28 Part I: Principles
Although the above-cited methods are appropri-
ate for quantitating the actual amounts of lipids
within a given sample, they do not offer the ability
to characterize the types of fatty acids within a mix-
ture. Gas chromatography, however, does offer the
ability to characterize these lipids in terms of their
fatty acid composition (Pike 1998). First of all,
mono-, di-, and triglycerides need to be isolated
individually if a mixture exists, usually by simple
adsorption chromatography on silica. The isolated
glycerides can then be hydrolyzed to release individ-
ual fatty acids, which are subsequently converted to
their ester form; that is, the glycerides are saponified
and the fatty acids thus liberated are esterified to
form fatty acid methyl esters. The fatty acids are
now volatile and can be separated chromatographi-
cally using various packed and capillary columns
using a variety of temperature-time gradients.
Separation of the actual mono-, di-, and triglyc-
erides is usually much more problematic than deter-
mining their individual fatty acid constituents or
building blocks. Although gas chromatography has
also been used for this purpose, such methods give
insufficient information to provide a complete tri-
glyceride composition of a complex mixture. Such
analyses are important for the edible oil industry for
process and product quality control purposes as well
as for the understanding of triglyceride biosynthesis
and deposition in plant and animal cells (Marini
2000).
With HPLC analysis, Plattner et al. (1977) were
able to establish that, under isocratic conditions, the
logarithm of the elution volume of a triacylglycerol
was directly proportional to the total number of car-
bon atoms (CN) and inversely proportional to the
total number of double bonds (X) in the three fatty
acyl chains (Marini 2000). The elution behavior is
controlled by the equivalent carbon number (ECN)
of a triacylglycerol, which may be defined as ECN
CN Xn where n is the factor for double bond con-
tribution, normally close to 2.
The IUPAC Commission on Oils, Fats, and Deriv-
atives undertook the development of a method for
the determination of triglycerides in vegetable oils
by liquid chromatography. Materials studied includ-
ed soybean oil, almond oil, sunflower oil, olive oil,
rapeseed oil, and blends of palm and sunflower oils
and almond and sunflower oils (Marini 2000,
Fireston 1994). AOAC International adopted this
method for determination of triglycerides (by parti-
tion numbers) in vegetable oils by liquid chromatog-
raphy as an IUPAC-AOC-AOAC method. In this
method, triglycerides in vegetable oils are separated
according to their equivalent carbon number by
reversed-phase HPLC and detected by differential
refractometry. Elution order is determined by calcu-
lating the equivalent carbon numbers, ECN s and
CN 2n, where CN is the carbon number and n is
the number of double bonds (Marini 2000).CARBOHYDRATE ANALYSIS
Carbohydrates play several important roles in foods,
including among other things, imparting important
physical properties to the foods as well as constitut-
ing a major source of energy in the human diet. In
fact, it has been estimated that carbohydrates ac-
count for greater than 70% of the total daily caloric
intake in many parts of the world (BeMiller and
Low 1998).
Carbohydrates found in nature are almost exclu-
sively of plant origin, with at least 90% of them
occurring in the form of polysaccharides (BeMiller
and Low 1998). Interestingly, although the most
carbohydrates are in the form of polysaccharides,
starches are about the only polysaccharide that is
digestible by humans. The vast majority of polysac-
charides are therefore nondigestible, and they have
been divided into two classes, soluble and insoluble,
which form what is commonly called dietary fiber.
For decades total carbohydrate was determined
by exploiting the tendency of carbohydrates to
condense with various phenolic-type compounds in-
cluding phenol, orcinol, resorcinol, napthoresorcinol,
and -naphthol (BeMiller and Low 1998). The most
widely used condensation was with phenol, which
offered a rapid, simple, and specific determination
for carbohydrates. Virtually all types of carbohy-
drates, mono-, di, oligo-, and polysaccharides, could
be determined. After reaction with phenol in acid in
the presence of heat, a stable color is produced that
can be read spectrophotometrically. A standard
curve is usually prepared with a carbohydrate simi-
lar to these being measured.
Although the above method was, and still is, used
to quantitate the total amount of carbohydrate in a
given sample, it does not offer the ability to deter-
mine the actual types and/or building blocks of