolism, is targeted for analysis. We can see the prevalence of triacylglycerols in the diet in liver, and also the importance of membrane phospholipids in the liver. This figure shows chromatograms of a standard.
Lipids and settling particles collected off the coast here and lipids in the mysid collected near the same depth. Here, chromatograms have been processed through plotting software. Being carbon-rich with a very high energy value, lipids are an important component of the productivity of shelf areas, which are particularly important for carbon cycling.
More surface primary production reaches the seabed in shallower water. We found that small mysid had the highest lipid concentration in our samples. Wax and sterile esters are combined here.
And there is peak splitting due to the presence of high levels of polyunsaturated fatty acids. The rapidity with which the Chromarod Iatroscan TLC-FID system provides synoptic lipid class data from small samples makes it a powerful tool for screening marine samples prior to performing more detailed chromatographic analyses. Such analyses usually require release of component compounds from lipid extracts and derivatization to increase volatility.
