delta
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can be added to the product. Phenolic antioxidants include buty-
lated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
propyl gallate (PG), and tert-butyl hydroquinone (TBHQ). These
four, plus the tocopherols, act as theprimaryantioxidants found in
foods and beverages produced in theUS. The structures of BHA,
BHT, and PG—the three most common phenolic antioxidants—
are shown in Figure 4. Phenolic antioxidants, such as BHT, are
regulated by the FDA, and can be added to many products at
levels up to 200ppm based on the fat content.
Phenolic antioxidants can be analyzed by reversed phase HPLC
using a Pinnacle II
™
C18 column and an acidifiedmobile phase.
As with the previous methods, an acidicmobile phase is used to
Structures of three commonly-used phenolic antioxidants.
Figure 4
BHA
BHT
PG
LC_0198
1
2
3
4
0
2
4
6
8
10
12
14
16
18min.
PeakList:
conc.:
(ppm)
1. propyl gallate 168
2. TBHQ
182
3. 2-BHA+ 3-BHA 197
4. BHT
193
Sample:
Inj.:
10µL
Conc.:
see peak list
Solvent:
methanol
Column:
Pinnacle
II
™
C18
Catalog #:
9214565
Dimensions: 150 x 4.6mm
Particle Size: 5µm
Pore Size:
110Å
Conditions:
Mobile Phase: A= 1% acetic acid
B=methanol
Time A B
(min.) (%) (%)
0 50 50
4 50 50
10 10 90
25 10 90
26 50 50
Flow:
1.0mL/min
Temp.:
30°C
Det.:
UV@280nm
Phenolic antioxidants can be quantitated easily using aPinnacle II
™
C18 column andUV detection at 280nm.
Figure 5
Structures of tocopherols—these compounds readily oxidizewhen exposed to light or oxygen.
(the activity is as follows:
δ> γ >β>α
)
Figure 6
suppress ionization of the analytes. The HPLC separation of
BHA, BHT, PG, andTBHQusingUVdetection at 280nm shows
how effectively these compounds can be separated using the
Pinnacle II
™
C18 column (Figure 5).
“Natural” antioxidants, such as tocopherols and tocotrienols, are
used to inhibit lipidoxidationand topromotegeneral health in the
consumer.Thesecompounds are foundnaturally inproducts such
as fats and oils.When used as additives, however, they are regu-
lated.Antioxidants suchas tocopherolscanbechallenging toana-
lyze, because they readily oxidizewhen exposed to light or oxy-
gen. The structures of four different tocopherols are shown in
Figure 6. The analysis of these tocopherols by normal phase
gamma
beta
alpha
