Download Proximate Analysis

Alternative Lipid Classification Scheme
Courtesy of G. S. Smith
CLASSIFICATION OF LIPIDS: Chemically, the lipids are either esters of fatty acids or
substances capable of forming such esters. The term "lipid" or "fat" may, however, be
encountered in general usage to characterize a wide variety of compounds that are insoluble
in water and soluble, to varying extent, in "fat solvents" or "organic solvents" such as: ether
(diethyl ether), chloroform, alcohol (methanol, ethanol, etc.), acetone, benzene, and
"petroleum ether" (usually a mixture of alkanes, especially hexane, "Skellysolve B", the
fraction boiling at about 60-68oC).
I.
SIMPLE LIPIDS: Esters of fatty acids and glycerol or other
alcohols
A. Fats and Oils
B. Waxes
II. COMPOUND LIPIDS: Esters of fatty acids which, on hydroloysis,
yield substances in addition to fatty acids and an alcohol.
A. Phospholipids (Phosphatides): Lipids which, on hydrolysis,
yield fatty acids, phosphoric acid, an alcohol (usually,
but not always, glycerol), and a nitrogenous base:
1. Lecithins - nitrogenous base is choline.
2. Cephalins - nitrogenous base is ethanolamine, serine, etc.
3. Sphingolipids - nitrogenous base is sphingosine.
B. Glycolipids (Cerebrosides): Lipids which, on hydrolysis, yield fatty acids, a complex
alcohol, and a carbohydrate. They contain nitrogen but no phosphoric acid.
C. Sulfolipids: complex lipids which contain sulfur, usually in addition to nitrogen and/or
phosphorus.
PREPARATION AND ANALYSIS OF LIPIDS exerpts from Lovern, Chemistry of Lipids of
Biochemical Significance.
Extraction "Lipids by definition are soluble in the "fat" solvents, such as ether, alcohol and
chloroform. Their extraction from tissues, however, is complicated by at least three factors:
a)
much of the lipid may be linked in some form of combination with protein or
carbohydrate, and these complexes are usually insoluble in fat solvents.
b)
c)
some lipids are only soluble in a limited range of fat solvents; and,
some fat solvents are also good solvents for certain non-lipid constituents of tissues. A
further, but in practice less troublesome, complication is that the original wet tissue
cannot be efficiently extracted with many otherwise good solvents, and must first be
dried.
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There is no difficulty in extracting triglycerides from fat-rich tissues, e.g. adipose tissue or oil
seeds. In general, triglycerides are not bound into lipoprotein or lipocarbohydrate complexes, and
this can certainly be taken for granted with the overwhelmingly greater part of the triglycerides
of fat-rich tissues. (The same is probably true for waxes.) Moreover, triglycerides are easily
soluble in practically all fat solvents, an exception being cold alcohol. Thus the wet tissue may
be extracted with successive batches of a solvent such as acetone, which first extracts the water,
and then the triglycerides. Alternatively the tissue may be dried, e.g., by freezedrying/lyophilizing/, and extracted with any desired solvent, e.g., light petroleum/petroleum
ether," heavily contaminated with non-lipids, particularly if a fairly selective solvent such as
light petroleum is used, either for the original extraction or for re- extraction of the first extract.
Phospholipids, sphingolipids and sterols are usually present in tissues in 'bound' form /or
partly so/. There is a great volume of published work on various methods of ensuring complete
extraction of these lipids....
It has long been known that ethanol is able to liberate much of the protein-bound lipid.
Although ethanol is not a good solvent for some lipids, e.g. the 'cephalin group', its solvent
power is much greater when it also contains such lipids as lecithin. It can also be increased by
addition of ether or benzene. Another favorite mixed solvent is methanol and chloroform.
It is a frequent practice to extract the wet tissue solely with such a solvent mixture. The
alcohol component removes water and liberates bound lipids. Thus Bloor. ....first used a 3:1
ethanol:ether mixture for the extraction of blood lipids, and this solvent has since found favour
for extraction of all kinds of tissues. There is no unanimity as to the relative merits of hot or cold
extraction, nor as to the period of extraction. ....the ratio of ethanol to ether could be varied over
a wide range without affecting the results..../some/prefer a 1:1 chloroform:methanol
mixture....ethanol:ether and chloroform:methanol have been shown to give higher yields.
Benzene:ethanol mixtures have found favour in extracting vegetable phosphatides…./ a 2:1
mixture is suitable/.
Acetone, like alcohol, possesses considerable lipid-freeing powers, but since it is a poor
solvent (at least when cold) for phosphatides and sphingolipids it is not used in the way just
described for alcohol. Acetone is frequently used, however, as a dehydrating solvent, to be
followed by some better general lipid solvent such a light petroleum or chloroform. Used in this
way, acetone will remove, besides water, the triglycerides, sterols and sterol esters of the tissue,
thus ensuring that virtually the only lipids present in the subsequent extracts are phospholipids
and sphingolipids....
It should be noted that acetone does extract a certain proportion of phospholipid material,
particularly when this is accompanied by large proportions of acetone-soluble lipids such as
triglycerides and sterols. In the absence of electrolytes, lecithin and similar substances are
appreciably soluble in cold acetone. Some workers add a electrolyte, commonly magnesium
chloride to the acetone to reduce this solvent effect, but it is not thereby completely
eliminated…. Hot acetone is a good solvent for cerebrosides....
Purification. The problem of non-lipid contaminants has a two-fold origin: a) many of the
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solvents, e.g., acetone and alcohol, especially with wet tissue, are quite effective extractants for
many of the non-lipid constituents of tissues, e.g., urea, amino acids, various nitrogenous bases,
sugars, etc.; and b) substances normally insoluble in fat solvents are readily soluble in the
presence of phospholipids.... Removal of contaminants carried into solution by phospholipids is
particularly difficult, partly because of the tendency of phospholipids to form complexes with all
manner of substances....
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