BLBS102-c02 BLBS102-Simpson March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come
2 Analytical Techniques in Food Biochemistry 29partially soluble in organic solvents. Lipids are widely dis-
tributed in nature and play many important biological roles,
including signaling (e.g., cholesterol), acting as structural mate-
rials in cellular membranes, and energy storage. The amphiphilic
nature of some lipids gives them the ability to form cellular
structures such as vesicles and liposomes within cellular aquatic
environments.
Analytically, lipid insolubility in water becomes an important
distinguishing characteristic that can be maximally exploited in
separating lipids from other nutritional components in the food
matrix such as carbohydrates and proteins (Min and Steenson
1998). Classically, lipids are divided into two groups based upon
the types of bonding between the carbon atoms in the backbone,
which influences the lipid’s physical characteristics. Fats having
a greater number of fatty acids with single carbon to carbon
bonds (saturated fatty acids) cause it to solidify at 23◦C (room
temperature). On the other hand, the fatty acids in oils have a
greater proportion of one or more double bonds between the car-
bon atoms (unsaturated fatty acids) and hence are liquid at room
temperature. Structurally, glycerides are composed primarily of
one to three fatty acids (a mixture of saturated and unsaturated
fatty acids of various carbon lengths) bonded to the backbone of
a glycerol molecule, forming mono-, di-, and triglycerides (the
most predominant), respectively. Animal fats in the milk (from
mammals), meat, and from under the skin (blubber), including
pig fat, butter, ghee, and fish oil (an oil), are generally more
solid than liquid at room temperature. Plant lipids tend to be liq-
uids (i.e., they are oils) and are extracted from seeds, legumes,
and nuts (e.g., peanuts, canola, corn, soybean, olive, sunflower,
safflower, sesame seeds, vegetable oils, coconut, walnut, grape
seed, etc.). Margarine and vegetable shortening are made from
the above plant oils that are solidified through a process called
hydrogenation. This method of solidifying oils increases the
melting point of the original substrate but produces a type of
fat calledtransfat, whose content can be as high as 45% of the
total fat content of the product. The process, however, is greatly
discouraged around the world as thesetransfats have a detri-
mental effect on human health (Mozaffarian et al. 2006).Trans
fats are so detrimental to human health that the amount oftrans
fat in food has been legislated even to the point of being banned
in some countries. In 2003, Denmark became the first country
in the world to strictly regulate the amount oftransfat in foods,
and since then many countries have followed this lead.
The total lipid content of a food is commonly determined us-
ing extraction methods using organic solvents, either singularly
or in combinations. Unfortunately, the wide relative hydropho-
bicity range of lipids makes the choice of a single universal
solvent for lipid extraction and quantitation nearly impossible
(Min and Steenson 1998). In addition to various solvents that
can be used in the solvent extraction methods, non-solvent wet
extraction methods and other instrumental methods also exist,
which utilize the chemical and physical properties of lipids for
content determination.
One of the most frequent and easiest methods to determine the
crude fat content in a food sample is the Soxhlet method, a semi-
continuous extraction method using relatively small amounts of
various organic solvents. In this method, the solid food sam-ple (usually, dried and pulverized) is placed in a thimble in the
chamber and is completely submerged in the hot solvent for
10 minutes or more before both the extracted lipid and solvent
are siphoned back into a boiling flask reservoir. The whole pro-
cess is repeated numerous times until all the fat is removed,
which usually requires at least two hours. The lipid content is
determined by measuring either the weight loss of the sample in
the thimble or by the weight gain of the flask reservoir. The main
advantage of this method is that it is relatively quick and specific
for fat as other food components such as proteins and carbohy-
drates are water soluble. Some preparation of the sample, such
as drying, pulverizing, and weighing, may be needed to increase
the extraction efficiency as extraction rates are influenced by the
size of the food particles in the sample, food matrix, etc. If the fat
component needs to be removed from the sample before further
analysis, the method also allows for the sample in the thimble
to remain intact without destruction.
Another excellent method for total fat determination is su-
percritical fluid extraction. In this method, a compressed gas
(e.g., CO 2 ) is brought to a specific pressure–temperature point
that allows it to attain supercritical solvent properties for the
selective extraction of a lipid from a food matrix by diffusing
through it (Mohamed and Mansoori 2002). This method per-
mits the selective extraction of lipids, while other lipids remain
in the food matrix (Min and Steenson 1998). The dissolved fat
is then separated from the compressed/liquefied gas by reducing
the pressure, and the precipitated lipid is then quantified as a
percent lipid by weight (Min and Steenson 1998).
A third often used method for total lipid quantitation is in-
frared, which is based on the absorption of infrared energy by fat
at a wavelength of 5.73 um (Min and Steenson 1998). In general,
there is a direct proportional relationship between the amount of
energy absorbed at this wavelength and the lipid content in the
material. Near-infrared spectroscopy has been successfully used
to measure the lipid content of various oilseeds, cereals, and
meats. The added advantage is that it maintains the integrity of
the sample, which is in contrast to the other previously reviewed
methods.
Although these three cited methods are appropriate to quan-
tify the actual amounts of lipids in a given sample, they are
not able to determine the types of fatty acids within a lipid
sample. In order to determine the composition of the lipid, GC
offers the ability to characterize these lipids in terms of their
fatty acid composition (Pike 1998). The first step is to isolate
all mono-, di-, and triglycerides needed if a mixture exists usu-
ally by simple adsorption chromatography on silica or by using
a one- or two-phase solvent–water extraction method. The iso-
lated glycerides are then hydrolyzed to release the individual
fatty acids and subsequently converted to their ester form, that
is, the glycerides are saponified and the liberated fatty acids
are esterified to form fatty acid methyl esters, that is, they are
derivatized. The method of derivatization is dependent upon the
food matrix, and the choice is an important consideration as
some methods can produce undesired artifacts. The fatty acids
are now volatile and can be separated chromatographically us-
ing various gases, various packed or capillary columns, and a
variety of temperature–time gradients.