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This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.
Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.
If seawater has been sampled, the first step usually involves separation into operationally defined 'dissolved' and 'particulate' factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.
Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).
The methods described here concern substances that are defined operationally as marine lipids. This definition is based on their amenability to liquid-liquid extraction in non-polar organic solvents, and it provides a convenient method for their separation from other substances in an aquatic matrix. Their hydrophobic nature facilitates their isolation from seawater or biological specimens, as well as their enrichment, and the removal of salts and proteins.
The measurement of lipid content and its composition in marine organisms has been of great interest in food web ecology, aquaculture nutrition, and food science for decades. Lipids are un....
NOTE: To clean glassware, instruments and filters for lipid analyses, wash them 3 times with methanol followed by 3 washes with chloroform, or heat them to 450°C for at least 8 hours.
1. Filtration procedure for seawater dissolved and particulate lipids
NOTE: The particular fraction of interest is operationally defined by the filtration procedure. In this case the pore size is 1.2 µm.
As the fastest growing food production sector, aquaculture is evolving in terms of technological innovations and adaptations to meet changing requirements. One of these is to reduce the dependence on wild-sourced fishmeal and fish oil, which provide feed ingredients for many aquaculture species. Terrestrial plant oils are being investigated as sustainable and economical replacements for fish oil in aquafeeds, and the liver is a target tissue for analysis because it is the primary site for lipid metabolism
The speed with which the TLC-FID system provides synoptic lipid class information from small samples makes TLC-FID an able tool for screening marine samples before undertaking more involved analytical procedures. Such analyses usually require release of component compounds from lipid extracts and derivatization to increase volatility in the case of gas chromatography. TLC-FID combined with GC-FID has been found to be a powerful combination for extracts of seafood and other foodstuffs14. For succes.......
The authors have no competing financial interests.
This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) grant number 105379 to C.C. Parrish. Memorial University's Core Research Equipment & Instrument Training (CREAIT) Network helped fund this publication.
....Name | Company | Catalog Number | Comments |
15 ml vials | VWR | 66009-560 | |
1-hexadecanol | Sigma | 258741-1G | |
1-Monopalmitoyl-rac-glycerol | Sigma | M1640-1g | |
2 ml vials | VWR | 46610-722 | |
25 mm glass fibre filters | Fisher | 09 874 32A | |
2ml pipet bulbs | VWR | 82024-554 | |
47 mm glass fibre filters | Fisher | 09 874 32 | |
5 3/4" pipets | Fisher | 1367820A | |
9" pipets | Fisher | 1367820C | |
Acetone | VWR | CAAX0116-1 | |
Agilent GC-FID 6890 | Agilent | ||
Calcium Chloride ANHS 500gm | VWR | CACX0160-1 | |
Caps for 2 ml vials | VWR | 46610-712 | |
chloroform | VWR | CACX1054-1 | |
Cholesteryl palmitate | Sigma | C6072-1G | |
Chromarod S5 | Shell USA | 3252 | |
Dichloromethane | VWR | CADX0831-1 | |
DL-a-phosphatidylcholine, dipalmotoyl | Sigma | P5911-1g | |
Ethyl Ether, ACS grade anhydr 4L | VWR | CAEX0190-4 | |
Glyceryl tripalmitate | Sigma | T5888-100MG | |
Hamilton Syringe 702SNR 25µl | Sigma | 58381 | |
Helium | Air Liquide | A0492781 | |
Hexane | VWR | CAHX0296-1 | |
Hydrogen regulator | VWR | 55850-484 | |
Iatroscan MK6 | Shell USA | ||
Kimwipes | Fisher | 066662 | |
Medical Air | Air Liquide | A0464563 | |
Medium nitrile gloves | Fisher | 191301597C | |
Nitrile gloves L | VWR | CA82013-782 | |
Nitrogen | Air Liquide | A0464775 | |
Nitrogen Regulator | VWR | 55850-474 | |
Nonadecane | Sigma | 74158-1G | |
Palmitic acid | Sigma | P0500-10G | |
Repeating dispenser | Sigma | 20943 | |
Sodium Bicarbonate 1kg | VWR | CA97062-460 | |
Sodium Sulfate Anhy ACS 500gr | VWR | CA71008-804 | |
Sulfuric acid | VWR | CASX1244-5 | |
Teflon tape | Fisher | 14610120 | |
tissue master 125 115V w/7mm homogenator | OMNI International | TM125-115 | |
TLC development tank | Shell USA | 3201 | |
UHP hydrogen | Air Liquide | A0492788 | |
VWR solvent repippetter | VWR | 82017-766 | |
VWR timer Flashing LED 2 channel | VWR | 89140-196 | |
Zebron ZB-Wax GC column | Phenomenex | 7HM-G013-11 |
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