Chem 460 Laboratory – Fall 2008
Experiment 2: Determination of the pKa of 4-Nitrophenol
Before Lab
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Review Section 2-2 in VV&P
Review sections 18-2, 18-3 and 19-1 in Harris's Exploring Chemical Analysis:
In this laboratory period you will determine the pKa of 4-nitrophenol. This experiment
incorporates several important techniques used in the biochemistry laboratory—solution
preparation, UV/Visible spectroscopy and data analysis using Excel.
Why investigate 4-nitrophenol? 4-nitrophenol and the closely related 2-nitrophenol are
often conjugated to substrate molecules. When used in certain enzymatic assays, the
nitrophenolate ion is released. This results in a yellow color that is easily detected by eye
and is quantifiable by spectrophotometry. Ultimately this can tell us how active a
particular enzyme is.
The Henderson-Hasselbalch equation tells us that the pKa of an acid is numerically equal
to the pH of a solution containing an equimolar amount of weak acid (HA) and its
conjugate base (A-). In Chem230 you determined the pKa of a weak acid by monitoring
the pH as you titrated it with a strong base. In many biochemical systems this strategy is
not practical, either because there is not a large enough quantity of the weak acid, or
because there are multiple acidic groups in the system. An alternative strategy is to find a
spectroscopic property that changes depending on whether the acid/base group of the
molecule is protonated or unprotonated. By placing the molecule of interest in a series of
solutions at different pH's and recording the spectral property, a plot of the spectral
property as a function of pH can then be used to determine the pKa of the group.
UV/Vis spectroscopy is one of the most common analytical tools in the biochemical
laboratory. When 4-nitrophenol is deprotonated there is a dramatic change in its
UV/visible spectrum. Thus, we can use UV/Vis spectroscopy to monitor the change in 4nitrophenol’s protonation state as a function of pH. We will then obtain the pKa of 4nitrophenol using Excel by fitting the experimental data you acquire in lab to a theoretical
line derived from the Hendersen-Hasselbalch equation.
A. Obtain UV/Vis spectra of protonated and deprotonated 4-nitrophenol
To begin, you need to acquire a UV/Vis spectrum from 200-700 nm for the protonated
and deprotonated forms of 4-nitrophenol. You have available: solutions of 1M HCl, 1 M
NaOH, and 1 mM 4-nitrophenol and 1 mL cuvets (should you use quartz or plastic?) The
final concentration of 4-nitrophenol should be such that the maximum absorbance is
approximately 1 AU. (Why?) Should you use the deuterium lamp, the tungsten lamp, or
both? What should you use as a blank?
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B. Select a wavelength to monitor and determine the extinction coefficients of
deprotonated and protonated forms at this wavelength
Determine what wavelength would be the best for monitoring the change from the
protonated to the deprotonated form of 4-nitrophenol. Discuss your choice with your
laboratory instructor. Then, at the wavelength you have chosen, make at least 3
independent measurements of the absorbance for both the protonated and deprotonated
forms. Calculate the extinction coefficients (a.k.a. molar absorptivity) for the
deprotonated and protonated forms at this wavelength.
C. Determine the absorbance of 4-nitrophenol in 0.1 M buffer at various pH's
For this experiment we will need 100 mM buffer solutions at pHs of 5, 6, 7, 7.5, 8, 8.5, 9
and 10. At the beginning of lab you and your partner will be assigned two of these pH's.
Come up with a plan for preparing 50 mL of buffer solution at each pH using the 0.5 M
stock solutions you made last week. Show your plan to your instructor before you start
making solutions.
For your spectrophotometry experiment you will need to prepare solutions that all have
the same concentration of 4-nitrophenol, but at different pH's. The simplest method for
doing this experiment is to add accurately 1-3 mL of the appropriate buffer into a cuvette
and then use this as a blank. (The amount of buffer needed will depend on the size of the
cuvette.) Then add a small amount of concentrated 4-nitrophenol to the cuvette and mix
thoroughly. The final concentration of 4-nitrophenol should be such that fully
deprotonated 4-nitrophenol at this concentration has an absorbance near 1 AU (using the
wavelength you determined in part B). What is the relative concentration of 4nitrophenol to buffer? Discuss your plan with your instructor before you start the
spectrophotometry experiments. Measure the absorbance of 4-nitrophenol at each of the
pHs using the wavelength you determined in part B.
D. Plot your data in Excel, estimate the pKa of 4-nitrophenol and use the
Henderson-Hasselbalch equation to generate a theoretical plot of the absorbance
as a function of pH
Enter your data into Excel and make a plot of Absorbance vs. pH. Show your plot to
your instructor and explain how you can estimate the pKa from it.
In Excel make a column containing pH values from 5 to 10 at 0.1 intervals (check with
your instructor if you don’t know how to do this quickly). In the adjacent column, enter a
formula that uses the Henderson-Hasselbalch equation, your values of the extinction
coefficients, and your estimate of the pKa to determine what the absorbance of 4nitrophenol should be at each of the pH values from 5 to 10. (Hint #1: Rearrange the
Henderson-Hasselbalch equation to solve for the fraction of 4-nitrophenol in the A- form
and multiply this by the total concentration of 4-nitrophenol. Hint #2: You will find it
useful to have a single box in the spreadsheet designated for the pKa—so ask your
instructor about $ notation if you don’t already know how to use it.)
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Overlay your data points with the theoretical curve (i.e put them on the same plot). In a
separate box in the spreadsheet, calculate the least squares variance (see Harris Ch 4,
pages 79-80). To do this, take the difference between your actual Absorbance and the
one calculated from your model, square this quantity and add each squared quantity
together. Then divide the sum by the number of measurements minus one (i.e. n-1). To
precisely determine the pKa of 4-nitrophenol (to two decimal places), minimize the least
squares variance by adjusting your pKa estimate by 0.10 pKa unit up or down, and see if
the least squares variance increases or decreases. If it is increases, then go the other way,
if it decreases keep going in that direction. Repeat until you have reached the minimum.
Lastly, determine how well the theoretical curve fits your data by calculating the
correlation coefficient R2 (information on how to do this will be presented in lab).
Produce a final plot in a format that would be acceptable in the journal Biochemistry.
Some copies of plots from Biochemistry will be in the lab for you to look at.
Report (50 pts)
Due by your next lab period:
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Your laboratory notebook pages corresponding to this experiment.
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The final plot of your data superimposed on the theoretical fit—this should be in a
format acceptable in the journal Biochemistry. Include a detailed figure caption that
allows the reader to interpret it correctly.
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Email your instructor the Excel file containing your data, theoretical model, least
squares and R2 calculations.
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