Spectroscopy and PNP

CHE315Section 03Professor

Submitted By: Submitted to:

October 21, 2011

2

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) PNPAn 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 λ max of 360 nm at an absorbance of 0.315 and the data results for the basic PNP absolute spectra shows a λ max of 400 nm at an absorbance of 0.515.

PNP + pH10 PNP + pH5λ Abs. λ Abs.340 0,1183 340 0.248350 0.178 350 0.034360 0.237 360 0.135370 0.317 370 0.086380 0.412 380 0.065390 0.486 390 0.039400 0.515 400 0.034410 0.49 410 0.0135420 0.0412 420 0.033430 0.291 430 0.024440 0.019 440 0.0215450 0.101 450 0.025460 0.04 460 0.024470 0.07 470 0.022480 0 480 0.018490 0.01 490 0.017500 0.012 500 0.024

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

Readings PNP + pH10 Readings PNP + pH10

1 0.081 1 0.081

2 0.084 2 0.084

3 0.083 3 0.083

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

340 360 380 400 420 440 460 480 5000

0.025

0.05

0.075

0.1

0.125

0.15

0.175

0.2

0.225

0.25

0.275

0.3

Wavelength (nm)

Abs

orba

nce

(360

nm

)

λmax = 360 nmAbs. = 0.135

340 360 380 400 420 440 460 480 5000

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Wavelength (nm)

Abs

orba

nce

(360

nm

)

Figure 1: Absolute Spectra for Acidic PNP Figure 2: Absolute Spectra for Basic PNP

λmax = 400 nmAbs. = 0.515

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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.

340 360 380 400 420 440 460 480 5000

0.06

0.12

0.18

0.24

0.3

0.36

0.42

0.48

0.54

0.6

Basic PNP Form

Acidic For PNP

Wavelength (nm)

Abs

orba

nce

(360

nm

)

Isobestic Point = 345 nm

λmax = 400 nmAbsorbanc e= 0.515

Figure 3: Absolute Spectra for Acidic and Basic PNP

Part B: Basic - Acidic Difference SpectrumA 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 λ max of 400 nm at an absorbance of 0.515.

λ (nm) Abs.340 0

00

350 000

360 0.1040.1040.104

370 0.2460.2460.246

380 0,3780,3780,378

390 0.4710.4710.471

400 0.5060.5080.507

410 0.4860.4860,486

420 0.4150.421

λ (nm) Abs.340 0350 0360 0.104370 0.246380 0.378390 0.471400 0.507410 0.486410 0.486420 0.417430 0.304440 0.206450 0.119460 0.06470 0.026490 0.083500 0.013500 0.013

Table 4: Basic - Acidic Difference Mean Data Results

320 340 360 380 400 420 440 460 480 500-0.020.020.06

0.10.140.180.220.26

0.30.340.380.420.46

0.50.54

Wavelength (nm)

Δ A

bsor

banc

e

Figure 4: Basic-Acidic Difference Spectra of PNP

λmax = 400 nmAbs= 0.515

4

0.416430 0.303

0.3030.306

440 0.2060.2070.206

450 0.1190.1190.119

460 0.0660.0570.057

470 0.0220.0250.026

480 0.0110.0130.014

490 0.0080.0090.008

500 0.0120.0130.013

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

Part II. Optical Assay of PNPPart A. Determination of the Extinction Coefficient for PNP(O-): Generation and Use of a Concentration

CurveAn 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, ε, 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.

λmax=360 nm

Absorbance Readings

1 2 310 μM 0.008 0.008 0.00920 μM 0.021 0.024 0.02130 μM 0.025 0.025 0.02550 μM 0.027 0.027 0.02775 μM 0.048 0.048 0.048Table 5: Optical Assay of PNP data results

Abs. Conc. (g/L)10 0.008320 0.02230 0.02550 0.02775 0.48

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

5

0 10 20 30 40 50 60 70 800

0.1

0.2

0.3

0.4

0.5

0.6

f(x) = 0.00405490813648294 xR² = 0.674163030443826

PNP [μM]

Abs

. (nm

) (36

0 nm

)

y = mx y = λmaxm = slopex = [PNP]

Figure 5: Optical Assay of PNP

Part B. Determination of an Unknown Concentration of PNP SolutionAbsorbance 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.

λ=360 nm Unknown "C"Readings Abs.

1 0.1772 0.1773 0.178

Mean 0.1773Table 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 λmax increases.

pH Abs. (nm) 5 0.052 0.051 0.0516 0.095 0.094 0.0957 0.15 0.149 0.1497.5 0.16 0.161 0.168 0.174 0.175 0.1738.5 0.197 0.195 0.1959 0.211 0.212 0.21210 0.251 0.251 0.251

pH

Abs. (nm) (mean)

5 0.0516 0.0957 0.1497.5 0.168 0.1748.5 0.1969 0.21210 0.251

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

Table 6: Absorbance as a function of pH data results

6

5 6 7 8 9 10-4.16333634234434E-17

0.05

0.1

0.15

0.2

0.25

0.3

pKa of PNP

pH

Abso

rban

ce (3

60 nm

)

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 PNP’s 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 Aλ = (ελ)(c)(l) where (A) is the absorbance at any wavelength, ελ is the molar absorptivity/extinction coefficient at a specific wavelength (λ), 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 ελ for a chemical species.

The first task was creating and looking at PNP’s absolute spectra in its basic (unprotonated) and acidic (protonated) form. Then PNP’s 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 λmax at 360 nm at an absorbance of 0.135. Whereas the basic absolute spectra showed a λmax 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 (ε) was the same for both species, wavelength and concentration stayed constant, the reaction’s net electric charge was zero on the absolute spectra, ΔAbs=0 on the acidic difference spectra. Also, the absorbance is 0.51 at λmax 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 ΔA = 0:

At every wavelength there is a difference in absorbance, ΔA:ΔA at every λ = Aλ,PNPO- - Aλ,PNPOH

ΔA at every λ = (ελ,PNPO-)([PNPO-])(L) - (ελ,PNPOH)([PNPOH)(L)If [PNPO-] = [PNPOH] then ΔA at every λ = (ε λPNPO- - ε λPNPOH)([PNPO-])(L)ΔA at every λ = (Δ ε λ)([PNPO-])(L)thus,

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When ε λPNPO- > ε λPNPOH, then ΔA > 0When ε λPNPO- = ε λPNPOH- then ΔA = 0When ε λPNPO- < ε λPNPOH- then ΔA < 0The 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, ε, 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.

9

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) PNPStep 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 (ε). 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 IPart B. Basic – Acidic Difference SpectrumStep #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 (ε) was the same for both species, wavelength and concentration stayed constant, the reaction’s net electric charge was zero on the absolute spectra, ΔAbs=0 on the acidic difference spectra.

The absorbance is 0.51 at λmax 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, ΔA:ΔA at every λ = Aλ,PNPO- - Aλ,PNPOH

ΔA at every λ = (ελ,PNPO-)([PNPO-])(L) - (ελ,PNPOH)([PNPOH)(L)If [PNPO-] = [PNPOH] then ΔA at every λ = (ε λPNPO- - ε λPNPOH)([PNPO-])(L)ΔA at every λ = (Δ ε λ)([PNPO-])(L)thus,When ε λPNPO- > ε λPNPOH, then ΔA > 0When ε λPNPO- = ε λPNPOH- then ΔA = 0When ε λPNPO- < ε λPNPOH- then ΔA < 0The difference of [PNPOH] (initial state) and [PNPO-] (final state) is what is changing at each wavelength.

Part II. Optical Assay of PNPPart 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 valuesEquation used: (Reading 1 + Reading 2 +Reading 3) / Total # of ReadingsSample Calculation from Raw Data:

ReadingsConc. 1 2 3

10 μM 0.008 0.008 0.009

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0.008 + 0.008 + 0.009 = 0.00830.0083/ 3 = 0.0027= Mean

Standard Deviation CalculationEquation used:

Sample Calculation from Raw Data:

ReadingsConc. 1 2 3 Mean

10 μM 0.008 0.008 0.009 0.00833

σ=√ (0.008−0.0083 ) ²+¿ (0.008−0.0083 ) ²+(0.009−0.0083 ) ² /3=¿¿0.00058

λmax=360nmReadings

Conc.( μM)

1 2 3 Mean SD

10 μM 0.008 0.008 0.009 0.00833 0.00058

20 μM 0.021 0.024 0.021 0.022 0.00173

30 μM 0.025 0.025 0.025 0.025 0

50 μM 0.027 0.027 0.027 0.027 0

75 μM 0.048 0.048 0.048 0.048 0

Step 8: Determine the extinction coefficient (ε) from your plot:From the Optical Assay of PNP graph:y = mxy = 0.004x360 nm = (0.004)(x)x = 9 x 10-5 MApply to Beer-Lambert equation:A= (ε)(c)(l)ε = A/cl.0018 = (ε)([9 x 10-5 ])(1)ε = 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 SolutionUsing 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 (λmax = 360 nm)

1 2 3 Mean

0.177 0.177 0.178 0.1783

From Concentration Standard Concentration Curve: ε = 2 x 10-9

Using Beer-Lambert Equation, solve for concentration, c:A= (ε)(c)(l)c = A / εc = 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 bufferλ = 330 nm

Readings PNP + pH10 buffer

1 0.081

2 0.084

3 0.083

Mean 0.826

c = A / εc = 0.826 / 2 x 10-9 = 4.13 x 10-10

Sample: PNP + pH5 bufferλ = 330 nm

Readings PNP + pH10 buffer

1 0.3

2 0.299

3 0.299

Mean 0.2993

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

Part III. Determine the pKa of PNP

Means of raw data:

pHAbs.

(nm) (mean)

5 0.051

6 0.095

7 0.149

7.5 0.16

8 0.174

8.5 0.196

9 0.212

13

10 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 PNPSample Calculation:λmax = 360 nm

Readings PNP + pH10 (Abs.)

1 0.117

2 0.119

3 0.119

0.117 + 0.119 + 0.119 = 0.3550.355 / 3 = 0.118

Part II. Optical Assay of PNPPart 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 µM PNP solution 0.1 mM stock:

Conc.(µM)

Stock PNP(µM)

dH20(mL)

= Sample PNP Conc.(mM)

Sample Volume

(L)

Stock Soln.(mL)

dH20(mL)

10 100 V1 = 10 0.0002 0.2 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.