• 11 •
Analyzing theHeat
Levels of Spicy Foods
Using anUltraC18HPLCColumn
As interest in spicy foods grows, so does the need
to test and classify products and rawmaterials for
their heat levels. In 1912, Wilbur Scoville developed
amethod for quantitating the heat content of foods.
A Scoville Heat Unit (SHU) is defined as the number
of parts sugar water needed to neutralize the heat
of one part sample extract (e.g., if the heat of a
cayenne pepper is 30,000 SHU, that means 30,000
parts of sugar water are needed to dilute one part
of cayenne pepper extract to the last point that hot-
ness can be detected). However, this test is some-
what subjective because it relies on the tasters’
palates and sensitivities. In addition, tasters can
handle only a limited number of samples at one
time before "fatiguing of the palate" occurs.
1
This
makes it difficult to process a large number of sam-
ples in a reasonable amount of time, and can affect
reproducibility of the tests.
There are seven generally recognized capsaicinoids,
three of which are shown in Figure 1. The capsaici-
noid content, and thus the heat level, depends on
the type of pepper, maturity, growing conditions,
and processingmethods. Starting in the 1970s, sev-
eral analytical methods for heat level measurement
were introduced. Of these, the HPLC procedures
have provided the greatest specificity while requir-
ing the least amount of sample preparation.
2
The
American Spice Trade Association (ASTA) and the
Association of Official Analytical Chemists (AOAC)
have both publishedmethods for the determination
of capsaicinoids by HPLC.
AOACMethod 995.03
3
specifies the separation of
the three target capsaicinoids responsible for the
heat in chili peppers. This separation uses reversed
phase HPLC on a C18 column followed by quantita-
tion using either UV or fluorescence detection. The
AOACmethod is performed isocratically with a
mobile phase consisting of 1% acetic acid in
water:acetonitrile (60:40). Standardization is per-
formed using synthetic capsaicin, N-vanillyl-
n
-
nonanamide, and the relative amounts of nordihy-
drocapsaicin, capsaicin, and dihydrocapsaicin are
calculated by applying the specified factors. Using
the appropriate calculations where 1ppm of total
capsaicinoids is approximately equal to 15 SHU, the
heat index then can be calculated.
Using a gradient elution program, an efficient sepa-
ration of the three target capsaicinoids is achieved
with the Restek Ultra C18 column (Figure 2). The
high percentage of organic at the end of the run
helps to elute any strongly adsorbing species pres-
ent in the samples and decreases analysis time. This
HPLC procedure provides an objectivemeasure-
ment of the heat level of a wide range of samples.
By using a Restek Ultra C18 column and a gradient
elution program, the analysis can be performed
quickly and reproducibly with onlyminimal sample
preparation. The selectivity and lot-to-lot repro-
ducibility of the Ultra C18 columnmake it an excel-
lent choice for heat level measurements.
Formore detail onHPLC detection of heat level,
including applications showing other products and
a table comparing hotness rankings by HPLC for
selected sauces, please request Applications Note
lit. cat.#59199.
nordihydrocapsaicin
dihydrocapsaicin
capsaicin
Figure 1
Structures of three capsaicinoids commonly
found in chili peppers
by RebeccaWittrig, Ph.D., Food, Flavor, & Fragrance Innovations Group Leader
2
4
6
8
10min.
Figure 2
The Restek Ultra C18 Column achieves fast, reproducible separation of capsaicinoids.
1. Bensinger, M. "HowHot is that 'Devil' Sauce?" Fiery Foods
Magazine (1997), Sept/Oct.
3. Chiang, G.H., Journal of Food Science (1986) 51 (2), 499-503.
2. AOACOfficial Methods of Analysis (2000), method 995.03.
✔
UltraC18 5µmHPLCColumns
For the complete HPLC column offering, refer toRestek’s
Annual Chromatography Products Catalog
(lit. cat.# 59960).
Length
1.0mm ID 2.1mm ID
3.2mm ID
4.6mm ID
30mm
9174531
9174532
9174533
9174535
50mm
9174551
9174552
9174553
9174555
100mm
9174511
9174512
9174513
9174515
150mm
9174561
9174562
9174563
9174565
200mm
9174521
9174522
9174523
9174525
250mm
9174571
9174572
9174573
9174575
for
more
info
Request Applications Note #59199
Sample:
habanero nugget extract;
Inj.:
10µL;
Conc.:
0.8g sample in 200mL;
Solvent:
ethanol;
Column:
Ultra C18 (cat.# 9174565)
Dimensions:
150x4.6mm;
Particle size:
5µm;
Pore size:
100Å
Mobile phase:
A: 1% acetic acid inwater
B: acetonitrile
Time (minutes):
%B
0
50
2
50
9
80
12
80
13
50
Flow:
1.0mL/min.;
Temp.:
ambient;
Det.:
UV@ 280nm
✔
Fast, reproducible separation
of capsaicinoids
✔
Onlyminimal sample
preparation needed
1. nordihydrocapsaicin
2. capsaicin
3. dihydrocapsaicin
1
2
3
LC_0160
