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The Effect of Time, Roasting Temperature, and Grind Size on The Effect of Time, Roasting Temperature, and Grind Size on
Caffeine and Chlorogenic Acid Concentrations in Cold Brew Caffeine and Chlorogenic Acid Concentrations in Cold Brew
Coffee Coffee
Niny Z. Rao
Thomas Jefferson University
Megan Fuller
Thomas Jefferson University
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Rao, Niny Z. and Fuller, Megan, "The Effect of Time, Roasting Temperature, and Grind Size on Caffeine and
Chlorogenic Acid Concentrations in Cold Brew Coffee" (2017).
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1
Scientific REPORts | (2017) 7:17979 | DOI:10.1038/s41598-017-18247-4
www.nature.com/scientificreports
The Eect of Time, Roasting
Temperature, and Grind Size on
Caeine and Chlorogenic Acid
Concentrations in Cold Brew Coee
Megan Fuller & Niny Z. Rao
The extraction kinetics and equilibrium concentrations of caeine and 3-chlorogenic acid (3-CGA) in cold
brew coee were investigated by brewing four coee samples (dark roast/medium grind, dark roast/
coarse grind, medium roast/medium grind, medium roast/coarse grind) using cold and hot methods.
3-CGA and caeine were found at higher concentrations in cold brew coee made with medium roast
coees, rather than dark roast. The grind size did not impact 3-CGA and caeine concentrations of
cold brew samples signicantly, indicating that the rate determining step in extraction for these
compounds did not depend on surface area. Caeine concentrations in cold brew coarse grind samples
were substantially higher than their hot brew counterparts. 3-CGA concentrations and pH were
comparable between cold and hot brews. This work suggests that the dierence in acidity of cold brew
coee is likely not due to 3-CGA or caeine concentrations considering that most acids in coee are
highly soluble and extract quickly. It was determined that caeine and 3-CGA concentrations reached
equilibrium according to rst order kinetics between 6 and 7 hours in all cold brew samples instead of 10
to 24 hours outlined in typical cold brew methods.
In 2015, domestic coee consumption in the United States reached an estimated 1.4 billion kg/year, making
it the second largest coee market in the world aer the European Union
1
. e majority of coee consumed
in the United States is prepared through various hot brewing methods, whereby the hot water solubilizes and
extracts numerous organic compounds from the roasted coee grounds. However, cold brew coee preparation
techniques have grown in popularity, both in at-home and consumer (or ready-to-drink, RTD) markets. Market
researcher, StudyLogic, estimates that coee shop sales of hot coee fell 3% in 2016, while cold brewed coee
sales were up nearly 80% over the previous year’s record
2
. Roast Magazine reports a 460% increase in retail sales
of refrigerated cold brew coee from 2015 to 2017, generating $38 million in 2017 alone
3
. In an eort to capitalize
on this rapidly growing market, Dunkin’ Donuts, Starbucks, and other commercial coee vendors have invested
in RTD cold brew coee beverages and are suggesting that colder, slower brewing processes alter avor, aroma,
and bioactive compounds
4
. Starbucks markets that cold brew coee is sweeter, smoother, with a more full-bodied
avor than conventionally brewed coee
5
. Dunkin’ Donuts claims that, “cold brew is less acidic and naturally
sweeter than regular coee, so it can easily be consumed black”
6
.
Cold brew coee, not to be confused with iced coee (which is hot brewed coee served over ice), is prepared
at room temperature (20 to 25 °C or colder) over a longer time period than traditional hot brewing methods,
typically steeping times range from 8 to 24 hours
7–10
. Brewing coee is an extraction process dependent on a
multitude of variables such as water volume, water temperature, diameter of the coee grind particles, the poros-
ity of the coee grind matrix, the pore network between coee grind particles, and brewing time. Temperature
oen signicantly inuences compounds aqueous solubility, so dierences in brewing temperatures may result in
signicantly dierent compositions in hot brew and cold brew coees. Additionally, the longer brewing times of
cold brew coee may aect the nal composition of cold brew coee if the diusion of the compounds across the
grind matrix is a kinetically limiting phenomenon.
An extensive body of literature exists detailing the chemistry of hot brewed coee, including quantifying the
caeine concentration as a function of hot water brewing method
11–14
. Bioactive chemicals such as chlorogenic
Department of Chemistry and Biochemistry, Thomas Jeerson University, East Falls Campus, Philadelphia, PA,
19144, USA. Correspondence and requests for materials should be addressed to M.F. (email: [email protected])
Received: 26 September 2017
Accepted: 7 December 2017
Published: xx xx xxxx
OPEN
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Scientific REPORts | (2017) 7:17979 | DOI:10.1038/s41598-017-18247-4
acids, caeine, and other dietary phenolic compounds that include caeoylquinic acids, dicaeoylquinic acids,
and feruloylquinic acids
15
are abundant in coee. ese chlorogenic acid compounds convey bitterness to coee
11
and are known to be active antioxidants that may cause health benets in coee drinkers
16–18
. e presence of
these bioactive compounds in the complex chemical composition of coee extracts have prompted numerous epi-
demiological studies to ascertain the degree and manner in which coee confers health risks and/or benets
19
to
the drinker. Researchers have observed both U-shaped
19–21
and J-shaped
22
associations between coee consump-
tion and risks of cardiovascular diseases. Inverse relationships have been found with coee consumption and
total mortality
23,24
, depression
25
, diabetes mellitus
26,27
and certain types of cancers
28–30
. Work by Bakuradze, et al.
showed compounds present in coee roast products, notably 5-caeoylquinic acid and caeic acid demonstrated
direct antioxidant activity in HT-29 (human colon) cells
31
. A recent review by Naveed et al. further highlighted
the therapeutic roles of chlorogenic acids in human health and called for further research in the area
32
. ese
studies oen focus on analysis of green coee beans, hot brewed coee consumption, or make no distinction
to the brewing method used. Research in hot brew coees show that both roasting temperature
33,34
and grind
size
35,36
aect the extraction kinetics and maximum extractable concentration of soluble compounds from coee
grinds, specically chlorogenic acids
37–40
. Increases in roasting temperatures correlate to a decrease in extractable
chlorogenic acid concentrations and to an increase in caeine concentrations
33
. However, because of the potential
chemical dierences between hot and cold brew coee, it is unknown if the new popular drink will convey similar
benets to its hot brew counterpart.
Despite the increasing popularity of cold brew coee, there is currently very little research published on the
chemistry or associated health risks and/or benets of cold brew coee. An exhaustive literature search revealed
very limited publications analyzing cold brew coee. In 2014, Kim and Kim reported that the avor of cold brew
coee may be more appealing to the Korean coee consumers aer 18 hours brewing time
41
. In 2017, Lane et
al. reported that caeine concentrations of commercially brewed cold brew coee was ~207 mg per 12 . oz
42
.
A third study by Shin in 2017 reported that the polysaccharides isolated from cold brew coee “may potentially
enhance macrophage functions and the intestinal immune system”
43
. To date, these publications represent the
majority of published research available on cold brew coee. Commercial vendors’ claims of lower acidity and
other taste and chemical attributes have yet to be veried by unbiased research.
Given cold brew coee’s signicant growth in the coee market and the potential importance of coee’s bio-
active compounds on human health, this research investigated the role of cold brewing methods on the kinetics
and equilibrium conditions of two compounds of interest: caeine and 3-chlorogenic acid (3-CGA). e pH of all
coees produced in this research was also measured to determine if cold brew coee does results in a less acidic
coee beverage. is work studied the extraction kinetics of caeine and 3-chlorogenic for both cold and hot
brew methods using single origin Arabica beans grown in the Kona Region of Hawai’i in order to determine the
eect water temperature and brewing time on the extraction kinetics and maximum equilibrium concentration
of these two bioactive compounds. Brewing methods employed in this work mimicked standard home-brewing
conditions to inform what, if any, dierences consumers can expect between hot and cold brew coees.
Results
Kinetics of cold brew coee extraction. Four coee samples were used in this study. See Table1 for
grind size distribution and roasting temperature characteristics for each of the samples.
e grain size distributions of the four samples show that the “medium” grind coees had wider particle
distributions, both containing about 5% of particles, by mass, that are larger than 3350 µm. e “coarse” grind
coees showed no 3350 µm portion, and have a narrower particle distribution with more than 70% of particles, by
mass, being retained on the 841 µm sieve. Coee beans are naturally porous. e pore space within each grain of
coee is considered the intragranular pores. e space between grains of coee is referred to as the intergranular
pores. Earlier studies found that particle distribution was vitally important to coee extraction, aecting both the
diusion of compounds through intragranular pores within grinds, as well as the uid ow between the grinds
(through the intergranular pore network)
14,44,45
. e importance of both intragranular and intergranular pore
space will be discussed further with respect to diusion limiting processes of compound extraction kinetics.
Sample Name (Roast - Grind) Grind Size (% by mass) Roasting Temperature
Medium - Medium
3350 µm - 5.7%
841 µm - 26.2%
400 µm - 53.3%
149 µm - 14.8%
215 –217 °C
Medium - Coarse
3350 µm - 0%
841 µm - 70.6%
400 µm - 23.1%
149 µm - 6.3%
215 –217 °C
Dark - Medium
3350 µm - 5.2%
841 µm -38.1%
400 µm - 45.4%
149 µm - 11.3%
223 –225 °C
Dark - Coarse
3350 µm - 0%
841 µm - 77.8%
400 µm - 17.5%
149 µm - 4.3%
223 –225 °C
Table 1. Summary of grind size distribution by percent mass (100.0 g of grinds used in each analysis) and
roasting temperature, as reported by the coee vendor.
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Scientific REPORts | (2017) 7:17979 | DOI:10.1038/s41598-017-18247-4
3-CGA. e compound, 3-CGA is freely soluble in water at room temperature
46
. Initial 3-CGA concentration
increased rapidly over the rst 180 minutes and slowed until reaching equilibrium at approximately 400 minutes
for all coee roasts and grinds (see Fig.1). Moroney et al.
45
attributed the initial fast extraction of soluble coee
compounds to the extraction of compounds from the surface and near-surface volume of the solid coee grind
matrix. e slower, longer time-scale extraction of additional CGA concentration, post 180 minutes, is likely due
to the mass transfer of the compound through intra-grain pores into intergrain pores, and ultimately into the bulk
liquid phase. e data collected in this current work follows the Spiro and Selwood
36
model well and suggests that
cold brew processes of 3-CGA extraction are governed by rst-order kinetics. A sample kinetic plot of ln (C
∞
/
[C
∞
− C]) for 3-CGA versus time is shown in Fig.2 where C
∞
is the equilibrium concentration of 3-CGA and C is
the concentration of 3-CGA at time t. Several sources
7–10
providing brewing instructions for cold brew coee rec-
ommend prolonged brewing times upwards of 12 to 24 hours. Any water/grind interaction longer than 400 min-
utes (6.7 hours) did not result in additional signicant extraction of 3-CGA. e mean concentrations of 3-CGA
at 400 and 1400 minutes were within one standard deviation of each other (see Table1). ese data suggest
3-CGA concentrations are inuenced by roasting temperature, but not grind size. Blumberg et al.
11
determined
that increased roasting temperatures resulted in degradation of chlorogenic acid precursors and lower extractable
total chlorogenic acid concentrations. e same study also observed that chlorogenic acids extracted quickly from
coee grinds, while 4-vinylcatechol oligomers showed strong retention to the coee grinds
11
. e longer steeping
times associated with cold brew coee may result in increased extraction of these catechol oligomers, which are
characterized by harsh bitter-tasting properties. Over-brewing cold brew coee may result in unpalatable extracts
due to these and other relatively slow-extracting compounds.
Caeine. Caeine, unlike 3-CGA, has a limited solubility at room temperature of 16 mg/mL
46
. However, all
four coees analyzed showed comparable extraction kinetics to those of 3-CGA. In all coees sampled, fast ini-
tial extraction was seen over the rst 180 minutes, with a slower rate of extraction aer 180 minutes. Similar to
Figure 1. Concentration of 3-CGA (top) and caeine (bottom) over time for (■) medium roast - medium
grind; (
●) medium roast - coarse grind; (
) dark roast – medium grind, (▲) dark roast - coarse grind. e
vertical line at 400 minutes represents the establishment of steady-state concentration for both 3-CGA and
caeine extractions.
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Scientific REPORts | (2017) 7:17979 | DOI:10.1038/s41598-017-18247-4
3-CGA, caeine also reaches nearly steady-state concentrations aer 400 minutes (see Fig.1). As with 3-CGA,
all samples followed a rst-order kinetic model. Spiro and Selwood
36
oered a thorough mathematical model for
the kinetics of caeine extraction at room temperature, and found that the diusion of caeine through the intra-
granular pore space to be the rate limiting step in the extraction process. is analysis concluded that extraction
times greater than 400 minutes do little to increase the caeine concentration of the resulting coee. Moreover,
caeine concentrations do not demonstrate the same sensitivity to roasting temperatures as 3-CGA, and all coee
roasts and grinds were found to have comparable caeine concentrations at equilibrium, with the exception of the
dark roast - coarse grind coee. e relatively high standard deviations are suspected to be caused by the hetero-
geneous grind size distributions from the commercially sourced beans. As the packaging was handled, settling of
ner particles may have caused inter-sample variability.
pH. Work by Andueza et al.
47
and Gloess et al.
48
both report there is no correlation between pH and perceived
acidity in the avor of coees. However, commercial coee vendors continue to relate acidity to coee taste when
marketing coee to consumers. e pH of coee studied in this work ranged from 5.40 to 5.63. Moon et al.
15
observed a correlation between total CGA concentrations and pH of coee extracts. However, data collected in
this work did not provide enough evidence to support the claim by Moon et al.
15
that coee samples containing
high concentration of 3-CGA tend to have high acidity or low pH.
Comparison of hot brew and cold brew coee. ere is a common marketing message that cold brew
coee is fundamentally dierent than hot brew coee. is may be attributed to acidity and/or caeine concen-
tration
49,50
. is work compared the same water-to-coee ratio using cold brew and hot brew extraction processes
to identify any dierences between the two methods with respect to 3-CGA and caeine concentrations. In the
coee extraction process, Moroney et al.
35
described two dierent extraction mechanisms that function on dier-
ent timescales. e fast extraction from the surface and near-surface matrix happens much more rapidly than the
diusion of compounds through the intragranular pore network to the grain surface. Because the time periods
for hot brew and cold brew are drastically dierent, 6 minutes vs. 1440 minutes respectively, the intragranular
diusion may limit the extractable concentration of soluble coee compounds in the hot brew, as compared to
the cold brew.
3-CGA. In Fig.3, the cold brew extraction of caeine and 3-CGA are shown for each of the four coee samples,
with the hot brew concentrations indicated by horizontal lines. Table2 shows the equilibrium concentrations of
3-CGA for the hot and cold brew coees along with the pH. In both hot and cold brew extractions, all samples
show comparable average 3-CGA concentrations and pH, regardless of water temperature. e CGA molecule is
not seen to be limited by the intragranular pore diusion processes, as observed with caeine extraction. CGA
is freely soluble in water, and this facilitates its extraction at both low and high temperatures. While grain size
did not impact the magnitude of 3-CGA concentrations, roasting temperature of the beans did show a noticeable
eect in both cold and hot experiments. In both hot and cold brew extractions, CGA was found in higher con-
centrations in medium roasts than in darks roasts, supporting the work of Trugo and Macrae
37
that suggests that
higher roasting temperatures decomposes CGA and results in lower extraction concentrations.
Caeine. Coarse grain samples, both medium and dark roast, showed a considerable deviation in caeine
concentrations between hot and cold brew extractions (Table3). In both samples, the cold brew coee was found
to have the higher concentration of caeine. Medium grain samples also showed higher concentrations of caf-
feine in cold brew extraction, however, the dierence was not statistically signicant. is result suggests that
the medium grind blends, in both hot and cold extraction, experienced nearly complete extraction during their
respective steeping times. e hot brew extraction saturated the intra- and intergranular pores and facilitated fast
Figure 2. First-order plot for the extraction of 3-CGA from medium grind - medium roast coee particles at
23.5 °C. R
2
= 0.983.
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Scientific REPORts | (2017) 7:17979 | DOI:10.1038/s41598-017-18247-4
diusion (6-minute steeping times) of caeine across the solid matrix to generate a bulk liquid phase with nearly
the same concentration of caeine as the cold brew coee generated in 400 minutes. Coarse grain samples, with
their higher relative proportion of particles in the 841 µm range, did not reach similar steady-state concentrations
in both hot and cold brews. e faster, hot water extraction was diusion limited, and likely did not allow the
full extraction of caeine across the larger radius particles. e longer brewing times for the cold brew samples
resulted in greater caeine extraction, allowing time for completion of the rate-limiting mass transfer step in the
extraction process.
Role of Grind Size and Roasting Temp in Cold Brew Coee. Further analysis of the data indicates that
the observed CGA and caeine concentration dierences between medium roast and dark roast are, in general,
substantial. Both CGA and caeine showed higher concentration in medium roast samples. Our data is in sup-
port of the works of Trugo, et al.
37
and Hečimović, et al.
51
, both suggest that higher roasting temperatures decrease
the concentration of CGA and caeine. e only exception is the observed dierence in concentration of caeine
Figure 3. Caeine (▲) and 3-CGA (●) concentration as a function of time for each of the four coee samples.
Horizontal lines represent each coee type’s hot water concentration for caeine and 3-CGA. Error bars
represent the range for each measurement.
Coee Sample
(Roast - Grind)
400 min 1440 min
3-CGA
Concentration (mg/L)
Caeine
Concentration (mg/L) pH
3-CGA
Concentration (mg/L)
Caeine
Concentration (mg/L) pH
Medium - Medium 480 ± 60 1060 ± 60 5.61 ± 0.01 510 ± 20 1180 ± 90 5.54 ± 0.02
Medium - Coarse 490 ± 30 1130 ± 50 5.47 ± 0.01 520 ± 40 1230 ± 60 5.40 ± 0.01
Dark - Medium 380 ± 10 970 ± 60 5.63 ± 0.01 390 ± 10 1080 ± 70 5.53 ± 0.01
Dark - Coarse 330 ± 50 930 ± 40 5.51 ± 0.02 360 ± 20 990 ± 30 5.41 ± 0.02
Table 2. Concentration of 3-CGA and caeine and pH of each cold brew coee sample aer 400 minutes and
1440 minutes of brewing time (Mean ± 95% Condence Interval, n = 6).
Coee Sample
(Roast - Grind)
3-CGA Concentration (mg/L) Caeine Concentration (mg/L) pH
Cold Brew
Method
Hot Brew
Method
Cold Brew
Method
Hot Brew
Method
Cold Brew
Method
Hot Brew
Method
Medium - Medium 510 ± 20 510 ± 30 1180 ± 90 1040 ± 70 5.54 ± 0.02 5.41 ± 0.02
Medium - Coarse 520 ± 40 460 ± 40 1230 ± 60 970 ± 70 5.40 ± 0.01 5.35 ± 0.03
Dark - Medium 390 ± 10 430 ± 30 1080 ± 70 1060 ± 70 5.53 ± 0.01 5.61 ± 0.02
Dark - Coarse 360 ± 20 340 ± 10 990 ± 30 840 ± 10 5.41 ± 0.02 5.48 ± 0.02
Table 3. Comparison of equilibrium 3-CGA and caeine concentrations (aer 1440 min) extracted using cold
and hot brew method along with pH (mean ± 95% Condence Interval, n = 6).
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when comparing medium roast – medium grind and dark roast – medium grind samples. Although the medium
roast samples showed higher concentration of caeine than dark roast samples, the observed dierence in con-
centration is insignicant due to large variations in the measurements.
Discussion and Conclusional Remarks
is work establishes that brewing times near 400 minutes are adequate to extract the majority of available caf-
feine and 3-CGA in medium and dark roast beans prepared at medium and coarse grinds. Moreover, coarse
grain samples, both medium and dark roast, showed a substantial increase in caeine concentrations than their
hot brew counterparts. No signicant dierences were seen in CGA concentrations between cold and hot brews.
Furthermore, the pH between cold and hot brews were comparable. is work suggests that any claims made
by coee vendors about the dierence in acidity or taste of cold brew coee is not due to variations in 3-CGA
concentrations. e results of this study validate earlier models proposed by by Moroney et al.
35,45
and Spiro and
Seldwood
52
and extend their ndings into cold brew extraction over long time periods.
Furthermore, our analysis indicates that the grind size does not have signicant impact the observed equilib-
rium concentrations for both CGA and caeine. When comparing samples with the same roasting temperatures,
the observed dierences in concentrations are largely within one standard deviation from another. Mathematical
modeling of coee extraction proposed by Moroney, et al.
35,45
suggested that the diusion of coee from the
intragranular pores to the intergranular pores is the rate limiting process. us, it takes longer for the extraction
process to reach equilibrium as the grind size increases. However, in the cold brew process, the extraction time
frame is on the order of hours instead of seconds. Such long extraction time scales allow for the slow diusion
from intragranular to intergranular pores, so this is not a rate determining process in cold brew methodologies.
We have noted that grind size was not well controlled in this study, as this work used commercially available cof-
fee without any size separation prior to extraction. Future work will dierentiate coee grinds by particle size to
further quantify the role of grind size in cold brew coee. Figure4 shows a graphical representation of a poorly
sorted and a well sorted coee grind. Grind size and grind sorting are both important parameters controlling
inter- and intragranular diusion.
Materials and Methods
Materials. Coee from the Kona region of Hawai’i was sourced from Kona Joe Coee, LLC (Kealakekua, HI).
is single origin Arabica (Kona Typica) coee was obtained roasted and ground from Kona Joe Coee follow-
ing their standard preparation processes. Four coee types were used in this study. Two roasting temperatures,
medium and dark, prepared at two grind sizes, medium and coarse were selected for this work. Both roasting and
grinding were done by Kona Joe Coee. Medium roast beans were roasted at 215 °C to 217 °C, Dark roast beans
were roasted at 223 °C to 225 °C. e beans were ground using a Mahlkönig DK-15 industrial grinder.
Standard stock solutions of 400 mg/L caeine and 3-CGA were made daily and diluted to establish calibration
curves for coee analysis. Both purchased from Sigma Aldrich (Milwaukee, WI). HPLC grade methanol was
obtained from Fisher Scientic (Nazareth, PA). Phosphoric acid (85% wt.) was obtained from Sigma Aldrich
(Milwaukee, WI) and diluted to 2.0 mM concentration using DI water. Filtered municipal tap water used in this
study. Analysis of this water, completed by Penn State University’s Agricultural Analytical Services Laboratory
found the water to have a total hardness of 174 mg/L and a pH of 7.5.
Methods. Particle size distribution. e particle size distributions of each grind were determined according
to ASTM C136/C136M-14 Standard Test Method for Sieve Analysis for Fine and Coarse Aggregates procedure.
Samples of 100.0 g of coee grinds were added to a sieve stack including sieve sizes #20 (0.841 mm mesh opening),
#40 (0.420 mm mesh opening), #100 mesh (0.149 mm mesh opening), and pan, to generate grain size distribu-
tions for each coee used in this study.
Cold brew experiments. e cold brewing process was carried out at room temperature (ranging from 21 °C to
25 °C over the experimental period) adapted from a home-brewing recipe suggested by e New York Times’
Figure 4. e graphical image of sorting eects on pore connectivity in coee matrix beds. Grain size
distribution inuences both the intragranular diusion process, as larger grains have greater diusion distances,
and the intergranular pore network, as poorly sorted grain beds have more tortuous pore networks.
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Cooking website
9
. A sample of 35.0 g of coee was placed in 350 mL of carbon-ltered municipal water. e coee
was contained in T-Sac
™
tea lter bag (size 4) and placed in a 32-ounce Mason jar tted with a screw-top lid. e
lter bag was used to reduce grind loss during sampling and ensure grinds remained submerged during steeping.
e coee/water mixture was sampled every 15 minutes for the rst hour, then every 30 minutes until hour 7, and
then once an hour until hour 12, a nal sample was taken at 24 hours. Samples collected aer the rst hour were
diluted (1:4) with DI water and ltered using HT Turyn (Pall) 25 mm diameter, 0.2 μm pore size membranes.
Fresh water was added to replace the volume sampled to maintain constant volume. is introduced a small dilu-
tion eect in the resulting solution. Additionally, even with the closed system, there was inevitably evaporation
over the 24 hour testing period. Coee received from Kona Joe Coee was not processed in any way prior to use,
to best match home-brewing conditions. Data presented are an average of triplicate experiments analyzed in
duplicates (n = 6).
Hot brew experiments. Hot brew extraction was conducted using the same coee to water ratio as was used in
the cold brew method. e water was heated to 98 °C and added to coee grounds in a traditional French press
carafe. e water and grounds were allowed to sit for 6 minutes before the lter was depressed and the coee
decanted. Since additional experiments showed that longer mixing times did not result in additional caeine or
3-CGA extraction, 6-minute extraction times were used for all hot brew experiments. Two samples were taken
from each hot brew and each experiment was performed in triplicate (n = 6).
Caeine and 3-CGA measurement. Caeine and 3-CGA were measured in both standard solutions and coee
extracts using an adapted methodology reported in GL Sciences Technical Note No. 67
53
. An Agilent 1200 Series
high performance liquid chromatography system (HPLC) was tted with a Supelco 5 µm column (15 cm × 4.6 cm)
(Supleco, Bellefonte, PA) run at 40.0 °C with a mobile phase mixture of 75% mobile phase A and 25% mobile phase
B (A: 95% 2.0 mM phosphoric acid and 5% methanol; B: 95% methanol and 5% 2.0 mM phosphoric acid). e
ow rate was 1.0 mL/min with an injection volume of 10.0 µL. Caeine and 3-CGA were detected using a diode
array detector at 280 nm and 325 nm respectively.
pH measurements. e pH of each brewed coee sample was measured with a Mettler Toledo FiveEasy
TM
F20
benchtop pH/mV meter.
Statistical Analysis. Two-tailed t-test and ANOVA were employed for determination of similarities in equilib-
rium concentrations of 3-CGA and caeine with consideration of the roast, grind size, and brewing method. e
output of the statistical analysis is included in the supporting information.
Data Availability. All data generated or analyzed during this study are included in this published article (and its
Supplementary Information les).
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Acknowledgements
e authors would like to thank Nicolas A. Parenti, Samantha Ryder, Nelly Tchato Setchie, and Maria Latorre
Socas for their contribution in data collection. is work would not have been possible without their oversight of
sample collection, HPLC analysis, and pH measurement. e authors would like to thank the generous funding
support provided by the Eileen Martinson ’86 Fund for the Undergraduate Capstone Experience at the omas
Jeerson University East Falls Campus. e authors would also like to thank Dr. Joseph Alban and Mr. Bruno
Prota at Kona Joe Coee for their insightful suggestions and discussions.
Author Contributions
M.F. contributed to the experimental design, execution. N.Z.R. conceived of the study, aided in experimental
design and execution. M.F. and N.Z.R. contributed equally to all versions of the manuscript.
www.nature.com/scientificreports/
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Scientific REPORts | (2017) 7:17979 | DOI:10.1038/s41598-017-18247-4
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18247-4.
Competing Interests: e authors declare that they have no competing interests.
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