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Kendall Claffey and Gabby Waldrop

Bio 541L-001

TA: Tori Peacock

January 26, 2021

Determining the pKa of P-Nitrophenol Using the Absorbance of Buffer Solutions with Differing

pH Values
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Purpose

The purpose of the experiment was to determine the pKa of P-Nitrophenol (PNP) using 8 buffer

solutions, each with a different pH, and a spectrophotometer to measure absorbance resulting in

the finding of maximum absorbance and pKa of PNP.

Background

To find the pKa of P-nitrophenol buffer solutions are needed. A buffer solution is a

solution where the pH is constant, or has little movement, when introduced to acids3. All buffer

solutions have a buffer range. The buffer range is the amount of variance in the pH of the

solution without becoming acidic or basic4. The pKa of a compound is directly related to the pH

of the compound5. The pKa and pH can be related to each other through the Hendersen-

Hasselbalch equation5. The Hendersen-Hasselbalch equation is pH= pKa + log ([A]/[HA])6. The

pKa can be found looking at the steepest slope of the curve between pH and absorbance6. At the

steepest slope of the curve pH=pKa of the solution6.

P-Nitrophenol (PNP) structure is C6H5NO3 and is a white/ yellow crystal compound2.

The dissociation reaction of PNP is displayed in the following image6.

The dissociation reaction is important because demonstrates what certain chemical’s effect on

the body will be7.

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Beer’s law is denoted by the equation A = elc and is useful to understand the amount of

energy of absorbed (A) using the molar attenuation coefficient (e), the path length (l) and the

solution concentration (c) being used8. To determine the absorbance of the solution a

spectrophotometer can be used7. The absorbance indicates how much solute is in the solution7.

Absorbance of different wavelengths can be found using a spectrophotometer1. After setting the

spectrophotometer to the maximum wavelength, different buffer solutions (with varying pH

values) can be placed in the spectrophotometer and the absorbance values can be measured1.

Therefore, the spectrophotometer can help determine the pH/pKa of a solution such as P-

nitrophenol1.

Materials and Methods

To begin the experiment, use 8 unknown buffer solutions1. Tear 8 separate pieces of pH

paper and place them on a paper towel1. Label the pieces of pH paper 1-81. Using a 20 µl

micropipette, place one drop of each buffer solution on one piece of pH paper1. Immediately

compare the color of the pH paper to the pH color chart1. Record each of the buffer solutions’

pH1.

To prepare the samples, label 8 test tubes 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.01. Using a

pipettor, place 1mL of P-Nitrophenol into each of the 8 test tubes1. Place 3mL of each buffer

solution into the glass test tube with the corresponding pH1. After each buffer solution is added

to its corresponding test tube discard of the pipettor tip1. Take 10 cuvettes and label them B

(blank), S (sample), 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.01. In the “B” cuvette add 2.0 mL of pH 10

buffer solution1. In the “S” cuvette add 0.5mL of 0.1 P-nitrophenol and 1.5mL of the buffer pH

10.0 solution1. In the following 8 cuvettes place 2.0mL of corresponding buffer solution into the

properly labeled cuvette1.

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To determine the max wavelength of P-nitrophenol, use the spectrophotometer1. On the

machine set the wavelength to 360nm using the button “go to lambda” and using the keys up and

down keys1. When done, press the “OK” button and place the B-cuvette in the first position and

the S-cuvette in the fourth position1. Shut the lid, press “zero” to blank1. Pull the handle to place

the S-sample into the correct position and record the absorbance observed1. Using the

wavelengths 360nm-430nm (every 10nm), find the max wavelength1. Use all 8 cuvettes and

label the wavelength for each of the cuvettes1. Be sure to zero the spectrophotometer after each

cuvette’s wavelength is recorded1. Read the results and determine the maximum wavelength1.

To determine the pKa of P-nitrophenol, use the spectrophotometer again1. Set the

machine to the maximum wavelength found in the previous step1. Remove the S-cuvette and the

B-cuvette1. Record absorbance found for each of the 8 cuvettes1. Read the results and determine

the pKa for P-nitrophenol (PNP)1. Using excel create a graph of absorbance vs. wavelength and a

graph of pH vs. absorbance1.

Results

Table 1

Table one includes data from 10nm increments of wavelengths between 360nm to 430nm. The

wavelengths show the corresponding observed absorbance values.

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Table 2

Table 2 includes data from 8 different pH buffer solutions at 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0

and the corresponding observed absorbance values.

Figure 1

Absorbance vs. Wavelength

0.45
0.4
0.35
Max
Absorbance (AU)

0.3
Wavelength=
0.25
400nm
0.2
0.15
0.1
0.05
0
350 360 370 380 390 400 410 420 430 440
Wavelength (nm)

Figure 1 indicates the maximum wavelength of the 8 buffer solutions. The figure shows the 8

wavelengths of 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm vs. the observed

absorbance. Maximum wavelength was found to be 400nm.

Figure 2
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Absorbance vs. pH
0.5

0.4

pKa= 7.4
Absorbance (AU)

0.3

0.2

0.1

0
0 2 4 6 8 10 12
-0.1
pH

Figure 2 indicates the pKa of P-nitrophenol. The pH of the 8 buffer solutions at 5.0, 6.0, 7.0, 7.5,

8.0, 8.5, 9.0, 10.0 vs. the observed absorbance values. The pKa of p-nitrophenol was found to be

7.4.

Discussion

The maximum wavelength of the graph was found by comparing the absorbance vs. the

wavelength1. The maximum wavelength had the highest absorbance value1. Table 1 showed the

values of absorbance found through the spectrophotometer at wavelengths 360nm, 370nm,

380nm, 390nm, 400nm, 410nm, 420nm, 430nm. Figure 1 was created using the data from table

1. Figure 1 showed a bell-shaped graph in which the maximum wavelength was easily

identifiable. In figure 1, the maximum wavelength in the experiment was found to be 400nm.

The spectrophotometer was set to the maximum wavelength and the cuvettes containing

the 8 buffer solutions with pH of 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0 were be placed in the

spectrophotometer1. Table 2 showed the data collected after setting the spectrophotometer to the

maximum wavelength, 400nm, and measuring the absorbance of pH values 5.0, 6.0, 7.0, 7.5, 8.0,

8.5, 9.0, 10.0. Figure 2 was created off of the data found in table 2. In figure 2, the graph showed
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the absorbance vs. the pH (pH=pKa6). The pKa was found at the steepest part of the slope of the

graph.

The pH of P-nitrophenol was found to be around 7.4. Although there is a range of pH for

P-nitrophenol, it was estimated to be around 7.079. The estimated value of 7.4 was relatively

close but slightly off. This could be due to experimental error. A possible experimental error

could be not having perfect measurements due to some solution being left in the pipettor tips.

Another potential error was when estimating the pKa using figure 2. The steepest part of the

slope was determined to be the pKa of p-nitrophenol (PNP)6. The steepest part of the slope was

hard to slope was difficult to get precisely accurate. This could have caused the experimental

pKa to be higher or lower which could have led to the pKa being closer to the expected value of

7.079.
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References

1. USC Biochemistry Department. 2021. Lab 1 Procedure: Determining the pKa of P-

Nitrophenol (PNP). [accessed 2021 Feb 8].

2. 4-Nitrophenol. National Center for Biotechnology Information. PubChem Compound

Database. [accessed 2021 Feb 8]. https://pubchem.ncbi.nlm.nih.gov/compound/4-

Nitrophenol

3. Clark J. buffer solutions. 2002 [accessed 2021 Feb 8].

https://www.chemguide.co.uk/physical/acidbaseeqia/buffers.html

4. Mott V. Introduction to Chemistry. Lumen. [accessed 2021 Feb 8].

https://courses.lumenlearning.com/introchem/chapter/buffer-range-and-capacity/

5. Reijenga J, van Hoof A, van Loon A, Teunissen B. Development of Methods for the

Determination of pKa Values. Analytical chemistry insights. 2013 Aug 8 [accessed 2021

Feb 8]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747999/

6. USC Biochemistry Department. 2021. Powerpoint: Determining the pKa of PNP.

[accessed 2021 Feb 8]

7. Wronka A. SPECTROPHOTOMETRIC DETERMINATION OF THE DISSOCIATION

CONSTANT OF p-NITROPHENOL Janicka M, Wiacek AE, editors. [accessed 2021

Feb 8]. http://www.katedrachf.umcs.lublin.pl/cwiczenia/Ang/PhysChem/Cw-13.pdf

8. Illustrated Glossary of Organic Chemistry - Beer's Law (Beer-Lambert Law). [accessed

2021 Feb 8]. http://www.chem.ucla.edu/~harding/IGOC/B/beers_law.html

9. Showing metabocard for 4-Nitrophenol (HMDB0001232). Human Metabolome

Database: Showing metabocard for 4-Nitrophenol (HMDB0001232). [accessed 2021 Feb

8]. https://hmdb.ca/metabolites/HMDB0001232
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Appendix