Capsaicin and Its Measurement
Stephen Koh
Zachary Swenson
New Explorations into Science, Technology and Mathematics
AP Chemistry
Periods 7 and 8
Abstract
Capsaicin is an organic molecule naturally found in many peppers. It is part of the vanilloid
group and is known for its ability to make food taste better, kill cancer cells, and act similarly to steroids
for horses. Currently, people employ the Scoville Scale to rank peppers according to their
pungency. This scale runs from 0 to 16 million Heat Units, where 0 indicates the lack of capsaicin and 16
million indicates pure capsaicin. The Scoville Scale is not very efficient with so many different values
and its linear scale. A more effective way to rank the pungency of peppers would be through use of the
pH scale, which has only 14 values, runs logarithmically, and is more universally recognized. In order to
find a relation between Scoville Heat Units and pH, I measured the pH of 5 different species of peppers
with varying Scoville Heat rankings. I then averaged the pH of the 10 trials for each species. Carrying
out a linear regression analysis and a T-Test analysis, I discovered that although the average pHs of the 5
different species of pepper are significantly different, there is a weak correlation between pH and Scoville
Heat Units. Thus, a pepper’s spiciness cannot be measured with the more “scientific” and universally
understood pH.
Introduction
In 1492, the Italian master navigator Christopher Columbus received massive funding from the
Spanish monarchs, King Ferdinand and Queen Isabella, including the purchase of 3 caravel ships, 3 fully
fledged crews, and months-worth of supplies for each. In the Biblical story of the Genesis, Joseph’s
brother sells him into slavery. The ancient Egyptians successfully preserved their rulers for
centuries. What do all of these events have in common? None of them would have occurred without the
availability or motivation of spices.
Not only did spices serve as an impetus, driving the development of trade networks, but it also
gained culinary usage in many cultures, independent of one another. Both Indian and Latin American
cuisines all rely heavily on spice, but they developed this proclivity long before contact between the two
nations could be made. Thus, there must be something about the flavor of spiciness that many humans
find appealing.
Capsaicin is the chemical primarily responsible for giving peppers their spicy taste. Perhaps
peppers evolved to include capsaicin in order to ward off mammals who would otherwise consume the
plant whole and prevent the pepper’s reproduction. Meanwhile, birds, who transport the seeds of pepper
plants, cannot taste capsaicin. It makes evolutionary sense for the animals who promote pepper
reproduction to not be affected by capsaicin’s pungency. The fact that humans love and actively seek
capsaicin containing products despite the flavor being developed to stave us off makes peppers and
capsaicin in particular interesting to study.
I am interested in three questions: How does the Scoville Scale (for measuring the spiciness of a
pepper) correspond with that same pepper’s pH? What effects do spicy foods have on people? Why does
spicy food have these effects?
Peppers with various ratings on the Scoville Scale will be tested for pH. The Scoville Scale
ranges from zero to sixteen million Scoville heat units. If a clear relationship can be established between
pH and scoville units, people will have a more “scientific” and universally understood way to compare
Literature Review
Peppers developed pungency to combat fungus from bugs that try to eat them. Peppers are able
to do this because their pungency is highly antimicrobial. This was likely a reason chilies were so
popular before the development of refrigeration. By adding spice to foods, you could eat them past their
“expiration” and discourage insects from eating them. Pungency also serves to prevent most bugs and
mammals from attempting to eat the pepper and destroying the seeds. Humans, however, serve as the
sole exception to this phenomenon as they willingly consume spicy peppers. In fact, 25% of humans eat
peppers every day. Birds, on the other hand, cannot taste the heat and are known to disperse seeds. Birds’
receptors are not lined up the right way likely because they developed before peppers did. Some relatives
of the chili (solanaceous), such as the nightshade, are poisonous. The real heat of a chili can be found
around the seeds, in order to protect the chili’s genetic material. If one were to bite the tip of a chili, it
would not be nearly as hot. Consequently, the area surrounding the seeds is least likely to be infected
with E. coli or other microbes. (NPR, 2008).
In order to stop the burning sensation after eating chili peppers, one should drink milk or
consume some other dairy product. Casein, a lipophilic phosphoprotein, found in milk is able to remove
capsaicin from its receptor binding site. Rubbing alcohol is also effective in stopping the burning
sensation. If peppers get in one’s eyes, the burning can be alleviated by rinsing with water or
saline. Especially pungent peppers can blister skin and thus should be handled with gloves. Some
ornamental types of chilies, such as the False Jerusalem Cherry, are poisonous. A ripe chili, one that has
its ideal level of capsaicin, makes a small crackling noise when pressed. Black or dark spots on a chili are
caused by overexposure of the chilies to direct sunlight. People who regularly consume chilies develop
tolerance to pungency over time. “Capsaicin D” and “Heet” are two capsaicin-based pain relief
products. Fish, like birds cannot taste capsaicin but may use it to give their scales a healthy
look. (NMSU, 2007).
Besides adding flavor and microbe-killing potency to food, chili peppers also help humans by
lowering blood pressure and increasing salivation. Dr. Paul Rozin of the University of Pennsylvania
hypothesizes that humans enjoy spicy foods for the pain. In fact, when Rozin gave volunteers chili
peppers and asked which they liked best, most chose the one that was so spicy they could barely tolerate
it. Rozin explains this as, “Humans and only humans get to enjoy events that are innately negative, that
produce emotions or feelings that we are programmed to avoid when we come to realize that they are
are profitable. A company called Blair’s, for example, advertises a product called the 16 Million Reserve,
which is pure capsaicin (named for its 16 million scoville heat unit rating). (Gorman, 2010).
Figure 1 – Skeletal Model of Capsaicin (ChemSpider)
Apparently capsaicin also serves to destroy prostate cancer cells. According to researchers from
the Samuel Oschin Comprehensive Cancer Institute at Cedars-Sinai Medical Center, human prostate
cancer cells experience apoptosis (cell death) when exposed to capsaicin. In mice, prostate cancer cells
exposed to capsaicin grew to only about 20% the size of those not exposed to capsaicin. This effect is
achieved by lowering the activity of the NF-kappa Beta mechanism which is related to
apoptosis. Oftentimes, cancer cells will target the genes responsible for cell death in order to gain
immortality. Capsaicin also altered androgen receptors to slow the growth of prostate cancer cells,
specifically PSA, a protein indicating prostate cancer in men. If found to be effective on a large scale,
capsaicin could have major implications for prostate cancer treatment, a disease that impairs more men in
the US than any other. (Vanderboom, 2006).
A different study at the Nottingham University found that capsaicin kills cancer through a
different method, attacking mitochondria in cancer cells. Vanilloids, the molecular type in which
capsaicin belongs, are known to cause apoptosis by binding to mitochondria in cancer cells without
harming nearby cells not affected by cancer. So far, capsaicin has been used to treat samples of human
lung cancer and pancreatic cancer. Because capsaicin is already prevalent in the diets of many without
having a malignant effect, it would be safe as a potential drug. Capsaicin is currently used in topical pain
relief drugs, raising the possibility of a topical treatment for skin cancers. Regardless of whether
capsaicin ends up being a cure for cancer, the concept of attacking cancer cells’ mitochondria will likely
prove to be important. (BBC, 2007).
While capsaicin might kill cancer in humans, it has other applications for different animals. For
example, capsaicin has hyper sensitizing effects on horses, providing pain relief. Because of this, horses
testing positive for capsaicin have been banned from equestrian sports. A common way to administer
the 2008 Summer Olympics, Norway, lost its bronze medal to Switzerland because its horses tested
positive for capsaicin. In the 2004 Summer Olympics, both Germany and Ireland lost gold medals for the
same reason. (BCC, 2008).
Figure 2 – Calotte Model of Capsaicin (Senese, 2010)
Flavor is influenced by how molecules fit into receptor sites. If the fit is too tight or the shape is
off, then the molecule may be perceived as more powerful. According to the stereochemical theory of
odor, a particular odor is perceived when a molecule goes into an olfactory receptor. Pungent molecules
like capsaicin, experience powerful attraction to the receptor with an abundance of electrons because the
molecules themselves are deprived of electrons. Depending on the shape of a molecule, an odor may be
perceived differently. A long hydrocarbon tail, for example, increases solubility in fats and makes the
smell stronger. Vanilloids, the molecular group to which capsaicin belongs, have discrete differences
between molecules which result in strong differences between flavors. Capsaicin, has a very low
volatility and is almost completely odorless. Capsaicin's pungent flavor is caused by its hydrocarbon tail,
giving it the ability to bind with a corresponding lipoprotein receptor. The tail also gives capsaicin the
ability burn more strongly by passing through lipid-heavy cell membranes. Slight differences in tail
structure changes the place where the burn is felt (tongue, lips, mouth, throat, etc.). Capsaicin is
perceived as spicy because it opens a pathway for calcium ions to move into cells, which releases a pain
signal. Capsaicin has a low water solubility because of its long hydrocarbon tail. Besides milk and
alcohol, vegetable oils are effective in combating the burning sensation (like dissolves like). In high
concentrations, capsaicin can be toxic, making it popular among the Mayans for producing smoke screens
and today for producing pepper sprays. On the flip side, capsaicin consumption is known to create
endorphins, resulting in generally good feelings and a (weak) addiction to spicy foods. (Senese, 2010).
Capsaicin, one of the two major capsaicinoids (the other being dihydrocapsaicin), is used to rank
the pungency of a pepper on the Scoville Scale. The Scoville Scale runs from 0 to 16 million Scoville
heat units (see appendix for Scoville rankings of various peppers). There are three main methods used to
determine the pungency of a capsaicin containing compounds. One is to have a taster rate how spicy a
Test, dilutes samples in a lab setting until tasters cannot detect any pungency. Although this is more
accurate than the aforementioned taste test, it is also very subjective and tasters cannot be used many
times (because of reduced sensitivity to capsaicin). The best and most objective method is
high-performance liquid chromatography (HPLC), which requires dried chilies to be ground up and subjected
to HPLC, so the exact amount of capsaicinoids can be measured. (Bosland, 2011).
Relating to measurement of pungency, there are various methods to measure pH. In addition to
indicators, an electronic (and more accurate) pH meter can be used. Before beginning measurements, a
pH meter should be calibrated in order to obtain maximum accuracy. This can be done by using two
standard buffer solutions, one at the lower bound of expected pH and one at the upper bound of expected
pH. This usually calls for one buffer at pH 4 and another at pH 10. The pH meter has three controls that
can be adjusted to the pH of the first buffer, the pH of the second buffer, and the temperature,
respectively. Another calibration method requires three buffers, one at pH 7, one closer to the expected
pH (either 4 or 10 generally), and the unchosen pH from the second calibration. Some pH meters have
built in temperature. The purpose of calibration is to relate voltage output with the pH scale (~.06
volts/pH unit). Between measurements, the probe could be washed with distilled or deionized water and
Methods and Materials
My experiment requires me to measure the pH of 5 different chili peppers with various Scoville
Heat Unit rankings.
Before beginning to actually measure the pH’s though, I must first make several
preparations. Many of these preparations involve precautions necessary to stop capsaicin from getting on
my hands because capsaicin, especially in high concentrations can result in severe burns on skin and
mucous membranes. In order to prevent this, I will use magnets to attach recording paper to the board,
put on a lab apron, lab goggles, and 2 layers of plastic gloves. Afterwards, I will need to calibrate the pH
meter I’ll be using by employing three buffer solutions (4,7,10).
After completing the necessary precautions, I can begin measuring pH, taking 10 trials per pepper
species and making sure to stick the pH meter near the peppers’ seeds (where capsaicin is present in its
expected Scoville Heat Units ranking). Between two trials, I must dip the probe of the pH meter in a
beaker of distilled water in order to semi-recalibrate it. After each measurement, I must record the pH
value given by the meter on the paper attached to the board at the front of the room.
At the end of the experiment, I need to take appropriate safety precautions like discarding the
peppers, pencil, gloves I used. I also need to rinse the scalpel, outside of beakers, outside of pH meter,
outside of distilled water bottle, desk, goggles, apron and anything else coming in contact with peppers
with rubbing alcohol. After returning all laboratory equipment to its proper place, I should wash my
hands with soap, warm water AND rubbing alcohol, remove the paper from the board, and exit the
Results
Type of
Pepper
Sweet
Peppers
Jalapeno
Pepper
Cumari
Pepper
Bird's
Eye Chili
Habernero
Pepper
Scoville Heat
Unit Range
0
3,500–
8,000
30,000–
50,000
50,000–
100,000
100,000–
350,000
Midpoint of
Scoville Heat
Unit Range
0
5,750
40,000
75,000
225,000
Measured pH
Trial 1
4.9
4.3
4.8
4.5
5.2
Measured pH
Trial 2
4.8
4.4
5.3
4.6
5.0
Measured pH
Trial 3
4.6
4.4
5.1
4.9
5.0
Measured pH
Trial 4
4.9
4.3
4.3
4.6
4.7
Measured pH
Trial 5
4.6
4.4
4.5
5.1
5.2
Measured pH
Trial 6
4.8
5.4
4.7
5.0
4.5
Measured pH
Trial 7
4.3
5.2
4.9
4.8
5.0
Measured pH
Trial 8
4.6
5.5
4.5
5.3
4.7
Measured pH
Trial 9
4.8
5.4
4.3
5.9
5.0
Measured pH
Trial 10
4.7
5.2
4.7
5.4
5.0
Table 2 - Calculator input for scatter plot below (notice use of Scoville range midpoint for “L1” and average pepper species pH for “L2”)
Discussion
There are a couple of ways to analyze the significance and implications of the data. The first
involves finding the equation of the regression line, its correlation coefficient and coefficient of
determination (r and r2 , respectively) and then plotting the regression line with the points. Below are
calculator displays for the first method.
Figure 3 - Calculator output for equation of regression line (line of best fit)
With a correlation coefficient this small (.557 out of a possible 1.000) and a coefficient of
determination this small (.310 out of a possible 1.000), there is little correlation between the two
variables. Taking a closer look yields the following:
Figure 4 - Calculator scatter plot with regression line (the horizontal line running through center of graph)
Notice how the regression line isn’t running through any of the five points, let alone near
them. This provides visual evidence that Scoville Heat Units and pH have little, if any, correlation
The second method for analyzing the significance and implications of the data involves using
inferential statistics, namely a Paired T-Test for the Difference Between Two Means. Here, the null
hypothesis is that the difference between the observed and expected means for all 5 pepper species’ pH is
equal to zero. The alternate hypothesis is that the difference between the observed and expected means
for all 5 pepper species’ pH is NOT equal to zero. Thus, we are dealing with a two-tailed test. The mean of the 5 pepper species’ mean pH is 4.84 and the standard deviation is .136. Plugging this into the
Figure 5 - Imputing stats necessary to run a Paired T-Test for the Difference Between Two Means. Notice that the U0, or the difference between the means for the null hypothesis is equal
to zero. Also notice that the alternate hypothesis, U, is set as not equal to U0, indicative of a
two-tailed test.
Pressing “Calculate” yields the following:
Figure 6 - Calculator output for results of Paired T-Test for the Difference Between Two Means. Here, the p-value is what we need to pay the most attention to.
According to the guidelines for a T-Test, because the p-value is so small (here p< .01), there is
evidence to reject the null hypothesis, H0, and conclude that the difference between means is statistically
significant. Basically, this tells us that the 5 different species of peppers have significantly different
average pHs taking their mean, standard deviation, and T-distribution curve into consideration.
Lastly, we must take my earlier assumptions into consideration. Because it’s unlikely that the
small sample of peppers I tested accurately represent the pH of their entire species and because the
Scoville Heat Units of the peppers were probably not at the midpoint of their respective ranges, a
correlation between Scoville Heat Units and pH is less likely.
Considering the results of the two methods and the above assumptions, the following conclusion
can be drawn: although the average pHs of the 5 different species of pepper are significantly different, there is a weak correlation between pH and Scoville Heat Units. Thus, a pepper’s
spiciness cannot be measured with the more “scientific” and universally understood pH.
A possible explanation for the lack of correlation between the two quantities is that capsaicin, a
nonpolar molecule, isn't soluble in water, a polar solvent, so no H+ ions are released. The detection of H+
ions by a pH meter follows the equation: H20 <--> H +
species of peppers may have been detected as being more "acidic" due to having a higher water content,
References
BBC. (2007, January 9). How Spicy Foods Can Kill Cancer. Retrieved May 16, 2013 from http://news.bbc.co.uk/2/hi/health/6244715.stm
BBC. (2008, August 21). Olympic Horses Fail Drug Tests. Retrieved May 16, 2013 from http://news.bbc.co.uk/sport2/hi/olympics/equestrian/7574220.stm
Bosland, Paul W. New Mexico State University. (2011, February 15). Measuring Chile Pepper Heat. Retrieved May 16, 2013 from http://aces.nmsu.edu/pubs/_h/H237/
ChemSpider. Capsaicin. Retrieved May 16, 2013 from Structure.1265957.html
Gorman, James. The New York Times. (2010, September 20). A Perk of Our Evolution: Pleasure in Pain of Chilies. Retrieved May 16, 2013 from
http://www.nytimes.com/2010/09/21/science/21peppers.html?_r=0
New Mexico State University. (2007, May 4). Chile Information Frequently Asked Questions. Retrieved May 16, 2013 from
http://web.archive.org/web/20070504035555/http://spectre.nmsu.edu/dept/academic.html ?i=127 4&s=sub
NPR. (2008, August 15). What Made Chili Peppers So Spicy?. Retrieved May 16, 2013 from http://www.npr.org/templates/transcript/transcript.php?storyId=93636630
ScienceLab.com. Capsaicin, Natural MSDS. Retrieved May 16, 2013 from http://www.sciencelab.com/msds.php?msdsId=9923296
Senese, Fred. General Chemistry Online!. (2010, February 15). Fire and Spice. Retrieved May 16, 2013 from http://antoine.frostburg.edu/chem/senese/101/features/capsaicin.shtml
Vanderboom, Russell. EurekAlert!. (2006, March 15). Pepper Component Hot Enough to Trigger Suicide in Prostate Cancer Cells. Retrieved May 16, 2013 from
http://www.eurekalert.org/pub_releases/2006-03/aafc-pch031306.php
Wikipedia. (2013, May 19). Capsaicin. Retrieved May 16, 2013 from http://en.wikipedia.org/wiki/Capsaicin
Wikipedia. (2013, April 22). pH Meter. Retrieved May 16, 2013 from http://en.wikipedia.org/wiki/PH_meter
Appendix
Procedure
Materials
· Buffer solution with pH 4
· Buffer solution with pH 7
· Buffer solution with pH 10
· pH meter
· distilled water
· Scalpel
· 4 large beakers
· Lab apron
· (Closed!) lab goggles
· Plastic gloves x2
· Rubbing alcohol
· Paper towel
· Various peppers of different Scoville Heat Unit measurements
· Disposable pencil
· Paper to write on
· Dry erase board magnets
· Camera (have observer take photos throughout)
Instructions
1) Use magnets to attach recording paper to board
2) Put on lab apron, lab goggles, and first layer of plastic gloves (capsaicin from pepper can burn if it
makes contact with skin or mucous membranes)
3) Pour buffer solutions and distilled water each in a separate large beaker (should be enough fluids in
each to be able to submerge probe of pH meter)
4) Calibrate pH meter with buffer solutions (pH 7 first, pH 4 next, pH 10 last)
5) Put on second layer of plastic gloves before handling first pepper
6) Cut open a sweet pepper with scalpel
8) Record measured pH on recording paper making sure not to let paper touch anything coming in
contact with peppers
9) Clean pH meter in beaker of distilled water and wipe off excess water
10) Repeat steps 6-9 with the sweet pepper 10 times (use others of the same species if possible)
11) Repeat steps 6-10 with all other species of peppers
12) Throw peppers in trash bin
13) Remove second layer of gloves and discard along with pencil
14) Rinse scalpel, outside of beakers, outside of pH meter, outside of distilled water bottle, desk, goggles,
apron and anything else coming in contact with peppers with rubbing alcohol
15) Remove first layer of gloves and discard
16) Return all laboratory equipment
17) Rinse hands with some rubbing alcohol before washing with soap and warm water
18) Remove paper from board and exit lab
