The Determination of the pKa of Red Cabbage - Mahidol University

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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.

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Figure 7. The nonlinear curve fitting of the pKa value of red cabbage in pH ranging from 5.0-8.0 at 612 nm.

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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.

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© 2011 The Chemical Educator, S1430-4171(11)0xxxx-x, Published xx/xx/2011, 10.1333/s00897112404a, xxxxxxaa.pdf

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The Determination of the pKa of Red Cabbage - Mahidol University

Chem. Educator 2011, 16, 1–3 1 The Determination of the pKa of Red Cabbage Anthocyanin by the Spectrophotometric Method and Nonlinear Curve Fitting ...

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