26 Part I: Principles
were made with the development of the Lowry
method (Lowry et al. 1951, Peterson 1979), which
combines the Biuret reaction with the reduction
of the Folin-Ciocalteau phenol reagent (phospho-
molybdic-phosphotungstic acid) by tyrosine and
tryptophan residues in the proteins. The resulting
bluish color is read at 750 nm, which is highly sensi-
tive for low protein concentrations (sensitivity 20–
100 g). Other methods exploit the tendency of pro-
teins to absorb strongly in the ultraviolet spectrum
(i.e., 280 nm), primarily due to tryptophan and tyro-
sine residues. Since the tryptophan and tyrosine con-
tents in proteins are generally constant, the absorb-
ance at 280 nm has been used to estimate the
concentration of proteins using Beer’s law. Because
each protein has a unique aromatic amino acid com-
position, the extinction coefficient (E 280 ) must be
determined for each individual protein for protein
content estimation.
Although these methods are appropriate for quan-
titating the actual amounts of proteins available
within a sample or commodity, they do not possess
the ability to differentiate and quantitate the actual
types of proteins within a mixture. The most cur-
rently used methods for detecting and/or quantitat-
ing specific protein components can be cataloged in
the fields of spectrometry, chromatography, elec-
trophoresis, or immunology or a combination of
these (VanCamp and Huyghebaert 1996).
Electrophoresis is defined as the migration of
charged molecules in a solution through an electri-
cal field (Smith 1998). Although several forms of
this technique exist, zonal electrophoresis (in which
proteins are separated from a complex mixture into
bands by migrating in aqueous buffers through a sol-
id polymer matrix called a polyacrylamide gel) is
perhaps the most common. In nondenaturing/native
electrophoresis, proteins are separated based on
their charge, size, and hydrodynamic shape. In dena-
turing polyacrylamide gel electrophoresis (PAGE),
an anionic detergent, sodium dodecyl sulfate (SDS),
is used to separate protein subunits by size (Smith
1998). Isoelectric focusing is a modification of elec-
trophoresis in which proteins are separated by charge
in an electrophoretic field on a gel matrix in which a
pH gradient has been generated using ampholytes.
Proteins will focus or migrate to the location in the
pH gradient that equals the isoelectric point (pI) of
the protein. Resolution is among the highest of any
protein separation technique and can separate pro-
teins with pI differences as small as 0.02 pH units
(Smith 1998, Chang 1998). More recently, with the
advent of capillary electrophoresis, proteins can be
separated on the basis of charge or size in an electric
field within a very short period of time. The primary
difference between capillary electrophoresis and
conventional electrophoresis (described above) is
that a capillary tube is used in place of a polyacry-
lamide gel. Unlike a gel, which must be made and
cast each time, the capillary tube can be reused over
and over. Electrophoresis flow within the capillary
also can influence separation of the proteins in capil-
lary electrophoresis (Smith 1998).
High performance liquid chromatography (HPLC)
is another extremely fast analytical technique that
possesses excellent precision and specificity as well
as the proven ability to separate protein mixtures
into individual components. Many different kinds of
HPLC techniques exist, depending on the nature of
the column characteristics (chain length, porosity,
etc.) and the elution characteristics (mobile phase,
pH, organic modifiers). In principle, proteins can be
analyzed based on the polarity, solubility, or size of
their constituent components.
Reversed-phase chromatography was introduced
in the 1950s (Howard and Martin 1950, Diercky
and Huyghebaert 2000) and has become a widely
applied HPLC method for the analysis of both pro-
teins and a wide variety of other biological com-
pounds. Reversed-phase chromatography is gener-
ally achieved on an inert column packing, typically
covalently bonded with a high density of hydropho-
bic functional groups, such as linear hydrocarbons
with 4, 8, or 18 residues in length, or the relatively
more polar phenyl group. In fact, reversed-phase
HPLC has proven itself useful and indispensable in
the field of varietal identification. It has been shown
that the processing quality of various grains depends
on their physical and chemical characteristics,
which are at least partially genetic in origin, and that
a wide range of qualities exists within varieties of
each species (Osborne 1996). The selection of the
appropriate cultivar is therefore an important deci-
sion for a farmer, since it greatly influences the
return he receives on his investment (Diercky and
Huyghebaert 2000).
Size-exclusion chromatography separates protein
molecules based on their size or, more precisely,