Spectrometric Determination of the Acid Dissociation Constant of Methyl Red

Felices Nino B.1, San Juan, Jose Miguel2

1 2

Department of Chemical Engineering, College of Engineering Department of Chemical Engineering, College of Engineering

University of the Philippines, Diliman, Quezon City, Philippines Date Due: September 24, 2013 Date Submitted: September 19,2013

The experiment aims to determine the acid dissociation constant of methyl red by applying spectrophotometric principles based on Beer-Lamberts Law for multicomponent system. The instrument used for the experiment is a UV-Vis Spectrophotometer. First, the molar absorptivity of the basic and acidic form of methyl red is determined at the wavelengths of maximum absorption of each component. Then, the absorbance of a mixture of the acidic and basic form of methyl red, of known pH is taken. With this, the concentrations of the two components in the system are determined. Finally, the acid-dissociation constant or Ka of methyl red is determined using these concentrations and the measured pH of the solution by the Henderson-Hasselbalch equation. The plot of pH against log([MR-]/[HMR]) was taken for the two data sets and the least squares y-intercept gave the value of the pKa.. The first data set produced a pKa value of 5.434 and Ka value of 4.542 x 10-6. The second data set produces a pKa value of 5.6423 and Ka value of 2.279 x10-6. In average, the whole data gave a Ka value of 3.412 x 10-6 producing 14.29% error. A standard deviation of 1.600 x 10-6 was obtained. The precise and accurate values obtained conclude that the spectrophotometric determination of methyl red is a precise and accurate method. Certain limitations of the method and other systematic errors committed in the experiment caused the deviation in the obtained values.

Keywords: Beer-Lamberts Law, UV-Vis Spectrophotometer, Acid-Dissociation Constant, Wavelength of Maximum Absorption, Absorbance

INTRODUCTION
Spectroscopy is a field of knowledge that deals with the interaction of radiation and matter. Different methods of analysis were made as an application of spectroscopy. These methods use the different regions of the electromagnetic spectrum. In the field of chemistry, spectroscopic methods are used in both the qualitative and quantitative analysis of different chemical compounds. A branch of spectroscopy is called spectrophotometry. It is the quantitative study of the amount of electromagnetic radiation absorbed, emitted or transmitted by a species, particularly in the visible, near-UV and nearinfrared regions of the electromagnetic spectrum. It can be used to determine the concentration of the unknown by using its ability to absorb radiant energy at specific wavelengths. Spectrophotometry employs an instrument called spectrophotometer which is composed of two main components: a spectrometer that produces light of a certain wavelength and a photometer which could measure the intensity of light. Samples were held in a small container called the cuvette. A beam of light is passed into the cuvette and the photometer inside the instrument measures the amount of light passing through it. The photometer then delivers a voltage signal into the galvanometer that indicates the absorbance of the sample being measured. Each molecular species absorb light at characteristic frequencies of the electromagnetic radiation. This process transfers energy to the molecule and results in the intensity of the incident electromagnetic radiation. This decrease in energy per unit area is called attenuation and absorption of light causes this process. Beer-Lamberts Law or simply Beers law describes quantitatively how the amount of

attenuation depends on the concentration of the absorbing molecules and the path length over which absorption occurs. When a parallel beam of monochromatic radiation passes through an absorbing solution of path length b and concentration c, the interaction between the photons and the particles of the absorbing solution reduces the radiant power from an initial value P0 to a value P. The transmittance T of the solution is measured by the fraction of incident radiation transmitted by the solution. (Eq. 1) Eq. 1 Transmittance of a Solution

What is detected by the UV-Vis Spectrophotometer is the percent transmittance of the solution. However, in quantitative analyses, it is more convenient to use the amount of radiant energy absorbed by the solution. This defines the absorbance. The absorbance is logarithmically related to transmittance by Eq. 2 Eq. 2 Absorbance of a Solution

What makes it more convenient to use than the transmittance of a solution is that because it is linearly related to the concentration of the absorbing species in a solution. This is clearly expressed by Beers Law. (Eq. 3) Eq. 3 Beer-Lamberts Law

Where A is the absorbance, a is the specific absorptivity constant, b is the path length of the radiation in cm and c is the concentration of the solution. When c is expressed in molarity, the proportionality constant a can be replaced by , the molar absorptivity.

The absorbance of a solution is related to the wavelength of the incident light. In employing quantitative analyses, the wavelengths of maximum absorption for the absorbing species are obtained. This is done by plotting the absorbance of a solution against the wavelength. This gives the absorption spectrum of a particular species. From this, the peak of the graph gives the value for the wavelength of maximum absorption. Also, the visible spectrum shown by Table 1 could be helpful in predicting the wavelength of maximum absorption of a chemical species. Table 1 Visible Spectrum Wavelength Region Absorbed(nm) 400-435 435-480 480-490 490-500 500-560 560-580 580-595 595-650 650-750 Color of Light Absorbed Violet Blue Blue-green Green-blue Green Yellowgreen Yellow Orange Red Color of Light Transmitted Yellow green Yellow Orange Red Purple Violet Blue Blue-green Green-blue

The acid-dissociation constant of the indicator, methyl red (4-dimethylaminobenzene-2carboxylic acid) can be studied using spectrophotometry since both the acidic and basic forms of methyl red absorb strongly in the visible region with minimal interferences from other substances. However, the absorption spectra of the basic and acidic forms of methyl red overlap and at the wavelength of maximum absorption; there is interference from the other. Let the acidic form of methyl red be HMR and the basic form be MR-. To analyze the mixture, the molar absorptivities of these forms of methyl red are first determined at the wavelength of maximum absorption of each component. Solutions containing only HMR are prepared and their absorbances at the two wavelengths of maximum absorption are determined. The same goes for MR-. Using a calibration curve, the molar absorptivities of each component is determined for each wavelength. Mixtures of solutions containing both HMR and MR- are then analyzed. Their absorbances were determined and the systems of equations below were used to determine the concentration of each component in the mixture. Eq. 5 and 6 At HMR

Beers law can also be applied in the analysis of multicomponent systems. Provided that there are no interactions among the various species in the multicomponent system, the total absorbance is the sum of the individual absorbances as described by Eq. 4 Eq. 4 Absorbance of Multicomponent System

At MR-

This relationship allows the determination of the concentrations of the individual components in a mixture even if there is a strong overlap in their spectra.

To determine the experimental Ka value, the plot of pH against log([MR-]/[HMR]) for each data set was taken. Linear regression was applied, obtaining an equation in the form of Eq. 7. The least squares y-intercept b was taken as the pKa value and the Ka value was calculated using Eq. 8.

Eq. 7 Henderson-Hasselbalch Equation

diluted to mark in a 50 mL volumetric flask with distilled water. 0.5442 g of NaOAc.3H2O crystals are dissolved in 100 mL of water to make a 0.040 M NaOAc solution. 12.50 mL of this solution is diluted to 0.010 M by transferring it to a 50 mL volumetric flask and diluting it the solution to mark. 25 mL of 1.0 M HOAc is produced and 1 mL of this solution is transferred to a 50 mL volumetric flask and diluted into mark with distilled water ti give a 0.020 M HOAc solution. The strong acid HCl is also prepared. 50 mL of 0.10 M HCl is produced. 5 mL of this solution is transferred to a 50 mL volumetric flask and diluted to mark using distilled water to obtain a 50 mL 0.010 M solution of HCl. From the standard methyl red solution, 5.00 mL was pipetted and transferred into a 50 mL volumetric flask. 5.00 mL of 0.100 M HCl solution was added and the resulting solution was diluted into mark inside the flask. It was covered and mixed thoroughly. Using a pH meter, the pH of the solution was checked. This solution was labeled SOLUTION HMR and was kept under a pH of approximately 2 to ensure that HMR is the only species of methyl red present in the solution. Again, from the methyl red standard solution, 5.00 mL was pipetted and transferred into a 50 mL volumetric flask. 12.50 mL of the 0.0400 M NaOAc solution was added and the resulting solution was diluted into mark inside the flask. It was covered and mixed thoroughly, and using a pH meter, its pH was also checked. This solution labeled SOLUTION MR-. It was ensured to be kept under a pH of approximately 8 to ensure that MR- dominates in the solution. Solutions containing HMR at different concentrations were prepared in a 50 mL

Eq. 8 The acidic form of methyl red, HMR is red in color and dominates the solution under the pH of 4.4. The basic form of methyl red MR- is yellow and dominates the solution under the pH of 6.2. The acidic form of methyl red is red in color. The expected wavelength of maximum absorption would lie in between 490-540 nm. And theoretically, the value is located at 520 nm. The basic form of methyl red is yellow in color. The expected wavelength of maximum absorption would lie between 400-450 nm. Theoretically, the value is located at 425 nm. The theoretical value for the Ka of methyl red is 3.981 x 10-6. The experimental value obtained is compared with the theoretical value using the relative deviation shown in Eq. 9.

Eq. 9 Relative Deviation

This study aims to quantitatively determine the acid-dissociation constant of methyl red using the principles of spectrophotometry in multicomponent systems. METHODOLOGY The methyl red stock solution is prepared. 0.05000 g of methyl red is dissolved in 30 mL of 95% ethanol in a 150 mL beaker. The solution is transferred to a 50 mL volumetric flask and is diluted to mark with distilled water. The methyl red standard solution is prepared from the stock solution. 2.50 mL of the stock solution is added with 25 mL of 95% ethanol is

volumetric flask and were labeled Solutions 1-3. Varying volumes of HMR and 0.010 M HCl were added for each solution. Table 2 HMR Only Solution Number 0.010 Solution Concentration M HCl HMR mL mL 1 4.96 15.04 1.40E-05 2 10.00 10.00 9.28E-06 3 15.04 4.96 4.60E-06 Solutions containing MR- at different concentrations were prepared in a 50 mL volumetric flask and were labeled as Solutions 4-6. Varying volumes of MR- and 0.010 M NaOAc were added. Table 3 MR- Only Solution 0.010 Solution Concentration Number M MR- mL NaOAc mL 4 4.96 15.04 1.40E-05 5 10.00 10.00 9.28E-06 6 15.04 4.96 4.60E-06 Solutions containing the standard methyl red solution were prepared in 50 to 100 mL. Different volumes of 0.0200 M HOAc and 0.040 M NaOAc were added. These solutions were labeled Solutions 7-10. Table 3 Multicomponent System of HMR and MRSolution Number 7 8 9 10 Std Methyl Red mL 6.00 6.00 6.00 6.00 0.0200 M 0.040 M HOAc NaOAc mL mL 1.20 12.80 2.40 11.60 4.80 9.20 7.20 6.80

using H2O as a reference cell. From the spectra obtained, the wavelengths of maximum absorption for the acidic and basic forms were determined by taking the peak of each plot. The wavelength of maximum absorption at HMR was labeled HMR and the wavelength of maximum absorption for MR- was labeled MR. The absorbance of the solutions 1-10 were obtained at HMR and using water as a reference cell. The pHs of solutions 7-10 were determined using a pH meter. After the experimentation, the glasswares were washed and the equipments were kept. Different reagents used were disposed properly. 95 % ethanol and methyl red solutions were disposed in the Organic Waste Container. NaOAc and the acid solutions were drained and flush into sink with a copious amount of running water. RESULTS AND DISCUSSION The aim of this experiment is to obtain a value for the acid-dissociation constant. For the experiment, two data sets were used. First, the wavelengths of maximum absorption for Solution HMR and Solution MR- were obtained. For the first data set HMR is 520.20 nm and MR is 426.00 nm. For the second data set HMR is 519.80 nm and MR is 433.40 nm. An example absorption spectrum is shown by Figure 1. Second, a calibration curve for the HMRcontaining and MRcontaining solutions were obtained to determine the molar absorptivity constant of each species at the two wavelengths of maximum absorption. Graphs 1-8 shows the calibration curves of each species at the two wavelengths of maximum absorption for the data sets provided.

Using a double-beam UV-Vis Spectrophotometer, the absorption spectra of Solution HMR and Solution MR- were obtained

Figure 1: Sample Absorption Spectrum: Green line: [HMR] Black line: [MR-]

DATA SET 1

0.3

Graph 1[HMR] vs Abs. at HMR

y = 19025x + 0.0031 R = 0.9998

Graph 3[MR-] vs Abs. at HMR

0.018 0.016 0.014 0.012 Absorbace 0.01 0.008 0.006 0.004 0.002 y = 1175.7x - 0.0006 R = 0.9972

0.25 0.2 0.15 0.1 0.05

Absorbance

0 0.00E+00

5.00E-06

1.00E-05

1.50E-05

0 0.00E+00

5.00E-06

1.00E-05

1.50E-05

[HMR]

[MR-]

Graph 2[HMR] vs. Abs at MR0.035 0.03 0.025 Absorbance 0.02 0.015 0.01 0.005 0 0.00E+00 Absorbance y = 2244.5x - 0.0015 R = 0.9992

Graph 4[MR-] vs Abs. at MR0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0.00E+00 y = 9191.9x - 0.0057 R = 0.9998

5.00E-06

1.00E-05

1.50E-05

5.00E-06

1.00E-05

1.50E-05

[HMR]

[MR-]

DATA SET 2

Graph 5[HMR] vs Abs. HMR

0.4 0.35 0.3 Absorbance Absorbance 0.25 0.2 0.15 0.1 0.05 0 0.00E+00 y = 25545x + 0.0005 R = 1

Graph 7[MR-] vs Abs. HMR

0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 0.00E+00

y = 1496.5x + 0.0094 R = 0.7501

5.00E-06

1.00E-05

1.50E-05

5.00E-06

1.00E-05

1.50E-05

[HMR]

[MR-]

Graph 6[HMR] vs Abs. MR0.08 0.07 0.06 Absorbance 0.05 0.04 0.03 0.02 0.01 0 0.00E+00 5.00E-06 1.00E-05 1.50E-05 y = 4916.6x - 0.0016 R = 1 Absorbance

Graph 8[MR-] vs Abs. at MR0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0.00E+00 5.00E-06 [MR-] 1.00E-05 1.50E-05 y = 10902x + 0.0088 R = 0.9954

[HMR]

The trendline of each graph can be written in the form y=mx+b. y corresponds to the absorbance, x corresponds to the concentration of the solution and b gives the indeterminate error from the analysis. The slope m of the graph is the molar absorptivity multiplied by the path length. Since the path length equals 1 cm, the value of m gives the molar absorptivity for a particular species at a particular wavelength. Recalling Eq. 5 and 6, the system of equation can be solved to obtain the concentration of HMR and MR- for each of Solutions 7-10. At HMR

The pHs of the solutions were plotted against the logarithm of the ratio of the concentration of MR- with HMR. The trend line was taken, and the least squares y-intercept b was taken to be the pKa. For each solution, we could also determine the pKa values using the Henderson-Hasselbalch Equation. This is provided b Tables 6 and 8. Eq. 7 Henderson-Hasselbalch Equation

Eq. 8

At MR-

Table 6 pKa and Ka Data Set 1 Solution Number 7 8 9 10 pKa 5.617 5.166 5.331 5.337 Ka 2.414E-6 6.828E-6 4.666E-6 4.607E-6

Cramers rule was used to solve the system of equations. Given equations ax+by=c and dx+ey=f, the determinant D is given by ae-bd. The value of x is determined by (ce-fb)/D and the value of y is determined by(af-dc)/D. Tables 4 and 5 give the value of the calculated molarities of HMR and MR-. Table 4 [HMR] and [MR-] Data Set 1 Solution 7 8 9 10 [HMR] 1.363E-5 1.391E-5 2.690E-5 3.044E-5 [MR-] 5.214E-5 5.991E-5 4.998E-5 2.798E-5

Table 6 pKa and Ka Data Set 2 Solution Number 7 8 9 10 pKa 6.0125 5.864 5.170 5.207 Ka 9.716E-7 1.368E-6 6.755E-6 6.213E-6

Table 5 [HMR] and [MR-] Data Set 2 Solution 7 8 9 10 [HMR] 2.888E-5 5.971E-5 1.610E-5 2.039E-5 [MR-] 4.420E-5 5.156E-5 4.329E-5 2.527E-5

Graphs 9 and 10 show the plot of pH against log([MR-]/[HMR]) for data sets 1 and 2, respectively.

Graph 9: pH vs log ([MR-]/[HMR] Data Set 1

6.3 6.2 6.1 6 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 0 y = 0.8031x + 5.434 R = 0.4381 6.3 6.2 6.1 6 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 0

Graph 10 pH vs log([MR-]/[HMR] Data Set 2

y = 0.243x + 5.6423 R = 0.0084

pH

0.2

0.4 log{[MR-]/[HMR]

0.6

0.8

pH

0.1

0.2

0.3

0.4

0.5

log{[MR-]/[HMR]

The first data set produced a pKa value of 5.434 and Ka value of 4.542 x 10-6. The second data set produces a pKa value of 5.6423 and Ka value of 2.279 x10-6. The average value or the mean of the two data sets were obtained using Eq. 10 Eq. 10 Mean An average value of 3.412 x 10-6 was calculated as the Ka produced by the experiment. The precision of each data set was obtained. The standard deviation is calculated for each set using Eq. 11.

Eq. 12 Range

The range of the values is 2.263 x 10-3. The produced standard deviation of the experiment is small. This means that the method used for the analysis can be said to be precise. Next, the accuracy of the values obtained was tested. The relative deviations of the experimental values for Ka were taken using Eq. 9. The theoretical value for the acid-dissociation constant of methyl red is 3.981E-6.

Eq. 9 Relative Deviation

Eq.11 Standard Deviation A standard deviation of 1.600 x 10-6 was obtained. The range of each data set was also obtained using Eq. 12

The average Ka value of the experiment gives a relative deviation of 14.29 %. Therefore, the method used for the determination of the aciddissociation constant of methyl red is said to be accurate. There are various sources of errors that can be encountered in the experiment. The use of marched cells for the experiment is important. If the cells holding the analyte and

blank solutions are not of equal path length and equivalent in optical characteristics, an intercept will occur in the calibration curve. This intercept value indicates the amount of indeterminate error. A source of error could be the resolution of the spectrophotometer which is limited by the purity and intensity of the monochromatic light and the sensitivity of the instrument at a certain wavelength. To avoid such deviations, the instrument is set at a wavelength band near the wavelength of maximum absorption so that molar absorptivity changes with wavelength. The intensity of the monochromatic light can also be ensured by checking up the power supply of the spectrophotometer. Another source of errors is the presence of stray radiation. It arises from the instrument as a result of scattering and reflection off surfaces of gratings, lenses or mirrors, filters and windows. This error makes the absorbance of the sample be less than the expected value, most significantly at high absorbance values. Filter instruments could minimize the errors from this cause. Ensuring that the cuvette is transparent and has no unnecessary marks like thumb marks on it can be also be done minimize its deviations. The temperature, to which the reading of the absorbance was carried out, needs to be maintained constant. This is due to the fact that a variation in temperature could cause a change in the value of any equilibrium constant by the vant Hoff equation. Eq. 13 Vant Hoff Equation ( )

solution should have been boiled to avoid interferences in the analyte. In the preparation of the solutions analyzed for spectrophotometry, some volumes are very fractional such 4.96 mL and 15.04 mL that were just taken into estimates. These estimations may have led into error. Judging from the values obtained, the accuracy and precision of the measured values can be considered significant. Hence, the values obtained are reliable. CONCLUSION AND RECOMMENDATION The aim of the experiment is the quantitative determination of the acid-dissociation constant of the indicator, methyl red using multicomponent method of spectrophotometric analysis. The first data set produced a pKa value of 5.434 and Ka value of 4.542 x 10-6. The second data set produces a pKa value of 5.6423 and Ka value of 2.279 x10-6. An average value of 3.412 x 10-6 was calculated as the Ka produced by the experiment. In testing the reliability of the measured values, the standard deviations were computed. A standard deviation of 1.600 x 10-6 was obtained. The calculated value for the standard deviations is small, concluding that the values produced are close to one another. Hence, the precision of the method of analysis is concluded. Other methods for the determination of the aciddissociation constant of methyl red could also be used. One method that could be employed is potentiometric titration. This type of analysis provides more reliable data than titrations using indicators, especially for colored and turbid solutions. Here, the potential of suitable indicator electrode is measured as a function of volume. A pH meter could be used here to directly determine pH since the analyte is an acid.

In the preparation of the methyl red stock solution, the solid must be ensured to have completely dissolved in the solution to avoid errors in the concentration of the samples. The NaOAc solution is basic and may contain carbonates that induce carbonate error. This

Calorimetric methods employing isothermal systems could also be employed for the aciddissociation constant of methyl red. REFERENCES [1] Skoog, Douglas, et. al. Analytical Chemistry: An Introduction, Thomson Learning Asia. 2000 [2] D.C. Harris, Quantitative Chemical Analysis, 7th ed. New York: W.H. Freeman and Company, 2007. [3]Institute of Chemistry. Quantitative Inorganic Analysis Laboratory Manual.University of the Philippines-Diliman. 2007 [4] . 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. APPENDICES WORKING EQUATIONS

Eq. 5 and 6 At HMR

At MR-

Eq. 7 Henderson-Hasselbalch Equation

Eq. 8

Eq. 9 Relative Deviation

Eq. 10 Mean

Eq.11 Standard Deviation Eq. 1 Transmittance of a Solution Eq. 12 Range

Eq. 2 Absorbance of a Solution

Eq. 3 Beer-Lamberts Law

Eq. 13 Vant Hoff Equation ( )

Eq. 4 Absorbance of Multicomponent System