Determination of Constant of Ionization of a Weak

Acid By Spectrophotometry
It is a frequent occurrence in science that a given parameter cannot be measured
directly, in which case related determinations must be taken and through
mathematical manipulations of the results, arrive at the desired measurement. A
common example of this is the measurement of velocity, which cannot be taken
directly, but rather distance and time must be measured, and then velocity can be
calculated.
The experiment that we will be conducting today is another good example. The
acid that will be used is Bromophenol Blue, a weak acid that has the added
characteristic of changing colors as it ionies! since ioniation is a function of p",
the degree ioniation can be measured at different p"#s, and using the
"enderson$"asselbalch equation, the %
a
of the acid can be calculated. These
measurements will be made by using spectrophotometry.
&pectrophotometry is perhaps the most widely used of all laboratory techniques,
because many substances of biochemical interest absorb light in the ultraviolet,
visible or near infrared region.
Two parameters influence the absorption of light' the wavelength of maximal
absorption, and the extent of absorption (extinction coefficient). The color of
substances is due to the fact that light is absorbed at wavelengths other than that
which are visible! e.g. an orange solution is visible, because blue light is
absorbed, and orange and red light are transmitted.
EXPERIMEN !B"ECI#E$
To determine the %
a
value of Bromophenol Blue ("B*) by spectrophotometry.
A% ABS!RPI!N SPECRA
In any spectrophotometric determination, the first step is always to find the
optimum wavelength for the absorption of the substance to be studied. This is
accomplished by taking readings of a sample of the substance at varying
wavelengths (+). The wavelength affording maximum absorption will be the
optimum one.
PR!CED&RE
,. &et up - test tubes. .abel ,$-. N!E' all your glassware must be totally clean/
0. Add reagents according to the following table.
*eagent Test Tube 1umber
, 0 2 3 4 5 6 -
7.,8 9itrate Buffer p" 0.3 0.- 2.0 2.5 3.7 3.3 3.- 4.0
m. :.7 :.7 :.7 :.7 :.7 :.7 :.7 :.7
,.4x,7
$2
"B* (m.) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0
:4; <thanol 7.- 7.- 7.- 7.- 7.- 7.- 7.- 7.-
2. =or spectophotometric determinations, the cuvettes must be very clean. The
same cuvette is used for all readings. In between readings, the cuvette is rinsed
with water, follow by a rinse with a small amount of the substance to be analyed.
*eadings are taken starting with the most diluted solution, and continuing to the
most concentrated.
3. 8easure the absorbance of tubes , and - from 237nm to 507nm at 07nm
intervals against a water blank. *emember to set the ero after every + change.
>etermine the optimum +. Attach a plot of these spectra.
+ Tube , Blank Tube -
?.>. ; Trans. ?.>. ; Trans.
237
257
2-7
377
307
337
357
3-7
477
407
437
457
4-7
577
507
4. 8easure the absorbance of tubes ,$- at the optimum + against a water blank.
@ero the instrument on water.
RES&'S$
Tube A ?.>. ; Trans Blank ( ) Tube A ?.>. ; Trans Blank ( )
, 4
0 5
2 6
3 -
B% SANDARD C&R#E
In every spectophotometric determination, a standard curve must be drawn. This
will allow the determination of unknown concentration by establishing a
comparison, either mathematically, or by using the graph.
,. &et up 6 test tubes and label ,$6.
0. Add reagents according to the following table'
Test Tube 1umber
*eagent , 0 2 3 4 5 6
7.,8 9itrate Buffer, p" 0.3 (m.) :.7 :.7 :.7 :.7 :.7 :.7 :.7
,.4x,7
$2
8 "B* (m.) 7., 7.0 7.3 7.5 7.- ,.7 $$$$
:4; <thanol 7.: 7.- 7.5 7.3 7.0 $$$$ $$$$
Bnknown (m.) $$$$ $$$$ $$$$ $$$$ $$$$ $$$$ ,.7
CA'C&'AE (E M!'AR C!NCENRAI!N !) (BR IN EAC( &BE
Tube 9oncentration Tube 9oncentration
, 0
2 3
4 5
6
*I#E A SAMP'E CA'C&'AI!N$
RES&'S$
Tube A ?.>. ; Trans Tube A ?.>. ; Trans
2. 9onstruct a standard curve with the data obtained from tubes ,75. .abel the
axes carefully. >raw the best straight line, up to the point at which significant
deviations are observed, represent the region in which Beer#s law is obeyed.
3. >etermine the concentration of the unknown from the absorbance of tube 6
and the standard curve.
4. Then calculate the concentration of your unknown also from the absorbance of
tube 2.
C% CA'C&'AI!N !) p+ #A'&E$
,. The indicator Bromophenol Blue ("B*), dissociates as follows'
"B* "
C
C B*
$
a) Drite the equilibrium constant equation for this dissociation'
b) Drite the corresponding "enderson$"asselbalch equation'
c) .et (T) be the total concentration of both indicator species'
T E B*
$
C "B*
d) >evelop a linear equation of the form'
y E ax C b
where y E p"! a E ,! b E p%! "int' look at the equation in (b)
e) Dhat is xF
f) Glot p" as a function of x'
g) >etermine p%. >etermine %a.
D% N!ES
The indicator consists of a mixture of the dissociated and the undissociated
species of "B*, their relative concentration, a function of p". At all times,
(Beer#s .aw).
A E < I c