Hi, I am extracting lipids from microalgae with a simplified single-step method, in which I use chloroform:methanol 2:1 as solvent; after adding 0.73% NaCl I have phase separation and I should recover the lower phase in which my lipids are. I tried with a glass Pasteur pipette but I think that also some of the upper phase and/or some cellular debris are accidentally taken, and I don't want it because then I will measure the lipids gravimetrically after solvents evaporation and this could influence the final weight. Any suggestion to avoid this problem? Thanks in advance.
You can use the filter paper to separate the cellular debris.
The upper methanol layer is usually removed using an automatic pipette or a pipette with a pear, but it must be done carefully. A small portion of lipids, you lose a separation of the water-methyl.
You can use a medical syringe probably, if you have a small amount of the extract.
Dear Lorenza
I have published two patents on this subject.
Please review
METHODS FOR PRODUCTION OF ALGAE DERIVED OILS
Application number: 20130210093
Abstract: The present invention comprises a method of producing algae derived oils comprising growing, stressing, and harvesting algae and then extracting total lipids from which specific oil fractions are purified.
Type: Application
Filed: June 14, 2011
Issued: August 15, 2013
Assignee: IO-Mega Holding Corporation
Inventors: Raveendran Pottathil, Sanjay Kumar Amrutrao Deshmukh
Single step transesterification of biodiesel feedstock using a gaseous catalyst
Patent number: 8497389
Abstract: Embodiments of the present application provide methods for processing biodiesel from feedstock using a single-step process. The methods can include, for example, use of a gaseous catalyst as part of the esterification/transesterification process. Embodiments of the present application also provide systems for the methods thereof.
Type: Grant
Filed: December 8, 2009
Issued: July 30, 2013
Assignee: Initio Fuels LLC
Inventor: Raveendran Pottathil
Gravimetric measuremnets are no longer accepted by the lipid community. Use a direct ie in situ methylation. If you must extract be ware that the result you obtain will be solvent dependent, see Guckert etal J. Microbiol methods 8: 139-149 ( 1988) I don't have an electronic copy, Sorry
KEC
Dear Lorenza,
As you are using a gravimetric method to assess the amount of lipids extracted then additional material collected during liquid-liquid extraction will influence your result. Have you considered using inorganic phosphate quantification as an alternative method to asses the amount of lipids extracted? This way any contaminant material (peptides, sugars, phenolics) present in the organic phase will not interfere with your result. The inorganic phosphate assay is very simple involving a digestion step, addition of molibdate salt for color development and colorimetric measurement at 797nm.
An alternative option could be performing an additional cleaning step with water to your extract.
Hope this helps,
Ana
Dear all
I agree with Ana on the gravimetric method. The method I published involve "direct in-situ esterification" of all types of lipids [FFA,PL and Neutral lipids" in one step [HCl;Methanol] and subsequent quantization through GLC
Let me know if you need any more than the published process
Dear Ana, do you have the detailed protocol for this inorganic phosphate quantification?
Dear Lorenza,
Pls email me on [email protected] and I'll send you the protocol.
Best regards, Ana
A few responses to points raised (sorry if any sounds unkind...):
Filter paper is a problem because you can lose material.
Gravimetric results are not sufficiently insightful these days, if you want to publish.
Fatty acid work is not really indicative of anything more than the fatty acid profile. It doesn't tell you about TAGs, nor about phospholipids.
GC is difficult to use for quantification because you need to have comprehensive internal standards, which is not necessarily easy to organise. So, you might want to be careful about this approach.
What's the application you wish to use this for?
We just want to compare different algal strains and select the best one for biomass production and/or lipid production.
Sounds interesting. I believe there have been one or two studies in this area already, though I guess not on the species/strains you are looking at. I'd be really interested to see profiling data as well as mass data on it--how long and how unsaturated the FAs are, and how much and which type(s) of phospholipid there are could highlight which species are good for which tasks, e.g. fuel as compared to food.
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