Thanks for that Peter. AOCS substituted cyclohexane for carbon tetrachloride in the iodine value determination.
I think the main issue is that if you extract fat from fish tissue you could skewer the results especially if you are going to do a fatty acid profile of the extracted fat since the phospholipids will be high in EPA and DHA.
I also did a Google search but somehow missed that particular paper. I will download it and read it over.
Again thanks.
Tony
----- Original Message -----
From: P Howgate
To: Anthony Bimbo ; Seafood Discussion Group
Sent: Tuesday, August 08, 2006 5:19 PM
Subject: Re: Bligh and Dyer Lipid Extraction Method
Dear Tony
I don't think that Isooctane can adequately replace chloroform in the B&D lipid extraction procedure. What constitutes a lipid is really an operational: substances that are soluble in organic solvents and not soluble in aqueous solvents. The lipids in fish muscle are themselves a mixture, but they predominately comprise 2 classes, triglyceride lipids and phospholipids. The triglycerides are free as globules of oil, but the phospholipids are bound to membranes. A non-polar solvent such as diethyl ether or the light alkanes used in the Soxhlet method will not split the phospholipid/membrane bond and that is why the Soxhlet method gives a lower value that the B&D. Even if the appropriate ratio of isooctane/methanol/water in the ternary mixture of the 3 solvents could be determined I do not think that the system will extract the polar lipids, i.e the phospholipids. If you are analysing fish with high lipid contents then isooctane or any other non-polar solvent would be suitable and even if the phospholipid are not extracted it would not mean a large error in the results. Phospholipids constitute about 1% of the total wet weight of muscle and this value does not differ much between species. However it is not necessary to use the B&D with fatty fish- the procedure of adding dehydrated sodium sulphate to the minced fish to absorb the water and extracting the mix with the solvent would be adequate.
B&D devised their procedure for the purpose of extracting lipids from fish with low total lipid contents, i.e. containing predominately phospholipids, and this is why they chose a polar solvent like chloroform. The trick in the B&D procedure is to get a mix chloroform and methanol in the correct proportions with the water in the sample to form a mono-phasic system then adding further water and chloroform to produce a biphasic system. You want to minimise the amount of methanol so that only lipids partition into the chloroform phase; too much means that the chloroform layer would contain methanol which in turn allow non lipids to partition into the chloroform. The original B&D paper - Canadian Journal of Biochemistry and Physiology, 1959, vol. 37, 911-917 - describes their experiments to optimise the ratios. A more recent paper - Smedes, F., Thomasen, T.K. (1996), Evaluation of the Bligh, Dyer lipid determination method, Marine Pollution Bulletin, 32, 681-688 - has re-evaluated the B&D procedure and the authors examined the principles behind the procedure. I do not want to try to summarise the paper, but the conclusions include 'The results obtained by applying the method of B&D for lipid determination are strongly operationally defined. The addition order of solvents is very important for the kinetics of the extraction; first dissolve, then extract.'. It seems that you modify the B&D procedure at your peril. The authors found that absorption of solvent on the residue, and the lipid it contains, though small is not insignificant, and they also concluded that the methanol content in the B&D mixture might not be optimal.
The answer to your question then is to find a polar solvent that can extract phospholipids and isooctane would not fit the bill, but all is not lost. After writing the above I Googled the web and came across a paper by Smedes with the title 'Determination of total lipid using non-chlorinated solvents' in The Analyst, 1999, 124, 1711-1718. Just what you want. The whole paper is available at http://pubs.rsc.org/ej/AN/1999/A905904K.PDF. The abstract is:
"The restrictions on the use of chlorinated solvents under the Montreal Protocol makes it necessary to develop an alternative method to the Bligh and Dyer lipid extraction as currently applied to marine tissues. Several different solvent mixtures were systematically tested as a replacement for chloroform. The presence of a polar solvent is a prerequisite in order to obtain phase separation between the aqueous and organic phases, but too high a concentration of solvent in the aqueous phase prevents the more polar lipids from being extracted. A high content of water in the organic phase can result in co-extraction of non-lipids. Several combinations of solvents may be able to extract lipids, but for reasons of safety and toxicity, a propan-2-ol-cyclohexane-water (8 + 10 + 11 v/v/v) mixture has been proposed. The method is not sensitive to a wide range of sample-phase volume ratios provided that the solvent compositions remain constant. Application to plaice, mussel and herring samples showed results that were in agreement with the extraction following Bligh and Dyer using chloroform and methanol."
Best wishes
Peter Howgate
----- Original Message -----
From: Anthony Bimbo
To: Seafood Discussion Group
Sent: Tuesday, August 08, 2006 7:26 PM
Subject: Bligh and Dyer Lipid Extraction Method
Hello Everyone:
An issue just came up in a project I am doing for a client. The Bligh and Dyer Lipid Extraction Method has been around for a long time and is used to extract the total lipid from, among other things, fish tissue. The solvent mix is chloroform/methanol/water.
Chloroform is no longer allowed or is allowed under very controlled conditions so many methods that have used chloroform have switched over to some other solvent. For example in the AOCS method for Peroxide Value in oils and fats, the chloroform has been replaced by Isooctane. Collaborative tests have shown that the new solvent works fine.
I am wondering if anyone knows if the same has been done with the Bligh and Dyer Fat Extraction Method. Will Isooctane in conjunction with methanol and water work with the lipid extraction from fish tissue? If not, are there any suggestions?
Maybe someone up in Halifax NS can ask Graham Bligh about this.
Thanks
Tony Bimbo
Technical Consultant- International Fisheries
55 Cedar Lane, PO Box 1606
Kilmarnock, VA 22482 USA
Tel /Fax 804-435-3915
Email apbimbo@verizon.net
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