2 Analytical Techniques 31tivity is paramount to their detection. Although
ultraviolet absorbance is the most common detec-
tion method, both fluorescence and electrochemical
detection are also used in specific cases. Refractive
index detection is seldom used for vitamin detection
due to its inherent lack of specificity and sensitivity.
During the 1960s, gas chromatography using
packed columns was widely applied to the determi-
nation of various fat-soluble vitamins, especially
vitamins D and E. Unfortunately, thin-layer chroma-
tographic and open-column techniques were still nec-
essary for preliminary separation of the vitamins, fol-
lowed by derivatization to increase the vitamins’
thermal stability and volatility (Ball 2000). More
recently, the development of fused-silica, open tubu-
lar capillary columns has revived the use of gas
chromatography, leading to a number of recent ap-
plications for the determination of fat-soluble vita-
mins, especially vitamin E (Marks 1988, Ulberth
1991, Kmostak and Kurtz 1993, Mariani and Bellan
1996). This being said, the method of choice for
determining fat-soluble vitamins in foods is HPLC
(Ball 2000). The interest in this chromatographic
technique is due to the lack of need for derivatiza-
tion and the greater separation and detection selec-
tivity this technique offers. Various HPLC methods
of analysis were introduced for the first time in the
1995 edition of the Official Methods of Analysis of
AOAC International; these include vitamin A in
milk (AOAC 992.04, 1995) and vitamins A (AOAC
992.26, 1995), E (AOAC 992.03, 1995), and K
(AOAC 992.27, 1995) in various milk-based infant
formulas (Ball 2000).
It should be noted that at present there is no uni-
versally recognized standard method for determin-
ing any of the fat-soluble vitamins that can be ap-
plied to all food types (Ball 2000).
PIGMENT ANALYSIS
Color is a very important characteristic of foods and
is often one of the first quality attributes used to
judge the quality or acceptability of a particular food
(Schwartz 1998). There are a vast number of natural
and synthetic pigments, both naturally occurring and
added to foods, that contribute to food color. Of the
naturally occurring pigments in foods, the vast ma-
jority can be divided into five major classes, four of
which are distributed in plant tissues and one in ani-
mal tissues (Schwartz 1998). Of those found in
plants, two types are lipid soluble (i.e., the chloro-
phylls and the carotenoids), and the other two are
water soluble (i.e., anthocyanins and betalains).
Carotenoids are found in animals but are not biosyn-
thesized; that is, they are derived from plant sources
(Schwartz 1998).
Several analytical methods have been developed
for the analysis of chlorophylls in a wide variety of
foods. Early spectrophotometric methods allowed
for the quantitation of both chlorophyll a and chloro-
phyll b by measuring absorbance at the absorbance
maxima of both chlorophyll types. Unfortunately,
only fresh plant material could be assayed, as no
pheophytin could be determined. This became the
basis for the AOAC International spectrophotomet-
ric procedure (Method 992.04), which provides
results for total chlorophyll content as well as for
chlorophyll a and chlorophyll b quantitation.
Schwartz et al. (1981) described a simple re-
versed-phase HPLC method for the analysis of
chlorophylls and their derivatives in fresh and pro-
cessed plant tissues. This method simplified the
determination of chemical alterations in chlorophyll
during the processing of foods and allowed for the
determination of pheophytins and pyropheophytins.
For carotenoid analysis, numerous HPLC meth-
ods have been developed, particularly for the spe-
cific separation of various carotenoids found in
fruits and vegetables (Bureau and Bushway 1986).
Both normal and reversed-phase methods have been
used, with the reversed-phase methods predominat-
ing (Schwartz 1998). Reversed-phase chromatogra-
phy on C-18 columns using isocratic elution proce-
dures with mixtures of methanol and acetonitrile
containing ethyl acetate, chloroform, or tetrahydro-
furan have been found to be satisfactory (Schwartz
1998). Detection of carotenoids usually range from
approximately 430 to 480 nm. Since -carotene in
hexane has an absorption maximum at 453 nm,
many methods have detected a wide variety of
carotenoids in this region (Schwartz 1998).
Measurements of anthocyanins have been per-
formed by determining absorbance of diluted sam-
ples acidified to about pH 1.0 at wavelengths be-
tween 510 and 540 nm. Unfortunately, absorbance
measurements of anthocyanin content provide only
for a total quantification, and further information
about the amounts of various individual anthocy-
anins must be obtained by other methods. Reversed-
phase HPLC methods employing C-18 columns