S.

COLE 1027197

Practical
Portfolio Report

Spectrophotometry report
Buffer solution report
S.COLE 1027197

MAKING STANDARD SOLUTIONS

INTRODUCTION TO SPECTROPHOTOMETRY
Aim
The aim of the experiment was to understand the basics of spectrophotometry and use this
knowledge to determine unknown concentrations of aqueous copper sulphate.

Introduction
The process of spectrophotometry is similar to absorption spectroscopy. Light within the
ultraviolet and visible region of the electromagnetic spectrum (200nm – 700nm) is beamed
at the sample is absorbed by the sample. Certain molecules absorb light at different
wavelengths so each molecule will have a different emission spectrum. If the wavelength at
which the molecule absorbs light is known, then using a spectrophotometer, levels of
absorbance for a specific can be found. A spectrophotometer works by measuring how
much light is absorbed by the molecule or compound. If the absorbance is low then the
amount of the molecule in the sample is low and vice-versa. This information can be used to
determine unknown concentrations by plotting graphs of absorbance values against known
concentrations.

Method
We were supplied with solutions of 0.1M copper sulphate and had to make 10cm 3 solutions
of with certain concentrations. We pipetted certain amounts of copper sulphate with certain
amounts of water to make the concentrations shown in table 1. These would be the known
concentrations to be plotted on a graph later. The spectrophotometer was set to a
wavelength of 807nm then a clean cuvette was pipetted with 3cm 3 of distilled water and
placed in the spectrophotometer. This acts as a blank and ensures that the absorbance due
to water in the copper sulphate solution is ignored. Once the spectrophotometry was
zeroed, the cuvette containing distilled water was replaced with a cuvette containing 3cm 3
of the lowest concentration of copper sulphate. The absorbance was measured and then
the cuvette with the next concentration up was put in side. This was done for all other
concentration and the results are shown in table 1.

Next we were supplied with two unknown concentrations of copper sulphate labelled
“unknown one” and “unknown two”. We took 4cm3 of each solution and diluted sample
“unknown one” with distilled water in a ratio of 1:1. The volume of the unknown solutions
and distilled water is shown in table 2. The spectrophotometer was zeroed again with 3cm3
of distilled water in a clean cuvette and the concentrated sample of unknown one was
placed in the spectrophotometer. Unlike the first section, where we worked up from lowest
concentration to highest concentration, after each cuvette we zeroed the machine as the
concentrations of each sample was unknown. The results can be seen in table 2.

A graph for table 1 was plotted showing absorbance (y-axis) against concentration of
standards mol dm-3 (x-axis) a line was plotted and the concentrations of the unknown
solutions were read of the graph using the respective absorbance.
 S.COLE 1027197

Results

Table 1 – Standard Curve Absorbance & Concentration Data

Volume of Standard (cm3) Volume of Water (cm3) Concentration (mol dm-3) Absorbance
10 0 0.1 mol dm-3 1.204
8 2 0.08 mol dm-3 0.769
6 4 0.06 mol dm-3 0.525
5 5 0.05 mol dm-3 0.327
4 6 0.04 mol dm-3 0.169
2 8 0.02 mol dm-3 0.134
0 10 0.00 mol dm-3 0.000

Table 2 – Unknown Solutions Absorbance & and Concentration Data

Unknown Volume of Unknown Volume of Concentration (mol dm-3) Absorbance
3 3
(cm ) Water (cm )
1 (concentrated) 4 0 0.115 mol dm-3 1.330
1 (diluted) 2 2 0.077 mol dm-3 0.648
2 4 0 0.076 mol dm-3 0.631
S.COLE 1027197

Discussion
Part one of the experiment involved learning how to use the spectrophotometer and
obtaining data from it. From table 1, a trend appears showing that as concentration
increases, absorbance too increases and vice versa; as concentration of the sample
decreases, so does the absorbance. From this data a standard curve was produced.
Theoretically, it should be a straight line as absorbance is proportional to the concentration;
however I obtained a line with a slight curve straightening out as the concentration went up.

For part two of the experiment, we had unknown concentrations of copper sulphate and
had to find the concentration using their absorbance reading. A line from the absorbance
reading was drawn and where it intersected the curve, a line was drawn down to the
concentration. The results in red in table show the concentrations of the unknown solutions.

Conclusion
From the experiment it is safe to say that as the concentration of a substance increases, the
absorbance also increases. However a straight line was not produced on the graph so it
can’t be said that there is a proportional relationship between the two factors. This could be
due to human error of handling the equipment. In the group everyone took part in making
the solutions and using the spectrophotometer, so different styles of handling the
equipment could have led to different results from the true value. It may have been better
to do the experiment alone but considering the time limit it wasn’t a practical option. Also
doing repeats would have giving more accurate results and may have resulted in obtaining a
straight line on the graph.
S.COLE 1027197

Investigate the behaviour of phosphate buffers in

response to NaOH
Aim
The aim of the aim experiment is to be able to use a pH meter correctly to obtain accurate
results and to determine when the buffer ceases to act as a buffer.

Introduction
A buffer solutions solution resists change in pH when small amounts of acids or bases are
added to it. Buffers are usually made up of a weak acid and its salt or a weak base and its
salt. In this case, however, we use sodium hydroxide (a strong base) and a phosphate buffer.
In order understand how a buffer works, the dissociation of acids and bases must also be
understood.

Strong acids fully dissociate giving up their hydrogen ion and weak acids only partially
dissociate. This means they are less likely to give up there hydrogen ion and stay in their
ionised form. The general equation for the dissociation of acids is:
HA + H2O => H3O+ + A-
Here the acid dissociates with water form hydronium ion and a conjugate base.

Strong bases fully dissociate accepting hydrogen ions and weak bases partially dissociates,
meaning they are less likely to bind to free hydrogen ions. The general equation for the
dissociation of bases is:
B + H2O => BH+ + OH-
Here the base dissociates with water forming a conjugate acid and a hydroxide ion.

Method
We were provided with a solution of 0.1M sodium hydroxide, distilled water and 20ml f
three different concentrations of a phosphate buffer (0.05M, 0.1M and 0.5M) for each of
the buffer solution the pH was first measured with a pH meter. Then before adding small
amounts of sodium hydroxide, the pH meter was rinsed with distilled water and then the pH
was measured again. After adding sodium hydroxide and taking down the pH, the pH meter
was rinsed with distilled water again before adding more sodium hydroxide. This was done
for all other solutions of the phosphate buffer. Similarly with distilled water, the pH was
measured and then sodium hydroxide was added to the solution. The pH was measured
again and before adding more sodium hydroxide, the pH meter was rinsed. The data
collected can be seen in the tables below.
S.COLE 1027197

Results
Table 1 0.1M of Sodium Hydroxide added to 20ml of 0.05M Phosphate buffer
Volume of NaOH added (ml) pH of the solution
0.0 6.93
0.1 6.94
0.2 6.96
0.4 7.01
0.6 7.04
0.8 7.08
1.0 7.11
2.0 7.29
3.0 7.49
4.0 7.84
5.0 8.75
6.0 10.48
7.0 10.93
8.0 11.21
9.0 11.35
10.0 11.48

Table 2 0.1M of Sodium Hydroxide added to 20ml of 0.1M Phosphate buffer

Volume of NaOH added (ml) pH of the solution
0.0 6.91
0.1 6.93
0.2 6.95
0.4 6.96
0.6 6.97
0.8 7.01
1.0 7.09
2.0 7.13
3.0 7.24
4.0 7.42
5.0 7.61
6.0 7.89
7.0 8.59
8.0 10.38
9.0 10.78
10.0 11.08
S.COLE 1027197

Table 3 0.1M of Sodium Hydroxide added to 20ml of 0.5M Phosphate buffer

Volume of NaOH added (ml) pH of the solution
0.0 6.93
0.1 6.92
0.2 6.93
0.4 6.90
0.6 6.95
0.8 6.93
1.0 6.97
2.0 6.94
3.0 6.95
4.0 6.98
5.0 6.97
6.0 7.01
7.0 7.00
8.0 7.02
9.0 7.02
10.0 7.06

Table 4 0.1M of Sodium Hydroxide added to 20ml of distilled water

Volume of NaOH added (ml) pH of the solution
0.0 7.05
0.1 10.48
0.2 10.92
0.4 11.26
0.6 11.34
0.8 11.45
1.0 11.54
2.0 11.86
3.0 12.00
4.0 12.05
5.0 12.12
6.0 12.17
7.0 12.21
8.0 12.25
9.0 12.27
10.0 12.29

Table 5 Volume of NaOH to increase pH by one unit

pH 7 to pH8 pH 8 to pH 9 pH 9 to pH pH 10 to pH pH 11 to pH
10 11 12
Graph 1 4.0 ml 1.0 ml 0.6 ml 1.2 ml n/a
Graph 2 6.3 ml 1.0 ml 0.6 ml 1.2 ml n/a
Graph 3 n/a n/a n/a n/a n/a
Graph 4 n/a n/a 0.1 ml 0.2 ml 2.1 ml
S.COLE 1027197

Discussion
From graph 1, the 0.05M phosphate buffer resists change of pH fairly well until 4ml had
been added to the buffer. The pH had increase by 0.93. It’s a fairly small change but in
certain situations it could be fatal such as in biological buffers. In this experiment, however, I
wouldn’t consider this as a significant change in pH in comparison to the volume of sodium
hydroxide added. After adding more sodium hydroxide, the pH starts to increase more
sharply. The increase in pH between the addition of 4ml and 5ml is almost equal to the
increase in adding just 4ml of sodium hydroxide. The pH of the buffer further increases
sharply upon adding more sodium hydroxide. After adding volumes more than 7ml of
sodium hydroxide the pH starts to level out at a pH of around 11. At this stage the buffer is
now alkali. From this, I would conclude that the buffer ceases to act as a buffer after adding
more than 4ml of sodium hydroxide.

From graph 2, the curve drawn for the 0.1M phosphate buffer is similar to the 0.05M buffer.
The pH of the buffer increases very slightly between 0ml of sodium hydroxide and 6ml of
sodium hydroxide. The increase in pH between these volumes is 0.93. Again, compared to
the volume added it’s a fairly small change. Upon adding one more millilitre results in a
sharp increase in pH of around 0.7. The curve follows a similar trend found in graph 1 and
the pH starts levelling out at 11 after 9ml of sodium hydroxide has been added. From this
graph I can conclude that the buffer stops acting like a buffer after volumes of more than
6ml of sodium hydroxide has been added.

Graph 3 has a different curve from the others. The 0.1M phosphate buffer after the
experiment only had a 0.13 increase in pH. This is a good example of a buffer as the pH
change is around 0.1. However, as mentioned for graph 1, this small change could prove
hazardous in certain situations such as biological buffers. Though for this experiment this
change in pH is acceptable. Graph 4 is the reaction of a strong base with water. The
equation for this reaction is:
NaOH + H2O => Na+ + OH- + H2O
Water here acts as weak acid donating an H+ proton and sodium hydroxide dissociates into
Na+ and OH+. The hydroxyl ion accepts the proton forming water, but now water becomes a
hydroxyl ion. This causes the solution having an alkali system.