Chem. Educator 2011, 16, 1–3
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The Determination of the pKa of Red Cabbage Anthocyanin by the
Spectrophotometric Method and Nonlinear Curve Fitting
Ampaporn Munmai† and Ekasith Somsook*,‡
†Institute for Innovative Learning, Mahidol University, 999 Buddhamonthon Sai 4 Rd. Salaya, Buddhamonthon,
Nakornpathom 73170, Thailand; ‡NANOCAST Laboratory, Center for Catalysis, Department of Chemistry and
Center for Innovation in Chemistry, Faculty of Science, Mahidol University, 272 Thung Phaya Thai, Rama VI
Rd. Ratchathewi, Bangkok 10400, Thailand, [email protected]
Received May 26, 2011. Accepted September 21, 2011.
Abstract: A linear plot based on the spectrophotometric method and Henderson-Hasselbalch equation has a
drawback of choosing the appropriate pH for acid and basic forms of the indicators which may lead to the
incorrect pKa values especially in the systems with many involved reactions at the equilibrium. Herein, a
nonlinear curve fitting based on the Henderson-Hasselbalch equation was proposed to solve this problem for the
determination of the pKa values of red cabbage anthocyanin solution.
Introduction
Materials and Methods
Red cabbage is useful for human health in preventing
cardiovascular diseases, some types of cancer [1–3],
headaches, gout, diarrhea, and peptic ulcers [4]. It has been
used for food and beverage colorants where its color can be
changed according to the pH value due to a pigment called
anthocyanin which the central core of anthocyanin is called an
aglycone core [5] as shown in Figure 1.
Red cabbage anthocyanin can be represented in various
structures based on R and R ' group in B ring of the aglycone
core which has been characterized to be cyanidine [3, 6, 7]. It
exists in a positive charge of oxonium ion called a flavylium
cation. It shows a red color in an acidic solution while it
becomes a colorless of pseudo-base form in a basic solution as
shown in Figure 2.
pKa is an important physical parameter to indicate the
acidity of molecules. Spectrophotometry is widely used in
chemical experiments to determine the ionization constant
(pKa) of many indicators [9–14]. For a simple method with one
acid form and one basic form, pKa may be determined by the
modified Handerson-Hasselbalch equation [9] according to the
following equation (1). The drawback of this method is that the
pKa may be determined incorrectly because the appropriate pH
of the acid and basic forms may not be chosen.
Materials. The red cabbage (Brassica oleracea L. var.
capitata f. rubra) of Brassicaceae family was purchased from a
local supermarket. The chemicals used in this experiment were
of analytical grade and also consist of hydrochloric acid
(Merck), and sodium hydroxide (Merck) used to adjust the pH
values.
Preparation of Phenolphthalein Solution. A 1% solution
of phenolphthalein in ethanol was used as a validation reagent
of indicator under this study. The phenolphthalein indicator
was prepared by dissolving 0.2 cm3 of 1% phenolphthalein in
ethanol with water in 250 cm3 volumetric flask according to
published methods [9]. Fifty milliliter of indicator was poured
into each of 100 cm3 beakers. The solutions were adjusted pH
values of 1.54–12.01 by dropwise addition of 0.1% and 1% of
HCl and also 0.1 mol/dm3 or 1 mol/dm3 NaOH solutions. The
pH values lower than 3.08 and more than 11.07 of indicators
were adjusted with concentrated HCl solution and NaOH
pellets.
Preparation of Methyl Orange Solution. A 0.1% methyl
orange was used as the validation reagent. The methyl orange
solution was prepared by dissolving 1.5 cm3 of 0.1% methyl
orange in water in a 250 cm3-volumetric flask according to
published methods [9]. 50 cm3 of solution was poured into
each of 100 cm3 beakers. Then, the solutions were adjusted pH
values by dropwise addition of 0.1% and 1% of HCl or 0.1
mol/dm3 and 1 mol/dm3 NaOH solutions. The pH values lower
than 2.30 and more than 11.03 of indicators were adjusted with
a concentrated HCl solution and NaOH pellets. The solution
volumes used for two indicators were listed in Table 1.
Preparation of Red Cabbage Stock Solution. The freshly
chopped red cabbage (605 mg) was heated in 500 cm3 of
distilled water for 5 minutes until the color faded and then
filtered the solution. The volume of final obtained solution was
adjusted to 500 cm3 (0.12%w/v) with distilled water. The red
cabbage stock solution was freshly prepared before using for
the determination of the pKa values.
Spectrophotometric Method. Visible absorbance spectra
were collected for phenolphthalein indicator, methyl orange
 A  AIn  
log 
  pK a  pH
 AHIn  A 
(1)
Herein, we proposed a nonlinear fitting based on the
modified Henderson-Hasselbalch for the determination of pKa
of red cabbage anthocyanin solution.
*
Address correspondence to this author.
Institute for Innovative Learning.
‡
NANOCAST Laboratory, Center for Catalysis.
†
© 2011 The Chemical Educator, S1430-4171(11)0xxxx-x, Published xx/xx/2011, 10.1333/s00897112404a, xxxxxxaa.pdf
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Chem. Educator, Vol. 16, 2011
Somsook et al.
Table 1. Solutions used to measure pKa of indicators
Indicators
0.1% Methyl orange
1% Phenolpthalein
3
Volume of indicator / cm
1.5
0.2
max / nm
510a
550a
Note: aReference 9
Figure 1. Chemical structure of anthocyanin aglycone core.
Figure 2. Chemical structure of the two forms of anthocyanin
aglycone cores [8].
Figure 3. The color of the red cabbage solutions at different pH
values.
Absorbance
0.8
pH
increases
0.4
1.44
1.51
1.60
1.71
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.21
4.40
4.60
4.80
5.01
5.22
5.41
5.60
5.82
6.00
6.20
6.40
6.60
6.80
7.00
7.21
7.40
7.80
8.02
8.20
0
400
500
600
700
Wavelength (nm)
800
Figure 4. Spectra of red cabbage solutions at different pH values. For
increasing of all pH values of red cabbage solutions, they were
increased from approximately 1.4 to 13.80, with increment 0.2
intervals.
indicator and red cabbage solutions at various pH values and
covered the wavelength from 400 to 800 nm using a HP-8453
Hewlett Packard UVVisible scanning spectrophotometer at
room temperature (31oC). The absorbances of phenolphthalein
and methyl orange solutions at various pH were measured,
respectively, at the λmax 550 and 510 nm.
A volume of 20 cm3 of the stock red cabbage solution was
diluted with water to 100 cm3 of solution (0.024% w/v) for the
further investigation. Similarly, 15 cm3 of 0.024% red cabbage
solutions were adjusted the pH values ranging from 1.44 to
13.80 with the increment of 0.2 intervals. The pH values were
measured with a Mettler Toledo InLab 413 pH meter using a
combined electrode. The pH meter was calibrated by using
three aqueous standard buffer solutions of pH 4.0, 7.0, and
10.0.
The Determination of pKa. The absorbances data of two
indicators and red cabbage were performed to determine the
pKa values by nonlinear plot assisted by Microsolf Excel
Solver based on the modified Henderson-Hasselbalch equation
(see the supplementary section). Also, the pKa values of red
cabbage anthocyanins by linear and nonlinear plots were
compared on the varying of the pH of acid and basic forms.
Results and Discussion
The red cabbage is an excellent origin of colorful phenolic
compounds, with the anthocyanins being the most abundant
class [3, 6, 7, 15, 16]. In this report, the colors of the red
cabbage solution were observed in the pH range of 1.4413.80
in which the solution (0.024 %w/v) was red, purple, blue,
green, yellow at pH 1, 4, 6.2, 7.2, 12.80, respectively, as
shown in Figure 3. This indicates that many reactions
involving protonation, deprotonation, hydration, and
nucleophilic substitution at equilibrium in the color formation
of red cabbage solutions. The spectra of red cabbage solutions
at different pH were shown in Figure 4.
Due to complicated reactions involving in the pKa
determination of red cabbage solutions, a linear plot based on
equation (1) may not be a good choice for solving this
problem. Here, a nonlinear fitting based on the modified
Henderson-Hasselbalch equation [13] for the determination of
pKa of red cabbage anthocyanin solutions (see more details in
the Supplementary section).
( pK a  pH ) 

10
 +X
Ameas = constant 


pK

pH
(
)
 1  10

a


(2)
Where Ameas = observed absorbance at specific wavelength
and X is absorbance of background. Then the determination of
the pKa values of red cabbage anthocyanins was carried out
using nonlinear curve fitting. The curve fitting of the red
cabbage solution by fitting the absorbance at 520 nm is shown
in Figure 5 and the pKa value was found to be 2.67. The
nonlinear curve fittings based on the Henderson-Hasselbalch
for phenolphthalein and methyl orange are presented in Figure
6 for validation of this method and. the pKa values for are 9.59
and 3.26 (ionic strength ≤ 0.05 mol/dm3), respectively, which
are close to reported data [9].
Moreover, this method was applied to measure the pKa value
in the pH values (5.0–8.0) and wavelength (612 nm) as
published in literature [8] and it was found that the pKa was
7.03 that was close to the reported pKa values (6.8–7.2) as
shown in Figure 7.
The further investigation was carried out by comparing the
linear and nonlinear methods based on Henderson- Hasselbalch
[9] equation as presented in Table 2. Upon choosing different
pH of the acidic and basic forms for the linear method could
lead to different pKa values. In contrast, the pKa values
obtained from the nonlinear method were close to the values as
reported in literatures [17, 18]. This shows clearly that the
limitation of the linear method and this could lead to the wrong
© 2011 The Chemical Educator, S1430-4171(11)0xxxx-x, Published xx/xx/2011, 10.1333/s00897112404a, xxxxxxaa.pdf
The Determination of the pKa of Red Cabbage Anthocyanin...
Table 2. Measurement of pKa values of red cabbage anthocyanin at
520 nm by linear and a nonlinear plot
pH range
Acid-Basic form
2.00–6.00
3.00–6.00
4.00–6.00
2.00–8.00
3.00–8.00
4.00–8.00
5.00–8.00
4.00–10.00
5.00–10.00
6.00–10.00
1.44–13.80
2.00–13.80
3.00–13.80
2–10
3–10
4–10
2–8
3–8
4–8
5–8
4–10
5–10
6–10
1.44–13.8
2–13.8
3–13.8
Linear plot
2.88
3.49
4.39
2.67
3.32
4.47
6.36
18.67
–62.67
– 0.58
0.80
– 1.04
– 0.53
pKa
Nonlinear plot
2.71
3.08
3.63
2.73
3.15
4.04
6.74
4.14
6.28
6.70
2.67
2.80
3.19
Chem. Educator, Vol. 16, 2011
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Conclusions
Due to various forms of anthocyanins in red cabbage, pKa
values can be difficult to be determined from the linear
method. Herein, we have demonstrated a simple laboratory
experiment that uses the spectrophotometric method coupled
with the nonlinear method to determine the pKa values of red
cabbage solutions. In this study, Microsolf Excel Solver is
useful for the experimental data analysis to plot nonlinear
regression which draws the relationship between observed
absorbance and pH based on the modified HendersonHasselbalch equation. The pKa values of red cabbage
anthocyanins from nonlinear regression plot are reliable and
reasonable of pKa values ranging from 2.67-6.74. In contrast,
pKa values from linear regression have shown some incorrect
values. As results of the comparison of linear and nonlinear
plot, nonlinear plots are effective for the pKa determination.
This simple experiment is recommended to be applied for
teaching chemistry laboratory.
Acknowledgements. The financial support from the
Institute for the Promotion of Teaching Science and
Technology (IPST), Center for Innovation in Chemistry
(PERCH-CIC) and the Office of the Higher Education
Commission and Mahidol University under the National
Research University Initiative are acknowledged.
References and Notes
Figure 5. Excel template for the determination of pKa of red cabbage
at 520 nm.
(a)
(b)
Figure 6. The nonlinear curve fitting of the pKa value of (a)
phenolphthalein and (b) methyl orange solutions.
1.
Steinmetz, K. A.; Potter, J. D. Journal of the American Dietetic
Association 1996, 96(10), 1027–1039.
2.
Cooke, D.; Steward, W. P.; Gescher, A. J.; Marczylo, T. European
Journal of Cancer 2005, 41(13), 1931–1940.
3.
Charron, C. S.; Clevidence, B. A.; Britz, S. J.; Novotny, J. A. Journal
of Agricultural and Food Chemistry 2007, 55(13), 5354–5362.
4.
Cheney, G. Journal of the American Dietetic Association 1950,
26(9), 668–672.
5.
Curtright, R. Journal of Chemical Education 1996, 73, 306–309.
6.
Wu, X.; Prior, R. L. Journal of Agricultural and Food Chemistry
2005, 53(8), 3101–3113.
7.
Dyrby, M.; Westergaard, N.; Stapelfeldt, H. Food Chemistry 2001,
72(4), 431–437.
8.
Chigurupati, N.; Saiki, L.; Gayser, C.; Dash, A. K. International
Journal of Pharmaceutics 2002, 241(2), 293–299.
9.
Patterson, G. S. Journal of Chemical Education 1999, 76, 395–398.
10. Tobey, S. W. Journal of Chemical Education 1958, 35, 514–515.
11. Brown, W. E.; Campbell, J. A. Journal of Chemical Education 1968,
45, 674–675.
12. Ramette, R. W. Journal of Chemical Education 1963, 40, 252–254.
13. Lai, S. T. F.; Burkhart, R. D. Journal of Chemical Education 1976,
53, 500.
Figure 7. The nonlinear curve fitting of the pKa value of red cabbage
in pH ranging from 5.0-8.0 at 612 nm.
14. Alter, K. P.; Molloy, J. L.; Niemeyer, E. D. Journal of Chemical
Education 2005, 82, 1682–1685.
15.
Arapitsas, P.; Sjöberg, P. J. R.; Turner, C. Food Chemistry 2008,
109(1), 219–226.
values of pKa values in the systems with many involving
reactions at the equilibrium.
16.
Wu, X.; Beecher, G. R.; Holden, J. M.; Haytowitz, D. B.; Gebhardt,
S. E.; Prior, R. L. Journal of Agricultural and Food Chemistry 2006,
54(11), 4069–4075.
Hazards. Sodium hydroxide and hydrochloric acid are
highly corrosive and cause serious permanent eye damage.
Rubber gloves and goggles should always wear while handling
the materials or solutions.
17.
Dangles, O.; Saito, N.; Brouillard, R. Journal of American Chemical
Society 1993, 115, 3125–3132.
18.
Mazza, G.; Brouillard, R. Journal of Agricultural and Food
Chemistry 1987, 35, 422–426.
© 2011 The Chemical Educator, S1430-4171(11)0xxxx-x, Published xx/xx/2011, 10.1333/s00897112404a, xxxxxxaa.pdf