8
ABSORPTION AND ANALYTICAL
SPECTROPHOTOMETRY OF INDICATOR DYES
3/03
(from Rendina, "Experimental Methods in Modern Biochemistry")
Reading Assignment: Experimental Biochemistry, pp.3-10. (Library reserve)
Part A - Color vs. Wavelength
When you set the wavelength control on a spectrophotometer it selects a
specific wavelength (or narrow band of wavelengths) by adjusting the position
of a a slit. The lamp in the instrument gives off white light that is first
passed through a diffraction grating (which separates the light into its
component wavelengths) and then a slit that selects the desired wavelength.
The proper wavelength (λ) of light must be selected for the absorbing chemical
that you are measuring.
A colored chemical in solution will absorb the
complement of the color that it appears. So, for example, a chemical that
looks green will absorb its complement, purple light, and transmit green light
thereby appearing green. To measure this chemical one would have to choose a
wavelength in the purple region of the spectrum (500-580 nm) where this
chemical absorbs light.
Typically the specific wavelength chosen is the
wavelength of maximum absorbance λmax).
PROCEDURE:
A Bausch & Lomb Spectronic 20 Spectrophotometer has been outfitted with a
cuvette containing a piece of chalk with a beveled top. As a result, the light
passing through the cuvette is deflected upward and its color can be observed
by looking into the mouth of the cuvette. Dimming the light will make it easier
to see the colors. Record the observed color for each wavelength starting at
420 nm and every 40 nm interval, up to 740 nm.
Part B - The Absorption Spectrum of Phenol Red
In this experiment you will measure light absorption by the indicator
dye, phenol red, at various wavelengths. From your experimental values for
absorbance you will construct a graph of the absorption spectra of both the
proton acceptor and donor forms of this weakly acidic dye.
An acid-base indicator such as phenol red (PR) can exist in two forms, as
a proton acceptor (PR-) and as a proton donor (HPR). Actually, phenol red can
exist in four different forms, depending upon pH. We are interested here in the
red and yellow species that undergo a color change in the pH range of its pKa.
HPR
yellow
<-------------->
H+
+
PRred
PROCEDURE:
Prepare three solutions as indicated in Table 1, using deionized water
and clean dry test tubes. Mix them well. You will use the recording
spectrophotometer to determine the absorption spectra, from 300 to 800 nm for
each form of phenol red.
First the instrument must be “blanked” using
deionized water. Use a small plastic cuvette and fill it about two thirds full,
being careful not to touch the cuvette on the flat sides, which is where the
light passes through. After blanking, pour out the water and fill the cuvette
with some of the solution in tube one. Record its spectrum from 300 to 800 nm
and print out a copy. The instrument can determine what the λ max is and the
absorbance at λmax.
These values should be printed out on the spectrum or
recorded into your lab notebook. After rinsing the cuvette, repeat the process
with the solution in tube 2. Write your name on the printouts.
Table 1
Tube number
blank
1
2
---------------------------------------------------------------------A. H2O, ml
1.0
1.0
B.
1 x 10-4 M phenol red, ml
C.
0.1 M phosphate buffer, pH 5.8, ml
D.
0.1 M borate buffer, pH 9.8, ml
1.0
1.0
8.0
8.0
The two curves represent the absorption spectra for the proton donor
(HPR) and proton acceptor (PR-) forms of phenol red. Note that the proton donor
form has an absorption maximum (λmax) between 400 and 450 nm, while the acceptor
form has an absorption maximum (λmax) between 500 and 600 nm. Note also that at
the absorption maximum for the acceptor form, the donor form absorbs light very
weakly (why is this important later?). The quantitative determination of a
colored material by a spectrophotometric method is usually conducted at the
absorption maximum for the compound under study (why?). You will make use of
this principle in the next part of the experiment when the pKa of phenol red is
determined.
Calculate the molar extinction coefficient of each form of phenol red at
the absorption maximum for each. Be sure that you take into account the
dilution of the dye and that you give the appropriate units for the extinction
coefficient.
Part C- Spectrophotometric Estimation of the pKa of Phenol Red
INTRODUCTION: As has been pointed out in the previous section, phenol red is a
weak acid and the proton donor form dissociates reversibly in solution into a
proton and proton acceptor form:
HPR
yellow
<--------->
H+
+
PRred
This dissociation can be expressed in terms of the usual HendersonHasselbalch equation:
[PR-]
(1)
pH = pKa + log
[HPR]
Let [T] = [PR-] + [HPR] = sum of the acceptor and donor forms of the acid,
which is the total quantity of the acid present. Then [HPR] = [T] - [PR-]
Substituting in equation (1) we obtain
[PR-]
(2)
pH = pKa + log
[T] - [PR-]
This is a linear equation of the form y = ax + b, where b = pK a;
[PR-]
a = 1; y = pH; and x = log
. As a consequence, if pH's
[T] - [PR-]
were plotted on the ordinate (Y axis)and the corresponding values for
[PR-]
log
were plotted on the abscissa (X axis), a straight line inter[T] - [PR-]
secting the vertical axis at the pKa value should be obtained.
In the preceding section, you have determined the absorption spectra of
the acceptor and donor forms of phenol red, and have found that the acceptor
form has an absorption maximum at about 550 nm. At this wavelength, the donor
form absorbed very little light, and the absorption at 550 nm can therefore be
assumed to be almost entirely due to the acceptor form (PR -). At 550 nm, the
absorption of light by phenol red follows the Beer-Lambert Law.
Beer-Lambert Law:
A = Єcl = ЄPR-l[PR-]
where, A = absorbance
Є = extinction coefficient
l = optical pathlength (i.e., the diameter of the cuvette)
c = [PR-] = concentration of the absorbing species; in this case
the concentration of the proton acceptor form of phenol red.
If the optical pathlength, l, is in units of centimeters, (cm), and the
concentration, c, is in units of moles/liter (M), Є is the molar extinction
coefficient. Therefore, if a series of measurements of the absorbance at 550 nm
for various known concentrations of PR- is plotted as ordinate versus the
corresponding values for concentration as abscissa, a straight line is
obtained. Using this standard curve it is possible, by measuring the absorbance
of a solution of the absorbing species, to determine the concentration of PRin a solution of unknown concentration, or to measure the proportion of the PRto HPR present in a solution in which the total concentration of indicator is
known. The determination of the concentration of PR- at several pH values in a
solution of the total dye concentration would yield a plot from which the pKa
of the indicator may be obtained.
PROCEDURE:
Prepare solutions containing the components indicated in Table 2. Set the
wavelength scale of the spectrophotometer to the absorption maximum of the
acceptor (PR-) form. First, without a cuvette in the instrument, set the zero
or “dark current” by adjusting the zero knob to read 0%T (or ∞Abs). Using a
clean glass cuvette filled at least two thirds with the blank solution, set the
instrument to read 100%T (or 0 Abs). Then determine and record the absorbance
of each of your sample tubes by pouring each into the same cuvette (you do not
need to rinse the cuvette if you go from lower to higher concentrations. Show
your results to the instructor before proceeding. You will then use Excel to
plot the absorbance on the Y-axis (ordinate) versus the molar concentration of
phenol red on the X-axis (abscissa). This is your standard curve. The program
can calculate the slope of the best-fit straight line (going through zero),
which will be the extinction coefficient (Є) for the proton acceptor (PR -)
form. After consultation with your instructor, eliminate any data points that
look incorrect and indicate what you’ve done in your lab notebook. Use 1.0 cm.
for the pathlength.
Table 2
blank
0.1M borate buffer, pH 9.8, ml:
8.0
1 X 10-4 M phenol red, ml:
H2O, ml:
Final molar concentration of
phenol red (calculate in your
notebooks for the prelab)
absorbance at λmax:
1
2
8.0
8.0
0.1
2.0
1.9
0.2
1.8
Tube Number
3
4
8.0
0.4
1.6
8.0
0.6
1.4
5
6
8.0
8.0
0.8
1.2
1.0
1.0
0
0
The pKa of phenol red is in the vicinity of 7.8-7.9. Prepare a series of
tubes containing the components indicated in table 3. Set the wavelength scale
of the spectrophotometer to the absorption maximum of the acceptor (PR-) form.
Transfer a sample of the blank to a cuvette and with the cuvette in place, set
the instrument to read zero absorbance.
Measure and record the absorbance values of the remaining tubes. Do not
throw the solutions away as you will need to determine the pH of each. Using
the Є you determined from your standard curve for the acceptor form of phenol
red, convert the absorbance values to concentrations of PR -. Calculate the
values of the quantities in rows K and L. Using Excel, plot the actual pH (row
M) on the ordinate versus the corresponding value form row L on the abscissa.
Have the program draw the best-fit straight line through the points obtained
and determine the Y-intercept. This point is the pKa of the indicator.
[PR-]
(Note:
At
the
intersection,
log
has
the
value
of
zero.
[T] - [PR-]
[PR-]
Consequently,
must be equal to one at this point. (As you already
[T] - [PR-]
know, pKa = pH when the proton donor and acceptor forms of a weak acid are
present in equal concentration.)
Report your results and show samples of each of your calculations in a table
with the following:
Table 3
blank
A. 0.1M phosphate buffer, pH 7.2, ml:
8.0
1
2
Tube
3
4
5
8.0
B. 0.1M phosphate buffer, pH 7.4, ml:
8.0
C. 0.1M phosphate buffer, pH 7.8, ml:
8.0
D. 0.1M borate buffer, pH 8.1, ml:
8.0
E. 0.1M borate buffer, pH 8.4, ml:
8.0
F. 0.1M borate buffer, pH 8.8, ml:
G. H2O, ml:
6
8.0
2.0
H. 1 x 10-4 M phenol red, ml:
I. absorbance at λmax (nm):
0
J. concentration of PR-, molar:
0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
[PR-]
K.
[T] - [PR-]
[PR-]
L.
log
[T] - [PR-]
M. actual pH's (obtained from
the pH meter)
Note: Construct the appropriate charts and tables and do calculations
in your lab notebook before you come to the lab.