2 Analytical Techniques 27their hydrodynamic volume, and it has become very
popular in recent years. Size-exclusion chromatog-
raphy utilizes uniform rigid particles whose uniform
pores are sufficiently large for the protein molecules
to enter. Large molecules do not enter the pores of
the column particles and are therefore excluded, that
is, they are eluted in the void volume of the column
(i.e., elute first), whereas smaller molecules enter
the column pores and therefore take longer to elute
from the column. An application example of size-
exclusion chromatography is the separation of soy-
bean proteins (Oomah et al. 1994). In one particular
study, nine peaks were eluted for soybean, corre-
sponding to different protein size fractions; one peak
showed a high variability for the relative peak area
and could serve as a possible differentiation among
different cultivars. Differences, qualitatively and
quantitatively, in peanut seed protein composition
were detected by size-exclusion chromatography
and contributed to evaluation of genetic differences,
processing conditions, and seed maturity. Basha
(1990) found that size-exclusion chromatography
was an excellent indicator of seed maturity. Basha
(1990) discovered that the area of one particular
component (peak) decreased with increasing maturi-
ty and remained unchanged towards later stages of
seed maturity. The peak was present in all studied
cultivars, all showing a “mature seed protein profile”
with respect to this particular peak, which was there-
fore called “Maturin.”
LIPID ANALYSIS
By definition, lipids are soluble in various organic
solvents but insoluble in water. For this reason, lipid
insolubility in water becomes an important distin-
guishing and analytical factor used in separating
lipids from other cellar components such as carbo-
hydrates and proteins (Min and Steenson 1998). Fats
(solids at room temperature) and oils (liquid at room
temperature) are composed primarily of tri-esters of
glycerol with fatty acids and are commonly called
triglycerides. Other major lipid types found in foods
include free fatty acids, mono- and diacylglycerols,
and phospholipids.
Fats and oils are widely distributed in nature and
play many important biological roles, especially
within cell membranes. In general, many naturally
occurring lipids are composed of various numbers of
fatty acids (one to three) with various chain lengths,
usually greater than 12 carbons, although the vast
majority of animal and vegetable fats are made up of
fatty acid molecules of greater than 16 carbons.
The total lipid content of a food is commonly
determined using various organic solvent extraction
methods. Unfortunately, the wide range of relative
hydrophobicity of different lipids makes the selec-
tion of a single universal solvent almost impossible
for lipid extraction and quantitation (Min and Steen-
son 1998). In addition to various solvent extraction
methods (using various solvents), there are nonsol-
vent wet extraction methods and other instrumental
methods that utilize the chemical and physical prop-
erties of lipids for content determination.
Perhaps one of the most commonly used and eas-
iest to perform methods is the Soxhlet method, a
semicontinuous extraction method that allows for
the sample in the extraction chamber to be com-
pletely submerged in solvents for 10 minutes or
more before the extracted lipid and solvent are
siphoned back into the boiling flask reservoir. The
whole process is repeated numerous times until all
the fat is removed. The fat content is determined
either by measuring the weight loss of the sample or
the weight of lipid removed.
Another excellent method for total fat determina-
tion includes supercritical fluid extraction. In this
method, a compressed gas (usually CO 2 ) is brought
to a specific pressure-temperature combination that
allows it to attain supercritical solvent properties for
the selective extraction of lipid from a matrix. In this
way specific types of lipids can be selectively ex-
tracted while others remain in the matrix (Min and
Steenson 1998). The dissolved fat is then separated
from the compressed, liquified gas by a drop in pres-
sure, and the precipitated lipid is then quantified as
percent lipid by weight (Min and Steenson 1998).
Another method often used for total lipid quanti-
tation is the infrared method, which is based on the
absorption of infrared energy by fat at a wavelength
of 5.73 m (Min and Steenson 1998). In general, the
more energy is absorbed at 5.73 m, the higher
the lipid content in the material. Near-infrared spec-
troscopy has been successfully used to measure the
lipid content of various oilseeds, cereals, and meats;
it has the added advantage of being nondisruptive to
the sample, in contrast to other previously reviewed
methods.