Methyl Red

EXPERIMENT 6 ACID DISSOCIATION CONSTANT OF METHYL RED A Laboratory Report in Partial Fulfilment of the Requirements in Chem117 Laboratory

DAGONDON, VANESSA OLGA CAGAMPAN, JOHN SULUMOR (Group 1, Cluster 1) With Blyth Angela Balgos Christine Debbie Shanne Angeles (Group 2, Cluster 1)

Chem117 Laboratory – Physical Chemistry II Section 1

Performed on March 24, 2016 Submitted on May 3, 2016

Mr. Arnold Gaje, RCh Laboratory Instructor

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ABSTRACT The experiment aims to determine the acid dissociation constant of methyl red through spectrophotometric measurements based on Beer-Lambert’s Law for mixtures and through the Henderson – Hasselbach equation. The absorbances were obtained using a UV – VIS spectrophotometer and the pHs were obtained using a pH meter. Two standards were prepared: the acidic and basic form of methyl red. The wavelength at the highest absorbance was determined for highest concentration of the two standards. After, the absorbances of the two sets of standards were recorded. Four external calibration curves were generated by plotting the absorbance against the concentration for the two standards at two wavelengths. Through these, the concentration of the HMR and MR in each sample was calculated. Using the Henderson – Hasselbach equation the pKa of methyl red was obtained in each sample. The average pKa was determined to be 4.86 ± 0.03. This value obtained 3.82% relative error when compared to a literature value of 5.05 ± 0.05 determined by number of workers (Tobey, 1958).

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INTRODUCTION Methyl red, a dimethylaminobenzene-2-caboxylic acid exist as a zwitter ion in aqueous solutions. In acid solutions methyl red exist in the form of HMR. When base is added, a proton is lost and methyl red exist as the yellow anion MR -. The equilibrium constant for the ionization of methyl red is: MR−¿ ¿ pK = pH - log ¿ ¿

(1)

In which the ionization constant at known pH values may be calculated from the measurements of the ratio of (MR-)/(HMR).

Fig.1. HMR and MR- forms of methyl red (Tobey, 1958). Spectrophotometry can be used to determine the ratio of (MR-)/(HMR) since the two forms of methyl red absorb strongly in the visible range. Once the absorption spectra of the methyl red in both acidic and basic solutions is determined using a spectrophotometric instrument, two wavelengths are selected for analyzing mixtures of the two forms. The two Page 3 of 18

wavelengths serves as an indication at which the acidic form of methyl red can have a very large absorbancy index compared to the basic form at the first wavelength, and the reversed situation at the other wavelength, this processed helps to assess if the using several concentrations of methyl red can determine if Beer’s law is obeyed (Bell, 1958). Beer-lambert’s law states that the total absorbance of a solution at any given wavelength is equal to the sum of the individual components of the solutions. Thus, the composition of a mixture of HMR and MR- may be calculated from the absorbance of A1 and A2 at the two wavelengths using: A1 = α1,HMR(HMR) + α1,MR-(MR-)

(2)

A2 = α2,HMR(HMR) + α2,MR-(MR-)

(3)

Where there are two species present in the solution. The absorption spectra of each species tends to overlap. In the experiment the objective is to prepare and measure the visible absorption spectra of acidic and basic forms of methyl red, thereby determining the wavelength of maximum absorbance of the two solutions, then to create a calibration curve for each of the solutions and molar absortivities at λmaz, to determine the relative amounts of acid and it conjugate base in solutions. And ultimately to determine the pKa or the acid dissociation constants by following the change in absorbance as a function of pH.

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METHODOLOGY Preparation of solutions. The stock solution of methyl red was prepared by dissolving a 1g of crystalline methyl red (MR) in 300 mL of 95% ethanol and then diluting it to 500 mL with distilled water. A standard solution of MR was used in the actual experiment, it was prepared by taking out 4 mL of the stock solution and adding it to 50 mL of 95% ethanol and was diluted to 100 mL with addition of water. A set of standard solutions was prepared. A 250 mL of 0.04 M NaOAc, 100 mL of 0.01 M NaOAc, 100 mL of 0.02 M HOAc, 25mL of 0.1 M HCl, and 100 mL of 0.01 M HCl. Two set solutions was prepared. Solution A was prepared by adding a 10 mL MR solution with 10mL of 0.1M HCl into a 100 mL volumetric flask and was diluted to 100 mL with addition of water. Solution B was prepared by adding 10 mL of MR solution with 25 mL 0.04M NaOAc and was diluted to 100 mL in a 100mL volumetric flask with water. Each of the solution was diluted into 0.75, 0.5, 0.25 %Concentration of their original concentration by using 0.1 M HCl and 0.04M NaOAc in a 100 mL volumetric flask. Determining the wavelength of maximum absorbance. Solutions A and B was placed in a cuvette and the absorbance of solution was measured between 350-600 nm to measure their maximum absorbance. And the absorbance was plotted for the two solutions and XA and XB was aslo determined. A series of solutions was prepared (solutions 1to 4) by adding varying amounts of 0.02 M acetic acid, 50 mL, 25mL, 10 mL and 5mL respectively to 10 mL of standard indicator solution

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buffered plus 25 mL of 0.04 M NaOAc solution and diluting the solutions with water up to 100 mL of the volumetric flask. Determining the molar absorptivities of [HMR] and [MR-] at λmax. The absorbance of the solutions from set A and set B was placed in a cuvette separately and the absorbance of every solution was determined using a UV-Vis. The absorbance was plotted against the concentration and the molar absortivities of [HMR] and [MR-] was determined using the equation A=εbc

ε=

Where

A bc

(4)

(5)

A = absorbance b = path length c = concentration ε = molar absorptivity

Determining the relative amounts of acid and its conjugate base. The pH level of the solutions 1 to 4 was determined using a pH meter to ensure it doesn’t reach a basic level. The relative amounts of acid and its conjugate base was also determing by

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measuring the absorbance of the solutions at different wavelength with maximum absorbance determined from the solutions A and B (Daniels et. al, 1970). RESULTS The following results reflect consecutively each of the objectives stated in order to achieve the ultimate goal of the experiment, to be able to determine the equilibrium constant for the ionization of methyl red, Ka, using spectroscopic measurements. The average value of Ka expressed as a p value was determined to be 4.86 ± 0.03. This value obtained 3.82% relative error when compared to a literature value of 5.05 ± 0.05 determined by number of workers (Tobey, 1958). Figure 1 shows the absorption spectra of the two solutions: A (acidic solution of Methyl Red, HMR) and B (basic solution of Methyl Red, MR -). The maximum wavelength for solution A is 520 nm and the maximum wavelength for the solution B is 428.5 nm.

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0.8 0.7 0.6 0.5 Absorbance

0.4 HMR 0.3

MR

0.2 0.1 0 350

400

450

500

550

600

650

Wavelength (nm)

Figure 1. Absorption spectra: Absorbances of HMR and MR- versus λ Absorbances at the two selected maximum wavelengths were measured for the two sets of standards prepared. Each of the standards consists of different concentrations of HMR and MR-: 0.25, 0.5, 0.75, and 1 ppm. The recorded absorbances measured at each wavelength were plotted against the concentrations of the standard. Figure 1 show four linear plots of absorbance against wavelength at 520 nm and 428.5 nm for the two standards solutions.

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0.8 0.7 f(x) = 0.69x 0.6 HMR @ 428.5nm

Linear (HMR @ 428.5nm)

MR @ 428.5nm

HMR @ 520nm

Linear (HMR @ 520nm)

0.5

Absorbance

0.4

Linear (MR @ 428.5nm) 0.3

f(x) = 0.29x 0.2 0.1520nm) Linear (HMR @ 0 0.2

MR @ 520nm

f(x) = 0.05x f(x) = 0.03x 0.3 0.4

0.5

Linear (MR @ 520nm)

0.6

0.7

0.8

0.9

1

1.1

Relative Concentration of Methyl Red

Figure 2. Absorbances of HMR and MR- at λ520 and λ428.5 versus concentration Four samples of unknown solution were prepared. The absorbances of these solutions were measured at the two selected wavelengths. The pHs of the samples were also obtained using a pH meter. Table 1 shows the summary of the experimental data obtained for each of the samples. Table 1. Experimental data for the samples Volume of

Absorbance at

Absorbance at

Solution 1 2

pH HOAc (mL)

λ520

λ428.5

50 25

0.66 0.578

0.1 0.124

4.06 4.37 Page 9 of 18

3 4

10 5

0.273 0.148

0.234 0.27

5.19 5.6

The slope of the linear plots at Figure 2 denotes the absorptivity of each solution at each wavelength. These values are used to calculate for the concentrations of MR - and HMR via Beer’s Law. Using the concentrations of MR- and HMR and the measured pH of each the samples, the pKa can be determined. Table 2 shows summary of the results for each of the samples. Table 2. Summary of Result for each sample

Solution

[HMR]

[MR-]

pKa

1

0.948

0.167

4.81

2

0.825

0.275

4.85

3

0.365

0.747

4.88

4

0.177

0.908

4.89

The pKa values obtained in Table 3 is averaged to be 4.86 ± 0.03. DISCUSSION The acid dissociation constant of Methyl red (MR) was determined using spectrophotometric measurements. Methyl red exists as zwitterion in aqueous solutions and has resonance structure between its acidic (HMR) and basic form (MR -). It was observed that its acidic form is pink in color while its basic form is yellow in color and thus both will have strong absorption peaks in the visible portion of the spectrum (Toby, 1958). The acid-dissociation constant can be calculated using: Page 10 of 18

+¿ −¿ MR ¿ ¿ H¿¿ ¿ K=¿

(6)

A more useful in determining the acid-dissociation constant is the Hender – Hasselbach shown in eq. 1. To determine the acid – dissociation constant K, the pH and the concentrations of the acidic and basic form of the MR must be known. In the experiment, the relative concentrations of HMR and MR- were determined using spectrophotometric measurements and the pH of the sample was determined using a pH meter. Four samples of methyl red solutions were analyzed. Two sets of standards were prepared: HMR and MR-. For each set of standard, calibration curves were generated by plotting the absorbances of the standards against the relative concentration. A UV-Vis spectrometer as used to determine the absorbances of the solutions. Before reading the absorbances of the standards and sample, the maximum wavelength at which these absorbances will be read was determined. Figure 1 shows the absorption spectra of the HMR and MR -. The absorption spectrum is a plot of the absorbance against the wavelength. The wavelength with highest recorded absorbance was selected both HMR and MR-. This was done to improve the sensitivity of the signal measured that is the absorbance can still be measure effectively with small changes in the concentration (Skoog et. al, 2000). The absorbances of the standards were measured at the two wavelengths selected. Figure 2 shows the four calibration curves generated from the two sets of standards. The principle behind this spectrophotometric determination of the concentrations of HMR and MR- is the beer’s law. Beer’s law states that the absorbance is equal to the concentration, pathlength and molar absorptivity. There is a linear relationship between the Page 11 of 18

absorbance and the concentration. Four calibration curves were generated by plotting absorbance against the concentration in each of HMR and MR- as shown in figure 2. The slope represents that absorptivity since the path length is 1 cm. The slope and the equation of the best fitted in each of the linear plots was determined using the least square methods. Beer’s law can also be applied in mixtures. Absorbance is additive and if the pathlength b is equal to 1 cm, it can be expressed as: −¿ MR ¿ ( ε λ ) ¿ ¿ A λ =[HMR] (ε λ )HMR +¿ 520

520

(7)

520

−¿ MR ¿ (ε λ ) ¿ ¿ =[ HMR]( ε λ ) HMR +¿ 428.5

Aλ

428.5

(8)

428.5

Since the absorbance of the sample is known, together with the Absorptivities which are just the slopes of the calibration curves, the values for [HMR] and [MR -] can be calculated. Moreover, the pH of the sample was determined using a pH meter. Table 1 summarizes the pH readings of each of the sample prepared. Using the Henderson – Hasselbach equation, the pKas of the samples were calculated and summarized in Table 2. The average of the obtained pKa values is 4.86 ± 0.03. This resulted to an error of 3.82% relative error when compared to a literature value of 5.05 ± 0.05 determined by number of workers (Tobey, 1958). This minimum value of relative error indicates that there is no systematic error in the analysis just random errors. There random errors might originate from the preparation of the standards, the absorbance readings, the fluctuation of temperature and from personal errors. Page 12 of 18

CONCLUSION Methyl red is a pH sensitive azo dye used as indicator in volumetric titrations. In aqueous solutions, methyl red exists as zwitterion with a resonance structure between its two forms: basic, MR- and acidic, HMR. In acidic conditions, it forms a pink solution while in basic conditions, it forms a yellow solution. Since these two forms absorb strongly in the visible range, the acid dissociation constant of methyl red can determined using spectroscopic methods. Four mixtures of HMR and MR were prepared to be analyzed. The pHs of the samples were measured using the pH meter. Also, four calibration curves were prepared using the two sets of standards, HMR and MR. The absorbances were read from a UV-VIS spectrophotometer in the two selected wavelengths. Beer’s law is obeyed in the experiment. Beer’s law states that the absorbance is equal to the absorptivity, path length and concentration. The path length of the of the cuvette used in the UV – Vis spectrophotometer is 1 cm. This makes the slope in the calibration curve be equal to the absorptivity. The absorbance of the the mixture samples were obtained. Since the absorbance is additive, two sets of equations were used to calculate the concentration of HMR and MR in each sample. The acid dissociation constant was computed using the Hneder – Hasselbach equation. The average acid dissociation constant determined in the experiment is 4.86 ± 0.03 which obtained a 3.82% relative error when compared to a literature value of 5.05 ± 0.05 determined by number of workers (Tobey, 1958). Errors considered in the experiment are random in nature. No systematic error has been detected and therefore the experiment is a success. REFERENCES Page 13 of 18

Atkins, P.; de Paula, J. Physical Chemistry Ninth Edition; Oxford University Press: Great Britain, 2010. Bell, C. K. Acids and Bases: Their Quantitative Behaviour; Methuen & Co., Ltd.: New York, 1952. D.C. Harris, Quantitative Chemical Analysis, 7th ed. New York: W.H. Freeman and Company, 2007. F. Daniels, J. W. Williams, P. Bender, R. A. Alberty, C. D. Cornwell, J. E. Harriman, “Acid Dissociation Constant of Methyl Red,” Experimental Physical Chemistry, McGraw-Hill, New York, NY, 1970, pp. 113-115. Skoog, Douglas, et. al. Analytical Chemistry: An Introduction, Thomson Learning Asia. 2000. Tobey, J. The Acid Dissociation Constant of Methyl Red. Journal of Chemical Education. 1958, 35, 514. ACKNWLEDGEMENTS We would like to express our deepest gratitude to our laboratory classmates and especially to our cluster for working with us diligently, to our instructor for guiding us during the experiment, and to the chemistry department for providing us the materials for the experiment.

CONTRIBUTION OF AUTHORS Each of the members of the group contributed accordingly in the making of this laboratory report: Cagampan authored the introduction and material and method sections; Page 14 of 18

Dagondon, on the other hand, was in charge of the data analysis, the compilation of results, discussion and also the conclusion; Cagampan wrote the abstract and proof – read the overall report. APPENDICES Appendix I. Tables and Figures Table 1. Raw Data for the Absorbance and wavelength for HMR and MR standard solution solution A (HMR) wavelength absorbance 400 0.017 403 0.018 406 0.02 409 0.023 412 0.027 415 0.03 418 0.035 421 0.04 424 0.047 427 0.053 430 0.061 433 0.071 436 0.082 439 0.093 442 0.106 445 0.121 448 0.138 451 0.155 454 0.173 457 0.194 460 0.217 463 0.241 466 0.265 469 0.291 472 0.32 475 0.349 478 0.377 481 0.404

Solution B (MR-) wavelength absorbance 400 0.245 403 0.26 406 0.264 409 0.269 412 0.273 415 0.275 418 0.277 421 0.279 424 0.28 427 0.28 430 0.28 433 0.277 436 0.276 439 0.274 442 0.273 445 0.27 448 0.266 451 0.261 454 0.256 457 0.249 460 0.241 463 0.232 466 0.221 469 0.21 472 0.198 475 0.185 478 0.172 481 0.16 Page 15 of 18

484 487 490 493 496 499 502 505 508 511 514 517 520 523 526 529 532 535 538 541 544 547 550 553 556 559 562 565 568 571 574 577 580 583 586 589 592 595 598

0.436 0.466 0.494 0.52 0.55 0.577 0.602 0.624 0.643 0.66 0.67 0.677 0.679 0.676 0.671 0.663 0.652 0.64 0.626 0.61 0.594 0.569 0.541 0.509 0.471 0.425 0.373 0.327 0.278 0.228 0.184 0.145 0.114 0.086 0.063 0.047 0.035 0.025 0.019

484 487 490 493 496 499 502 505 508 511 514 517 520 523 526 529 532 535 538 541 544 547 550 553 556 559 562 565 568 571 574 577 580 583 586 589 592 595 598

0.144 0.131 0.118 0.106 0.093 0.081 0.071 0.063 0.055 0.046 0.04 0.035 0.031 0.026 0.023 0.021 0.018 0.016 0.015 0.014 0.013 0.012 0.012 0.011 0.011 0.01 0.01 0.01 0.009 0.008 0.007 0.007 0.006 0.006 0.006 0.005 0.005 0.005 0.005

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Table 2. Absorbances taken at the selected wavelengths for HMR and MR standards Relative Concentration

MR-

HMR λ520

λ428.5

λ520

λ428.5

0.25

0.139

0.012

0.01

0.073

0.5

0.334

0.028

0.014

0.16

0.75

0.526

0.04

0.023

0.208

1

0.7

0.056

0.026

0.283

Appendix B: Sample Calculations 

Determination of absorptivity/slope of liner plot via least square method For the linear plot of absorbance recorded at 520nm against relative concentration of HMR:

( Σx )2 6.25 S xx =Σ x − =1.875− =0.3125 n 4 2

S yy =Σ y 2−

( Σy )2 2.8886601 =0.897553− =0.17590275 n 4 ❑

S xy =Σxy−

m= 

( ΣxΣy ) 4.2475 =1.29625− =0.234375 n 4

S xy 0.234375 = =0.6913 S xx 0.3125

Determination of the concentration of HMR and MR in sample via algebraic method, substitution For sample 1 only: (ελ520)HMR = 0.6913 (ελ520)MR = 0.0281 (ελ428.5)HMR = 0.0549 (ελ428.5)MR = 0.2865

−¿ MR ¿ ( ε λ ) ¿ ¿ A λ =[HMR] (ε λ )HMR +¿ 520

520

520

−¿ MR ¿ (ε λ ) ¿ ¿ =[ HMR]( ε λ ) HMR +¿ 428.5

Aλ

428.5

428.5

Solving for [MR] and [HMR]:

−¿ MR¿ ¿ ¿

−¿ MR × ( ε λ ) MR ¿ A λ −¿ [ HMR❑] =¿ ¿

520

520



Determination of pKa For sample 1 only:

−¿ MR¿ ¿ ¿ pK a= pH −log¿