Instrumental analysis of jalapeno and habanero peppers using atomic absorption, UV-vis,

HPLC, and voltammetry: a comparative study

Dennis Wrin
Coastal Carolina University, Department of Chemistry and Physics
Experiments designed and performed in collaboration with Justin Zuczek and Jessee Holmberg

Abstract
This experiment was designed and performed for the goal of comparing the
concentrations of particular analytes in habanero and jalapeno peppers via the employment of
various instruments. Specifically potassium using atomic absorption, capsaicin using HPLC,
ascorbic acid (vitamin C) using voltammetry, and iron using UV-Vis spectroscopy. It was
predicted that other than capsaicin content, both peppers would contain approximately the same
amount of each species. This was however shown not to be completely true. While the habanero
was found to contain more capsaicin than the jalapeno, 1.90 (+/- 0.04) mg/g versus 0.37 (+/0.04) mg/g respectively, the habanero was also found to contain much more potassium as well,
3.94 (+/- 0.02) mg/g compared to that of the jalapeno at 0.57 (+/- 0.02) mg/g. The habanero was
also found to have a bit more vitamin C, 5.9 (+/- 0.5) mg/g, when compared to the jalapeno at 3.7
(+/- 0.3) mg/g. As well as slightly more iron with 0.0077 (+/- 0.0004) mg/g in the habanero
compared to 0.0063 (+/- 0.0004) mg/g in the jalapeno.

Introduction
The goal of this experiment is to compare the concentrations of particular analytes in
habanero and jalapeno peppers via the employment of various instruments. Specifically
potassium using atomic absorption, capsaicin using HPLC, ascorbic acid (vitamin C) using
voltammetry, and iron using UV-Vis spectroscopy. Each experiment was individually designed
and carried out as sub-project, and then overall concentrations of each species is compared
laterally across both peppers.
Capsaicin in food is known for it being the species responsible for causing the spicy or
hot feeling from a pepper when it is ingested. Based on this information it is predicted that the
habanero will contain significantly more capsaicin than the jalapeno pepper. Vitamin C, iron, and
potassium are all part of the daily recommended nutritional values and are present in various
amounts in almost every food. Therefore it is hypothesized that both peppers will contain
statistically similar amounts of vitamin C, iron, and potassium.
Experimental
Potassium via atomic absorption. To create the calibration curve a range of standards was
created from a stock potassium chloride solution (500g/ml K). This stock solution was created
by dissolving 0.0954g of KCl into 100mL of DI water. This initial stock was then again diluted
tenfold by 10mL into 100mL of DI water to produce a 50g/mL potassium working stock
solution. This working solution was then used to produce a range of standard solutions by
diluting 1, 2, 6, 8, and 10mL aliquots to 100mL, the resulting concentrations being 0.5, 1.0, 3.0,
4.0, and 5.0 g/mL.

To prepare the pepper samples for testing in the atomic absorption instrument, 1.9862g of
habanero and 1.9831g of jalapeno peppers were measured out and added to a 250mL flask. Next
25.0 mL of 6M HCl was added to the flask and the contents were boiled for 5 minutes. After
allowing to cool, 10 mL of water was added to the flask then filtered by vacuum. The filtrate was
collected and diluted to 50.0mL. Then 10.0mL each of the diluted filtrates were added to 50.0mL
volumetric flasks, followed by 20.0mL 6M HCl and diluted with water to the mark. After the
initial run on the instrument the habanero pepper was not showing up within the projected range,
therefore a tenfold dilution of the sample was made by placing 10.0mL of the sample solution
into a 100.0mL flask and diluting to the mark. This new testable sample was run again through
the AA instrument.
Capsaicin via HPLC. To create the calibration curve a range of standards was created
from a stock solution (965ppm capsaicin). The stock solution was provided to the group by the
instructor. This initial stock was then diluted multiple times to produce a range of standard
solutions with resulting concentrations of 0.965, 9.65, 48.25, 96.5, and 965ppm. After data
collection the 0.965 results were concluded to be erroneous and they were removed from
analysis.
To prepare the raw pepper samples for testing in the HPLC instrument 10.0028g of
habanero and 10.0142g of jalapeno peppers were each crushed in a mortar and pestle with
30.0mL of acetonitrile for 30 minutes. Each mixture was then filtered via vacuum filtration and
the filtrate was collected and measured to 25.0mL. Next, 1.00mL of each concentrated filtrate
was diluted to 10.0mL and these became the working samples used in the solid-phase extraction
(SPE). The SPE column filter was conditioned using two column volumes of each consecutively;
methanol, acetonitrile, and DI water. Following this the 10.0mL of diluted extract was added to

the SPE column and allowed to run through. The capsaicin was then released from the filter and
collected via 4.0mL methanol followed by 1.0mL of methanol containing 1% acetic acid, this
5.0mL was the tested sample. All solutions (stock and unknown) were then set-up to run through
reverse-phase HPLC.
Iron via UV-Vis spectroscopy. To create the calibration curve a stock 0.001M ferric
chloride solution was prepared by dissolving 0.0065g of FeCl3 into 25.0mL of DI water. Then
0.5, 1.0, 1.5, 2.0, and 2.5mL aliquots of the stock were placed into 10.00mL volumetric flasks
and diluted to the mark with 2M HCl. The resulting solutions provided the needed range for
testing. Just prior to testing the standards, a constant volume of 5.00mL of 1.5M KSCN was
added to each standard, resulting in the necessary iron ion color needed for UV-Vis (as well as a
two-thirds decrease in initial molarity).
To prepare the raw pepper samples first 5.005g of habanero and 4.995g of jalapeno
pepper were each placed into a crucible and burned until ash, then each was washed with 10.0mL
of 2M HCl and filtered via vacuum. Filtrate was measured to 10.00mL. Just prior to testing
5.00mL of the filtrate was mixed with 5.00mL of 1.5M KSCN, resulting in half the concentration
in the testing sample.
Ascorbic acid via voltammetry. A standard solution of 0.05M ascorbic acid was required
to perform the standard addition and was prepared using 0.7999g ascorbic acid and 1.0mL of 3M
nitric acid into a 100.00mL volumetric flask, and diluted to the mark with DI water.
To prepare the peppers for testing via voltammetry first 5.009g of habanero and 4.9999g
of jalapeno was weighed out and chopped finely. Each sample was then placed into a mortar and
pestle and ground for about 20 minutes, while adding small amounts of DI water (not exceeding

100 mL). This mixture was then filtered by vacuum. The collected filtrates were added to
volumetric flasks and each diluted to 100.00mL. The initial cell contained 10.00mL of this
sample solution before any standard additions. Following this 1.00mL additions were made four
times, reading the peak in current after each addition.
Results
Potassium via atomic absorption. The habanero and jalapeno pepper were found to have
an absorbance of 0.5801 and 0.8436 respectively. When subject to the line of best fit for the
constructed calibration curve it is shown that the experimental concentration of the tested
habanero solution is 3.13g/mL and the jalapeno is 4.73g/mL. It is reminded though that the
tested sample of the habanero had a lower concentration than the jalapeno, but the tested sample
was diluted tenfold, therefore represents approximately one tenth of the mass. It can be seen then
that the raw habanero pepper will appear to contain a higher concentration of potassium than the
jalapeno. Through further analysis of the data this was proven to be true with the raw jalapeno
pepper containing 0.57 (+/- 0.02) mg/g of potassium and the raw habanero pepper containing
3.94 (+/- 0.02) mg/g. It is concluded then that the habanero pepper contains more potassium than
a jalapeno.
Capsaicin via HPLC. The habanero and jalapeno pepper were found to have a retention
peak area of 129995 and 27150 respectively. When subject to the line of best fit for the
constructed calibration curve it is shown that the experimental concentration of the tested
habanero solution is 152.35 ppm and the jalapeno is 29.51 ppm. Given this, it can be seen that
the raw habanero pepper will have significantly more capsaicin than the jalapeno. Through
further analysis of the data this was proven to be true with the raw jalapeno pepper containing

0.37 (+/- 0.04) mg/g of capsaicin and the raw habanero pepper containing 1.90 (+/- 0.04) mg/g. It
is concluded then that the habanero pepper contains more capsaicin than a jalapeno.
Iron via UV-Vis spectroscopy. The habanero and jalapeno pepper were found to have an
absorbance of 0.288 and 0.227 respectively. When subject to the line of best fit for the
constructed calibration curve it is shown that the experimental concentration of the tested
habanero solution is 3.45(E -5)M and the jalapeno is 2.80(E -5)M. Taking into consideration the
concentration of the tested sample was half that of the initially prepared. Given this, it can be
seen that the raw habanero pepper will have slightly more iron than the jalapeno. Through further
analysis of the data this was proven to be true with the raw jalapeno pepper containing 0.0063
(+/- 0.0004) mg/g of iron and the raw habanero pepper containing 0.0077 (+/- 0.0004) mg/g. It is
concluded then that the habanero pepper contains more iron than a jalapeno.
Ascorbic acid via voltammetry. After graphing the peak currents versus the concentration
of the cell after each standard addition the habanero and jalapeno pepper were found to have xintercepts of 0.00167 and 0.00104 (absolute values) respectively. And furthermore initial dilute
concentrations of ascorbic acid at those values. Taking into consideration the dilution of the cell
at initial condition, the concentration of ascorbic acid in the peppers can be determined. Given
this, it can be seen that the raw habanero pepper will have slightly more ascorbic acid than the
jalapeno. Through further analysis of the data this was proven to be true with the raw jalapeno
pepper containing 3.7 (+/- 0.3) mg/g of ascorbic acid and the raw habanero pepper 5.9 (+/- 0.5)
mg/g. It is concluded then that the habanero pepper contains more vitamin C than a jalapeno.

Conclusion
While it was originally hypothesized that the habanero would have more capsaicin than a
jalapeno, it was also predicted that for the other analytes (iron, potassium, and ascorbic acid) the
peppers would have approximately the same concentration. However, through the experimental
process it was concluded that the habanero pepper actually contained statistically more of each
species tested. The habanero was shown to contain significantly more potassium and capsaicin
per gram of raw pepper when compared to the jalapeno. It also contained slightly more of both
ascorbic acid (vitamin C) and iron. It was concluded that both peppers can be considered a good
source of vitamin C, however the habanero pepper is a much better source of potassium as well
as a much hotter pepper than the jalapeno.

References
1. Determination of Calcium, Iron, and Copper in Food by Atomic Absorption. Methods Based

on Electromagnetic Radiation. p.258.

2. Huang, J.; Mabury, S. Hot Chili Peppers: extraction, cleanup, and measurement of capsaicin.
Journal of Chemical Education. 2000, 12. 1630.
3. Adams, P. Determining iron content in food by spectroscopy. Filtrates and residues.1995, 7,
649-651.
4. Measuring vitamin C in fruit juice by voltammetry using standard addition. Exploring
Chemical Analysis.

0.9000
f(x) = 0.17x + 0.06
R = 0.98

0.8000
0.7000
0.6000
0.5000

Absorbance (AU)

0.4000
0.3000
0.2000
0.1000
0.0000

Concentration K (g/mL)

Figure 1. This graph shows the constructed calibration curve for the potassium standards
absorbance versus concentration for potassium detection via AA. Also shown are the
experimental absorbance values for the habanero and jalapeno peppers and their placement on
the curve. Noted is that although the tested samples for the jalapeno pepper has a higher

concentration of potassium than that of the habanero, the raw habanero pepper actually has a
higher concentration due to the tenfold dilution performed on the tested sample.

140000
120000
100000
80000

f(x) = 837.22x + 2442.95

R = 1

Response (Area)
60000
40000
20000
0

20

40

60

80

100

Concentration (ppm)

120

140

160

Figure 2. This figure shows the graph of the response (area under the retention peak) versus the
concentration of the standard and the generated calibration curve of capsaicin detection via
HPLC. Also shown are the experimental response values for the habanero and jalapeno peppers
and their placement on the curve. It can be seen that the habanero sample has a much higher
concentration of capsaicin than that of the jalapeno.

1.6
1.4

f(x) = 9429.42x - 0.04

R = 0.99

1.2
1

Absorbance (AU)

0.8
0.6
0.4
0.2
0
0.00E+00

5.00E-05

1.00E-04

1.50E-04

2.00E-04

Concentration (M)

Figure 3. This figure shows the graph of absorbance versus the concentration of the standards
and the generated calibration curve of iron detection via UV-Vis spectroscopy. Also shown are
the experimental response values for the habanero and jalapeno peppers and their placement on
the curve. It can be seen that the habanero sample has a much slightly concentration of iron than
that of the jalapeno.

0
0

f(x) = 0.01x + 0
R = 1

0
0
0

Current (A)

0
0
0
0
0
0

0.01

0.01

0.01

Concentration of Ascorbic Acid spike (M)

0.01

0.01

0
0
f(x) = 0.01x + 0
R = 0.98

0
0
0

Current (A)

0
0
0
0
0
0

0.01

0.01

0.01

Concentration of Ascorbic Acid spike (M)

0.01

0.01

Figure 4. This figure shows both graphs for the standard addition of the jalapeno (top) and
habanero (bottom) using voltammetry. The curve is constructed using the peak of the current
observed for a particular potential versus the concentration of the cell after each standard
addition. Extrapolation of the line of best fit to find the absolute value of the x-intercept gives the
dilute concentration of the cell initially. Applying the dilution factor gives the concentration of
the peppers.