buffer and indicator

About the manual

in the 1st step(preparation of solutions), I have to prepare 8 kind of
solutions with different PH. Are they all buffer solution? Why are we
making 8 kind of solution with different PH value?

In 2n step (PH measurement)---Do not simply dilute yours with acid or
base to adjust the PH since this will change the indicator
concentration, which must remain the same for all solutions.

Why does the indicator concentration have to be the same?

In 3rd step, “If the %T reading at 400nm is below 20%, the solutions
that is in the cuvette should be diluted” why?

“Read the %T at 20-nm intervals,……every5nm” Don’t
understand what he is trying to tell?

“Make a quick plot of the absorbance vs wavelength in ur
notebook so you can determine what the wavelength_max for this
solution” why do I need to know the max. wavelength?

In 5th step, “ also, you should scan the wavelength to 630nm instead
of stopping at 600nm” “for best result s, use the same sample cell
to assure identical path lengths”

Don’t understand what he is trying to say?

5) In step 6, Why choose the first where HIn strongly absorbs (and
In- does not) and the second where In- strongly absorbs (and HIn does
not)?

About the spreadsheet

Pink space: Reason of plotting the graph. Don’t need to plot the
graphs…just explain each the meaning under each graph, because I
don’t know why I’m doing this.

Blue spaces: Do the data make sense to you? Can you tell me the meaning
of the numbers? From B50 to B70, the values seem keep going up. FromB85
to B105, the value seems go up and down. From B119 to B136, the values
seem go up and down. Does it mean anything?

Buffers and Indicators
1

Buffers and Indicators

INTRODUCTION
In this experiment, spectrophotometry is employed to measure the pKa of bromocresol green, an acid-base indicator. See
Appendix A for a review of this technique. The indicator (HIn) is a monoprotic organic acid and we can represent its
dissociation as follows:
HIn + H2O H3O+ + In-

the equilibrium expression for such a dissociation can be written
[In - ]
pH = pK HIn + log
[HIn]

This can be rearranged to give
[In - ]
log = pH - pK HIn
[HIn]

This is in the slope-intercept form of an equation for a straight line,

y = mx + b

[ In - ] [ In - ]
where y is log , m = 1 , and b = -pK HIn . Hence, if log is plotted vs pH , the slope is 1, the intercept is
[ HIn] [ HIn]
-pK HIn , and the line should cross the pH axis at pH = pK HIn (Fig. 1). At the latter point [In - ] = [HIn] , and hence the
log of this ratio of terms is zero, making pH = pK HIn




.

Figure 1 Plot to determine pK HIn
Buffers and Indicators
2
[In - ]
The ratio can be determined spectrophotometrically. First, a solution of bromocresol green is prepared in acidic
[HIn]
solution (low pH ) where essentially all the indicator is in the HIn form. The absorption spectrum is then taken over a
range of wavelengths. From a plot of Absorbance vs , the wavelength of maximum absorbance, max , for HIn is
determined. Similarly, a second solution is prepared in basic solution (high pH ) where essentially all the indicator is in the
In- form. Again, an absorption spectrum is taken over a range of wavelengths from which a max for In- is determined.

Buffered solutions with pH values on either side of the pK HIn of bromocresol green are then prepared and the absorbances
measured at the two selected wavelengths. The solutions contain the same total concentration of indicator, [HIn] + [In-],
but the ratios vary with pH . Figure 2 shows a typical plot of absorbance vs pH at the wavelength of maximum absorbance
for the In- species. The terms used in this figure are as follows:

Aa = absorbance of HIn
Ab = absorbance of In-
A = absorbance of the specific mixture being measured




Figure 2 Plot of absorbance of In - vs pH at max


From the graph it is evident that
[In - ] A - A a
=
[HIn] A b - A

Note that Aa and Ab, the extrapolated values from the graph are the same for all calculations at max for In-. Only A (the
absorbance of a specific mixture) changes from flask to flask.

If the wavelength used is the one at which HIn shows maximum absorbance, the curve will be similar to that shown in Fig.
2, except that it will start at a high absorbance and curve down to a low absorbance value at high pH . That is the resultant
curve will be the mirror image of Figure 2.
Buffers and Indicators
3

PROCEDURE
The stockroom will prepare the indicator for you (0.10 g of bromocresol green (C21H14Br4O5S) is dissolved in
approximately 100 mL of 95% ethanol, neutralized to pH of 7.0 with 1 M NaOH and quantitatively diluted in a 250-mL
volumetric flask to the mark with the ethanol solvent).

Solutions of 0.10 M acetic acid, 0.20 M sodium acetate will be provided for you. You will have to prepare on your own the
0.10 M HCl, 0.10 M and 1.0M NaOH solutions.

1. Preparation of Solutions. Due to limited numbers of volumetric flasks, you need to transfer these into beakers or
Erlenmeyer flasks as you prepare them. Pipet 2-mL of the bromocresol green (bcg) solution into each along with the
appropriate amounts of the other components to make 50-mL of the following buffers:

Flask Instructions Problems
1 Mix 48-mL of 0.10 M HCl with 2-mL of bcg. What is the expected pH?
2 Mix 48-mL of 0.10 M HAc with 2-mL What is the expected pH?
indicator
3 Make 48-mL of pH = 3.7 buffer using 0.10 M What volumes of HAc and NaAc are needed?
HAc and 0.20 M NaAc.
4 Make 48-mL of pH = 4.0 buffer using the 0.10 What volumes of HAc and NaAc are needed?
M HAc and the 0.20 M NaAc.
5 Make 48-mL of pH = 4.8 buffer using 0.10 M What volumes of HAc and NaOH are needed?
HAc and 0.10 M NaOH.
6 Make 48-mL of pH = 5.4 buffer using 0.10 M What volumes of HCl and NaAc are needed?
HCl and 0.20 M NaAc.
7 Make 48-mL of pH = 5.8 buffer using 0.10 M What volumes of HAc and NaAc are needed?
HAc acid and 0.20 M NaAc.
8 Mix 48-mL of 0.20 M NaAc with 2-mL bcg What is the expected pH?

2. pH Meaqsurement. Dilute each solution to the mark and mix thoroughly. Measure and record the pH value of each
solution using a pH meter. Appendix C explains how to use the pH meter. Appendix F explains the theory of how the
electrodes measure pH. Recalculate and prepare any incorrect solution. Do not simply dilute yours with acid or base to
adjust the pH since this will change the indicator concentration, which must remain the same for all solutions.

3. The Use of Spectrometer. Review how to use the Spec 20 (see Appendix A). Make certain it has warmed up at least 20
minutes before use. Whenever a new wavelength is chosen, you must first adjust the zero reading (with the sample holder
empty and closed) using the front left knob. The 100%T must then be adjusted using a sample "blank" (water in this case)
using the front right knob. Check the zero again. Check the 100%T again and adjust if necessary. Now put in your sample
and read the %T scale (this one is linear and easier to read). To convert this reading to absorbance, use the relationship

100
A = log or A = 2 - log %T
%T

For consistency of path length, select a pair of well-matched test tubes or cuvettes - use one for the water blank and the
other for all sample measurements. Be certain to wipe all drops and fingerprints off of the cell with a fine tissue and be
consistent about which side of the cell faces forward. See your TA for more information. A slight realignment of the cell
will give different values.

4. Absorption Spectrum of HIn. To determine the absorption spectrum of bromocresol green at low pH (the spectrum of
HIn), measure the percent transmittance of solution 1 from 400 to 600-nm, using water as a reference. The pH of this
solution is sufficiently low that most of the indicator is in the HIn form. If the percent transmittance reading at 400-nm is
below 20%, the solutions that is in the cuvette should be diluted. Read the percent transmittance at 20-nm intervals, except
in the vicinity of the minimum %T (or maximum absorbance) in the spectrum, where readings should be taken every 5-nm.
Make a quick plot of the absorbance vs wavelength in your notebook so you can determine what the max is for this
solution.
Buffers and Indicators
4

5. Absorption Spectrum of In-. To determine the absorption spectrum of bromocresol green at high pH, measure the
percent transmittance of solution 8 in a similar manner as directed above except that if %T at 400-nm is below 30%T,
dilution should be made. Also, you should scan the wavelength to 630-nm instead of stopping at 600-nm. For best results,
use the same sample cell to assure identical path lengths. Make a quick plot of the absorbance vs wavelength in your
notebook so you can determine what the max is for this solution. In the report, however, these two plots must be generated
by the spreadsheet and both plots must appear on the same graph with the horizontal axis being the wavelength from 350nm
to 650nm, the left axis is absorbance for HIn, and left axis, In-.

6. max for HIn and In-. Using the spectra obtained above, select the two max wavelengths at which further absorbance
measurements will be made. Choose the first where HIn strongly absorbs (and In- does not) and the second where In-
strongly absorbs (and HIn does not).

7. A vs pH. Now measure the percent transmittance of each of the remaining six solutions at the two wavelengths selected.
[In - ]
Prepare a graph of absorbance vs pH at each max (see Fig. 2). Determine the ratios in solutions 2 to 7 as explained
[HIn]
[ In - ]
above and shown in Fig. 2. Plot log vs pH (Fig. 1) and obtain a value of pKHIn from the graph at each max. These
[ HIn]
values should be the same.

8. Report the individual pKHIns and average value of the pKHIn for bromocresol green. Include all of the graphs in your
report.

9. Waste Treatment. Combine all of the solutions used in this experiment into your own single large container. Measure
the pH, neutralize to between 6 and 8, and pour down the drain. (What are the chemicals, at neutral pH which you have just
discarded?)


Have you
1. wipe down all benchtops, including the sink, hood, and balance areas?
2. return all stock chemicals to their proper locations?
3. return all equipment to your drawer or general supply area?
4. lock your desk drawer?