BLBS102-c02 BLBS102-Simpson March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come
30 Part 1: Principles/Food AnalysisThe separation of the actual mono-, di-, and triglycerides is
usually much more problematic than determining their individ-
ual fatty acid constituents or building blocks. Although GC has
also been used for this purpose, such methods result in insuffi-
cient information about the complete triglyceride composition
in 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).
Using HPLC analysis, Plattner et al. (1977) were able to estab-
lish that, under isocratic conditions, the logarithm of the elution
volume of a triacylglycerol (TAG) was directly proportional to
the total number of carbon atoms (CN) and inversely propor-
tional 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) or a TAG, which may be
defined as ECN=CN−X.nwherenis the factor for double
bond contribution, normally close to 2.
The IUPAC Commission on Oils, Fats, and Derivatives un-
dertook the development of a method for the determination of
triglycerides in vegetable oils by liquid chromatography. Mate-
rials studied included various oils extracted from plant materi-
als such as soybeans, almonds, sunflowers, olives, canola, and
blends of palm and sunflower oils, and almond and sunflower
oils (Fireston 1994, Marini 2000). The method for the determi-
nation of triglycerides (by partition numbers) in vegetable oils by
liquid chromatography was adopted by AOAC International as
an official IUPAC-AOC-AOAC method. In this method, triglyc-
erides in vegetable oils are separated according to their ECN
by reversed-phase HPLC and detected by differential refrac-
tometry. Elution order is determined by calculating the ECNs,ECN=sand CN− (^2) n, where CN is the carbon number andn
is the number of double bonds (Marini 2000).
CARBOHYDRATE ANALYSIS
Carbohydrates comprise approximately 70% of the total caloric
intake in many parts of the world, being the major source of
energy for most of the world (BeMiller and Low 1998). In foods,
these macromolecules have various important roles, including
imparting important physical properties to foods such as sensory
characteristics and viscosity.
The vast majority of carbohydrates are of plant origin mostly
in the form of polysaccharides (BeMiller and Low 1998). Most
of these polysaccharides are non-digestible by humans, the only
digestible polysaccharide being the starch. The non-digestible
polysaccharides are divided into two groups, soluble and insol-
uble, forming what is referred to as dietary fiber. Each of these
two groups has specific functions not only in the plant tissues
but also in human and animal digestive systems, subsequently
influencing the health of the entire organism.
For many years, the total carbohydrate content was deter-
mined by exploiting their tendency to condense with phenolic-
type compounds, including pheno, orcinol, resorcinol, napthore-
sorcinol, andα-naphthol (BeMiller and Low 1998). The most
widely used condensation reaction was with phenol, which was
used to determine virtually all types of carbohydrates including
mono-, di-, oligo-, and polysaccharides. The analytic method
is a rapid, simple, and specific determination for carbohydrates.
After reaction with phenol in acidic conditions in the presence of
heat, a stable color is produced, which is read spectrophotomet-
rically. A standard curve is usually prepared with a carbohydrate
similar to the one being measured.
Although the above method was, and is still, used to quan-
tify the total amount of carbohydrate in a given sample, it does
not offer the ability to determine the actual types of individ-
ual carbohydrates in a sample. Earlier methods such as paper
chromatography, open column chromatography, and thin-layer
chromatography have largely been replaced by either HPLC
and/or GC (Peris-Tortajada 2000). GC has become established
as an important method in carbohydrate determinations since
the early 1960s (Sweeley et al. 1963, Peris-Tortajada 2000) and
several unique applications have since then been reported (El
Rassi 1995).
For GC analysis, carbohydrates must first be converted into
volatile derivatives, the most common derivatization agent be-
ing trimethylsilyl (TMS). In this analytic technique, the aldonic
acid forms of carbohydrates are converted into their TMS ethers,
which are then injected directly into the chromatograph having
a flame ionization detector. Temperature programming is uti-
lized to maximally optimize the separation and identification of
individual components. Unlike GC, HPLC analysis of carbo-
hydrates requires no prior derivatization of carbohydrates and
gives both qualitative (identification of peaks) and quantitative
information of complex mixtures of carbohydrates. HPLC is an
excellent method for the separation and analysis of a wide va-
riety of carbohydrates ranging from the smaller and relatively
structurally simpler monosaccharides to the larger and more
structurally complex oligosaccharides. For the analysis of the
larger polysaccharides and oligosaccharides, a hydrolysis step is
needed prior to chromatographic analysis. A variety of different
columns can be used with bonded amino phases used to sepa-
rate carbohydrates with molecular weights up to about 2500 kDa
depending upon carbohydrate composition and their specific sol-
ubility properties (Peris-Tortajada 2000). The elution order on
amine-bonded stationary phases is usually monosaccharide and
sugar alcohols followed by disaccharides and oligosaccharides.
Such columns have been successfully used to analyze carbohy-
drates in fruits and vegetables as well as processed foods such
as cakes, confectionaries, beverages, and breakfast cereals (Be-
Miller and Low 1998). With larger polysaccharides, gel filtration
becomes the preferred chromatographic technique as found in
the literature. Gel filtration media such as Sephadex and Bio-
Gel have successfully been used to characterize polysaccharides
according to molecular weights.
MINERAL ANALYSIS
A (dietary) mineral is any inorganic chemical element in its
ionic form, excluding the four elements, namely carbon, nitro-
gen, oxygen, and hydrogen. These minerals are important as they
are required for a wide range of physiological functions, includ-
ing energy generation, enzyme production, providing structure
(skeleton), circulation fluids (blood), movement (muscle and