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Introducti
on
Procedur
e
Discussion
Answers to
Questions
Conclusion
Recommendatio
ns
INTRODUCTION
Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light as a beam
of light passes through sample solution. The basic principle is that
each compound absorbs or transmits light over a certain range of
wavelength. This measurement can also be used to measure the
amount of a known chemical substance. Spectrophotometry is one
of the most useful methods of quantitative analysis in various fields
such as chemistry, physics, biochemistry, material and chemical
engineering and clinical applications.
MATERIALS
• 0.1 M Sodium Diphosphate
• 0.1 M Sodium Phosphate
• 0.1 M Sodium Hydroxide
• Concentrated HCl
• 0.05% methyl red indicator
• Distilled Water
APPARATUS
• 25-mL Beaker (12 pieces)
• 1-mL graduated pipette (2 pieces)
• 10-mL graduated pipette
• 25-mL volumetric flask
• Suction bulb
• Wash bottle
• Hitachi U-500
• pH meter
SPECTROPHOTOME
TER
SETUP magnetic
stirrer
solution
hot plate
pH meter
PROCEDURE
To adjust the pH, add 0.1M HCl
to alkaline solutions and 0.1 M Make use of solution 1 and 10
NaOH to acidic solution. Adjust first, rinse the cuvette with
also the pH of the solutions 2-9, solution 1 three times and fill
with appropriate intervals so the cuvette 2/3 of its capacity.
that the pH of the 10 solutions Do this with solution 10 also.
should have 1-13 pH levels.
1.500
1.000
0.500
Abs-sol1
Abs-sol10
DIFF
0.000
300 400 500 600 700 800 900 DIFF
-0.500
-1.000
-1.500
-2.000
WAVELENGTH
GROUP 1 AND 3
GROUP 2 10
6
λ1 = 401 nm
4
λ2 = 503 nm
2
Absorbance
0 S1
300 400 500 600 700 800 900 S10
-2 dif
-4
-6
-8
-10
Wavelength
GROUP 2
Solution No. pH as adjusted Absorbance at λ1 ,A1 Absorbance at λ2,A2
1 1 0.351 2.78
12
10
0
-1 -0.5 0 0.5 1 1.5
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GROUP 4
2
1.5
0.5
0
300 400 500 600 700 800 900
-0.5
-1
-1.5
-2
1 1 1.815621833 0.094568267
10 13 0.102102867 1.0111468
GROUP 4
HIn In pKa
12
15.843 1.08 3.476413667
10
20.892 1.1099 4.734696166
8
7.146 1.089 5.647035132
pKa
6
pH
4.7306 12.045 10.15410942
1.5
1
Absorbance (1)
Absorbance (10)
Diference
0.5
0
300 400 500 600 700 800 900
-0.5
-1
GROUP 5
Samp No. / Name pH as adjusted Abs(521.0) Abs(422.0)
1 1 3.0964022 0.3176492
10 13 0.5675883 3.1320194
GROUP 6
4
Abs 1
0 Abs 10
300 400 500 600 700 800 900 Dif
-1
-2
-3
-4
GROUP 6
Solution No. pH adjusted Absorbance at 503, A1 Absorbance at 503, A1
1 1 3.1449049 3.1662043
9 12 0.1983772 0.1029489
10 13 0.1370171 0.0377663
GROUP 7
2
1.5
0.5
Abs
0 abs 13
300 400 500 600 700 800 900 dif
-0.5
-1
-1.5
-2
RECOMMENDATIONS
• Calibrate the pH meter immediately
• Turn on the computer and the spectrophotometer
before you start the experiment
• Use a magnetic stirrer to mix the solution faster when
adjusting the pH
ANSWERS TO QUESTIONS
1. You will observe that the two colors of the indicator at the extreme pH
values give different scanning curves. Comment on the relationship between
the color of the substance and the λmax.