Spectroscopy and PNP

CHE315 Section 03 Professor Submitted By: Submitted to: October 21, 2011

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RESULTS
Part I. Measuring the Absorbance Spectra of the Acidic and Basic Forms of para-Nitrophenol (PNP)
Part A: Absolute Spectra for Acidic (phenol) and Basic (phenolate) PNP
An absolute spectra was generated for the acidic and basic form of PNP. See Fig. 1 and Fig. 2. See Table 1 for the absorbance data results and Table 2 for the absorbance mean values of the data results. See the Appendix for detailed calculations. The data results for the acidic PNP absolute spectra shows a max of 360 nm at an absorbance of 0.315 and the data results for the basic PNP absolute spectra shows a max of 400 nm at an absorbance of 0.515. PNP + pH10 Abs. 0,1183 0.178 0.237 0.317 0.412 0.486 0.515 0.49 0.0412 0.291 0.019 0.101 0.04 0.07 0 0.01 0.012 PNP + pH5 Abs. 0.248 0.034 0.135 0.086 0.065 0.039 0.034 0.0135 0.033 0.024 0.0215 0.025 0.024 0.022 0.018 0.017 0.024 Readings PNP + pH10 Readings PNP + pH10

340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

1 2 3

0.081 0.084 0.083

1 2 3

0.081 0.084 0.083

Table 2: Absolute Spectra Means Data Results of acidic and basic PNP

Table 1: Absolute Spectra Data Results of acidic and basic PNP

0.3 0.275 0.25 Absorbance (360 nm) 0.225 0.2 0.175 0.15 0.125 0.1 0.075 0.05 0.025 0 340 360 380 400 420 440 460 480 500 Wavelength (nm) max = 360 nm Abs. = 0.135 Absorbance (360 nm) 0.525 0.5 0.475 0.45 0.425 0.4 0.375 0.35 0.325 0.3 0.275 0.25 0.225 0.2 0.175 0.15 0.125 0.1 0.075 0.05 0.025 0 340 360 380 400 420

max = 400 nm Abs. = 0.515

440

460

480

500

Wavelength (nm)

Figure 1: Absolute Spectra for Acidic PNP

Figure 2: Absolute Spectra for Basic PNP

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A plot of the acidic spectra and the basic spectra on the same plot determined the pI to be 345 nm. See Fig. 3.
0.6 0.57 0.54 0.51 0.48 0.45 0.42 0.39 0.36 0.33 0.3 0.27 0.24 0.21 0.18 0.15 0.12 0.09 0.06 0.03 0 340 360

max = 400 nm Absorbanc e= 0.515

Absorbance (360 nm)

Basic PNP Form

Isobestic Point = 345 nm

Acidic For PNP

380

400

420

440

460

480

500

Wavelength (nm)

Figure 3: Absolute Spectra for Acidic and Basic PNP Part B: Basic - Acidic Difference Spectrum
A basic-acidic difference spectra was generated, see Fig. 4, using the acidic form of PNP as the reference and generated a spectra of the basic form of PNP. See Table 3 for the basic-acidic difference spectra data results and Table 4 for the mean value of the data results. See Appendix for detailed calculations. The data results for the acidic difference absolute spectra shows a max of 400 nm at an absorbance of 0.515.

(nm) 340

350

360

370

380

390

400

410

420

Abs. 0 0 0 0 0 0 0.104 0.104 0.104 0.246 0.246 0.246 0,378 0,378 0,378 0.471 0.471 0.471 0.506 0.508 0.507 0.486 0.486 0,486 0.415 0.421

(nm) 340 350 360 370 380 390 400 410 410 420 430 440 450 460 470 490 500

Abs. 0 0.104 0.246 0.378 0.471 0.507 0.486 0.486 0.417 0.304 0.206 0.119 0.06 0.026 0.083 0.013
Absorbance

0.54 0.5 0.46 0.42 0.38 0.34 0.3 0.26 0.22 0.18 0.14 0.1 0.06 0.02 -0.02

max = 400 nm Abs= 0.515

320

340

360

380

400

420

440

460

480

500

Wavelength (nm)

Figure 4: Basic-Acidic Difference Spectra of PNP

500 0.013 Table 4: Basic - Acidic Difference Mean Data Results

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0.416 0.303 0.303 0.306 0.206 0.207 0.206 0.119 0.119 0.119 0.066 0.057 0.057 0.022 0.025 0.026 0.011 0.013 0.014 0.008 0.009 0.008 0.012 0.013 0.013

430

440

450

460

470

480

490

500

Table 3: Basic-Acidic Difference Spectra of PNP Data Results

Part II. Optical Assay of PNP

Part A. Determination of the Extinction Coefficient for PNP(O -): Generation and Use of a Concentration Curve
An Optical Assay of PNP was generated, see Fig. 5, by measuring absorbance values of 5 different concentrations of PNP and dH2O. From this data, the extinction coefficient, , was determined. See Table 5 and 6 for data values for the basic-acidic difference spectra data results and Table 4 for the mean values of the data. See Appendix for detailed calculations. The data results for the optical assay of PNP gives a linear equation of y = 0.0041x and an R2 of 0.5523.

max=360 nm 10 M 20 M 30 M 50 M 75 M

Absorbance Readings 1 0.008 0.021 0.025 0.027 0.048 2 0.008 0.024 0.025 0.027 0.048 3 0.009 0.021 0.025 0.027 0.048

Abs. 10 20 30 50 75

Conc. (g/L) 0.0083 0.022 0.025 0.027 0.48

Table 5: Optical Assay of PNP data results

Table 6: Optical Assay of PNP mean values of data results

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0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 10 20 30 40

Abs. (nm) (360 nm)

y = 0.0041x R = 0.5523

y = mx y = max m = slope x = [PNP] 70 80

50

60

PNP [M]

Figure 5: Optical Assay of PNP Part B. Determination of an Unknown Concentration of PNP Solution
Absorbance measurements of an unknown concentration PNP sample C in solution yielded a concentration of 8.915 x 10-9 M. See Table 7 for data results and Table 7 for mean of data results. See Appendix for detailed calculations.

=360 nm Readings 1 2 3 Mean

Unknown "C"
Abs. 0.177 0.177 0.178 0.1773

Table 7: Absorbance data results of an Unknown Concentration of PNP Solution

Part III. Determine the pKa of PNP

A plot of absorbance as a function of pH, see Fig. 6, was generated by taking absorbance values of 8 different PNP solutions that were at 8 different pH levels. See Table 6 and 7 for data values for the basic-acidic difference spectra data results and Table 4 for the mean values of the data. See Appendix for detailed calculations. The data results show that as pH increases, absorbancy at max
increases.

pH 5

7.5

8.5

10

Abs. (nm) 0.052 0.051 0.051 0.095 0.094 0.095 0.15 0.149 0.149 0.16 0.161 0.16 0.174 0.175 0.173 0.197 0.195 0.195 0.211 0.212 0.212 0.251 0.251 0.251

pH 5 6 7 7.5 8 8.5 9 10

Abs. (nm) (mean) 0.051 0.095 0.149 0.16 0.174 0.196 0.212 0.251

Table 6: Absorbance as a function of pH mean data results

Table 6: Absorbance as a function of pH data results

pKa of PNP
0.3 Absorbance (360 nm) 0.25 0.2 0.15 0.1 0.05 0 5 6 7 pH 8 9 10

Figure 6: Plot of absorbance as a function of pH

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DISCUSSION This experiment used UV-Visible spectrophotometry (UV/VIS) to study spectral properties of a chemical compound, namely para-nitrophenol (PNP), by exploiting its intrinsic physical properties and observing the its interactions with different wavelengths of light. The manner in which the compound reacts to the treatment of different UV/VIS wavelengths reveals information that can be quantitatively and qualitatively analyzed for determination and identification of chemical compounds and biochemical species. The overall objectives of this experiment was to study how PNPs spectral properties changed as a function of pH. The relationship of the interaction of a chemical species with wavelengths of light is explained by the Beer-Lambert law. It expresses the linear relationship between absorbance and concentration of a chemical species interacting with and absorbing UV/VIS wavelengths with the equation A = ()(c)(l) where (A) is the absorbance at any wavelength, is the molar absorptivity/extinction coefficient at a specific wavelength (), c is concentration of the chemical species and l is the path length of the sample (usually 1 cm). use this equation for the determination of the concentration of material in a solution, for the calculation of the expected absorbance of a known concentration of the sample solution, or for the calculation of the value of for a chemical species. The first task was creating and looking at PNPs absolute spectra in its basic (unprotonated) and acidic (protonated) form. Then PNPs difference spectra was created and compared to the basic-acidic absolute spectra. The acidic and basic absolute spectra demonstrated classic behavior for absorbance by the two species and agrees with theory. If there is an increase in pH, concentration of H+ decreases, absorbance increases and a higher pH solutions absorb at high wavelengths. If there is a decrease in pH, concentration of H+ increases, absorbance decreases and lower pH solutions absorb light at lower wavelengths. The acidic absolute spectra showed a max at 360 nm at an absorbance of 0.135. Whereas the basic absolute spectra showed a max at 400 nm at an absorbance of 0.515. A plot of the absolute acidic absolute spectra and the basic spectra on the same plot determined the isobestic point, or pI, to be 345 nm. The basic-acidic difference plot confirms agreement with theory that the basic and acidic spectra behaves as they should in the specified environment. Comparing these two spectra to the acidic-basic difference spectra. Comparing the absolute basic-acidic spectra to the difference spectra shows that where the isobestic point for the Absolute Basic spectra is where the wavelength of the difference spectra fell below zero. Thus, at pI, when molar absorptivity () was the same for both species, wavelength and concentration stayed constant, the reactions net electric charge was zero on the absolute spectra, Abs=0 on the acidic difference spectra. Also, the absorbance is 0.51 at max for the difference spectra and the absolute basic spectra. Additionally, at 480 nm, when the absorbance of the basic species went to zero on the absolute spectra, the absorbance on the difference spectra increased by approximately 0.06 of and then decreased to zero, while the basic species on the absolute species increased a small amount. The relationship between the Beer-Lambert and the shape and absorbance values of the difference spectra range explains why some absorbance values are greater than 0, some are less than 0, and why at one wavelength A = 0: At every wavelength there is a difference in absorbance, A: A at every = A,PNPO- - A,PNPOH A at every = (,PNPO-)([PNPO-])(L) - (,PNPOH)([PNPOH)(L) If [PNPO-] = [PNPOH] then A at every = ( PNPO- - PNPOH)([PNPO-])(L) A at every = ( )([PNPO-])(L) thus,

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When PNPO- > PNPOH, then A > 0 When PNPO- = PNPOH- then A = 0 When PNPO- < PNPOH- then A < 0 The difference of [PNPOH] (initial state) and [PNPO-] (final state) is what is changing at each wavelength. Clearly the acidic-basic difference spectra demonstrates agreement with Beer-Lambert law and shows how pH is a function of wavelength. Generating an optical assay of PNP allowed for the determination of the extinction coefficient for PNP(O-) which in turn, unknown concentration can be calculated. The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. The data results for the optical assay of PNP gives a linear equation of y = 0.0041x and an R2 of 0.5523 from which the extinction coefficient, , was calculated and was found to be 2 x 10-9. The data yielded usable result from which the unknown concentration of the PNP solution was calculated at 8.915 x 10-9 The linear equation generated from the optical assay shows the linear relationship between absorbance and concentration and is in accordance with the Beer-Lambert law and the R2 value is within acceptable range, with the closer to 1 the better. Overall, this experiment was successful and accomplished the objectives set by the lab manual. The data results were not indicative of any errors results (spectrophotometer, cuvettes, pipettes, operator error, etc.) that caused noticeable deviations from the expected, with the exception of the unknown concentration of the PNP solution.

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OBSERVATIONS

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APPENDIX Questions from Lab Manual for Spectroscopy and PNP
Part I. Measuring the Absorbance Spectra of the Acidic and Basic Forms of para-Nitrophenol (PNP) Part A: Absolute Spectra for Acidic (phenol) and Basic (phenolate) PNP
Step 10: The isobestic point, or Ip, in a reaction with a single species and a reagent is the point where the removal of the first proton is almost complete and the removal of the second proton has just began. The compound is present mostly as a zwitterions and its net electric charge is zero. It is the mean of pK1 and pK2 and gives the pH at the pI. When two chemical species are present they absorb light at a specific wavelength to the same degree. Also at that point, the absorbance of the two species at the specific wavelength stays constant, thus being related linearly by stoichiometry, such that the absorbance is the same for a particular wavelength and the concentrations are constant. Additionally, it is a point where at a specific wavelength, two species have the same molar absorptivity (). This can be useful to verify accuracy of a wavelength of a spectrometer. The spectra of a compound is measured at two different pH values, one above the pKa of the compound and one below the pKa of the compound. Use of isosbestic points have many practical applications. They can be used as reference points in chemical kinetics for the study of reaction rates because the absorbance at the wavelengths used are constant throughout the entire reaction. They can also be used to verify the accuracy of the wavelength of a spectrophotometer by measuring the spectra of a standard substance at two different pH conditions, one above the pKa of the substance and below).

Part I Part B. Basic Acidic Difference Spectrum

Step #5: The difference spectra resembles the absolute spectra. Where the isobestic point for the Absolute Spectra for Basic and Acidic PNP is where the wavelength of the difference spectra fell below zero. Thus, at pI, when molar absorptivity () was the same for both species, wavelength and concentration stayed constant, the reactions net electric charge was zero on the absolute spectra, Abs=0 on the acidic difference spectra. The absorbance is 0.51 at max for both spectra. At 480 nm, when the absorbance of the basic species went to zero on the absolute spectra, the absorbance on the difference spectra increased by approximately 0.06 of and then decreased to zero, while the basic species on the absolute species increased a small amount. Step #6: At every wavelength there is a difference in absorbance, A: A at every = A,PNPO- - A,PNPOH A at every = (,PNPO-)([PNPO-])(L) - (,PNPOH)([PNPOH)(L) If [PNPO-] = [PNPOH] then A at every = ( PNPO- - PNPOH)([PNPO-])(L) A at every = ( )([PNPO-])(L) thus, When PNPO- > PNPOH, then A > 0 When PNPO- = PNPOH- then A = 0 When PNPO- < PNPOH- then A < 0 The difference of [PNPOH] (initial state) and [PNPO-] (final state) is what is changing at each wavelength.

Part II. Optical Assay of PNP Part A Determination of the Extinction Coefficient for PNP(O -): Generation and Use of a Concentration Curve
Step 5: Calculate the mean and the standard deviation: Mean Calculation of absorbance values Equation used: (Reading 1 + Reading 2 +Reading 3) / Total # of Readings Sample Calculation from Raw Data: Readings Conc. 10 M 1 0.008 2 0.008 3 0.009

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0.008 + 0.008 + 0.009 = 0.0083 0.0083/ 3 = 0.0027= Mean Standard Deviation Calculation Equation used:

Sample Calculation from Raw Data: Readings Conc. 10 M ( 1 0.008 ) ( Readings 1 Conc. ( M) 10 M 20 M 30 M 50 M 75 M 0.008 0.021 0.025 0.027 0.048 0.008 0.024 0.025 0.027 0.048 0.009 0.021 0.025 0.027 0.048 0.00833 0.022 0.025 0.027 0.048 0.00058 0.00173 0 0 0 2 3 Mean SD 2 0.008 ) ( 3 0.009 Mean 0.00833 ) 0.00058

max=360nm

Step 8: Determine the extinction coefficient () from your plot: From the Optical Assay of PNP graph: y = mx y = 0.004x 360 nm = (0.004)(x) x = 9 x 10-5 M Apply to Beer-Lambert equation: A= ()(c)(l) = A/cl .0018 = ()([9 x 10-5 ])(1) = 2 x 10-9 The extinction coefficient is a measure of how strongly the sample absorbs light at a specific wavelength. A linear regression determined the slope and the y-intercept of the "line of best fit" from which the extinction coefficient was calculated. The linear regression "captured" the data trend of the scattered data plot that does not look linear. Linear regression linearized the data and determined the best fit function of the original data. Forcing the fit through the origin, zero, helps the Linear regression function to find the line that minimizes the sum of the squares of the vertical distances of the points from the line. R2 shows how close the measuring points are to a straight line, i.e., best fit.

Part B Determination of an Unknown Concentration of PNP Solution Using the standard curve
Step #4: Calculate the concentration of the unknown PNP that is in the cell (the 4 mL) diluted solution)

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Absorbances of PNP of an Unknown Concentration of PNP Solution Readings (max = 360 nm) 2 0.177

1 0.177

3 0.178

Mean 0.1783

From Concentration Standard Concentration Curve: = 2 x 10-9 Using Beer-Lambert Equation, solve for concentration, c: A= ()(c)(l) c=A/ c = 0.1783 / 2 x 10-9 = 8.915 x 10-9 Step #5: Calculate the concentration of the original unknown that was used (the PNP that the sample was made from ). Sample: PNP + pH10 buffer = 330 nm Readings PNP + pH10 buffer

1 2 3 Mean

0.081 0.084 0.083 0.826

c=A/ c = 0.826 / 2 x 10-9 = 4.13 x 10-10 Sample: PNP + pH5 buffer = 330 nm Readings PNP + pH10 buffer

1 2 3 Mean

0.3 0.299 0.299 0.2993

c=A/ c = 0.2993 / 2 x 10-9 = 1.50 x 10-10

Part III. Determine the pKa of PNP

Means of raw data: pH 5 6 7 7.5 8 8.5 Abs. (nm) (mean) 0.051 0.095 0.149 0.16 0.174 0.196

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9 10 0.212 0.251

If there is an increase in pH, concentration of H+ decreases, absorbance increases and higher pH solutions absorb at high wavelengths. If there is a decrease in pH, concentration of H+ increases, absorbance decreases and lower pH solutions absorb light at lower wavelengths. Le Chatelier's principle: When acid is added, the species is being protonated and pH decreases and equilibrium is forced to the left. When base is added, the species is being deprotonated and pH increases and equilibrium is forced to the right.

CALCULATIONS Part I. Part A: Means of raw data for Absolute Spectra for Basic and Acidic Form of PNP Sample Calculation:
max = 360 nm Readings 1 2 3 PNP + pH10 (Abs.) 0.117 0.119 0.119

0.117 + 0.119 + 0.119 = 0.355 0.355 / 3 = 0.118

Part II. Optical Assay of PNP Part A Determination of the Extinction Coefficient for PNP(O-): Generation and Use of a Concentration Curve
Calculation for preparation of PNP solutions of different concentrations: Equation Used: (M1)(V1)=(M2)(V2) Sample Calculation to prepare a 2 mL 10 M PNP solution 0.1 mM stock: Conc. (M) 10 Stock PNP (M) 100 dH20 (mL) V1 = Sample PNP Sample Conc. Volume (mM) (L) 10 0.0002 Stock Soln. (mL) 0.2 dH20 (mL) 1.8

Solve for x. x = 0.2 mL to which 1.8 mL dH20 was added to the 0.2 mL PNP for a total volume of 2.0 mL

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References Biochemistry 315 Laboratory Manual. John Jay College of Criminal Justice. Spectroscopy and PNP Skoog, West, Holler, Crouch, 2004. Fundementals of Analytical Chemistry, Sixth Edition. Thomson Brooks/Cole . India. pp. 710-730 and 784-810. Engel, Thomas, 2005. Quantum Chemistry and Spectroscopy, First Edition. Benjamin Cummings. L. G., Jr. Wade, 2005, Organic Chemistry, Pearson Prentice Hall. pp. 692-697.