EXPERIMENT 5

SPECTROPHOTOMETRIC DETERMINATION OF pKa

 OUTLINE
EXPERIMENT 5

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
NaOH to acidic solution. Adjust
also the pH of the solutions 2-9,
with appropriate intervals so
that the pH of the 10 solutions
should have 1-13 pH levels.
After preparing the 10
solutions, immediately adjust
the pH of the solutions 1 and
10, solution 1 should have a
pH of 1 while 10 should have
the pH of 13
Make use of solution 1 and 10
first, rinse the cuvette with
solution 1 three times and fill
the cuvette 2/3 of its capacity.
Do this with solution 10 also.
After adjusting the pH of
solutions 1-10, do a
wavelength scan of these
solutions using
spectrophotometer.
PROCEDURE
Record the wavelength, Measure the absorbance of the other
absorbances of solutions 1 and eight solutins using only the gathered
10. find the difference of the λ1 and λ2 as wavelengths
two absorbances then plot
absorbance vs. wavelength.

Place the cuvettes, 1- distilled

water, 2- solution1 and 3- Note the maximum and
solution 10. scan the samples minimum point on the graph
recording the absorbance of label this as λ1 and λ2.
the solution from 350nm to
800 nm at 10nm intervals.
 Sodium Sodium Distilled Concentr 0.1M
Indicator
Solution: Phosphat Diphosph Water ated HCl NaOH pH
(mL)
e (mL) ate (mL) (mL) (mL) (mL)
1 (dilute
to 25 1 0 0 4 0.2 -- 1
mL)
2 1 5 0 -- -- -- 2
3 1 5 1 -- -- -- 3.43
4 1 5 5 -- -- -- 4.86
5 1 10 5 -- -- -- 6.29
6 1 5 10 -- -- -- 7.72
7 1 1 5 -- -- -- 9.15
8 1 1 10 -- -- -- 10.52
9 1 0 5 -- -- -- 12
10 1 0 0 -- -- 24 13
DISCUSSION
GROUP 1 AND 3
ƛ1=401 nm
DIFF VS. WAVELENGTH ƛ2=521 nm
2.000

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

2 2.01 0.201 3.052

3 3.45 0.212 1.633

4 4.43 0.985 1.683

5 6.25 2.264 0.81

6 7.64 3.295 1.242

7 9.16 3.084 1.202

8 10.54 3.248 1.263

9 11.98 3.111 1.118

10 12.76 2.988 0.881

GROUP 2
Solution No. pH as adjusted [Hln] [In-] pKa

2 2.01 0.081 0.017 -0.678 1.993

3 3.45 0.048 0.018 -0.417 3.867

4 4.43 0.046 0.028 -0.216 4.646

5 6.25 0.004695 0.075 1.203 5.047

6 7.64 0.008914 0.109 1.087 6.553

7 9.16 0.009646 0.102 1.022 8.136

8 10.54 0.01 0.108 1.033 9.507

9 11.98 0.006615 0.103 1.192 10.788

GROUP 2 14

12

10

f(x) = 3.75x + 4.95

R² = 0.75
8
pH

0
-1 -0.5 0 0.5 1 1.5

��� ([��−])/([���])
GROUP 4
2

1.5

0.5

0
300 400 500 600 700 800 900

-0.5

-1

-1.5

-2

ABS 1 ABS10 DIFF

GROUP 4
Solution No. pH as adjusted Absorbance at Absorbance at

1 1 1.815621833 0.094568267

2 2.31 1.584293333 0.2287485

3 3.46 2.0892179 0.473059633

4 4.83 0.7146009 0.826530867

5 6.3 0.178738467 1.0585813

6 7.7 1.7930059 0.094528967

7 9.15 0.1089349 1.116399867

8 10.56 0.110994167 1.204472967

9 11.99 0.1080185 0.930251033

10 13 0.102102867 1.0111468
GROUP 4
HIn In pKa

18.156 1.021 2.249994432 14

12
15.843 1.08 3.476413667
10
20.892 1.1099 4.734696166
8
7.146 1.089 5.647035132

pKa
6

1.787 17.93 5.298544263

4

10.586 0.9453 8.749162232 2

8.265 11.164 9.01942303 0

0 2 4 6 8 10 12 14

pH
4.7306 12.045 10.15410942

2.288 9.303 11.380833

0.9457 10.1115 11.9709378

GROUP 5
2.5

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

2 2.66 1.6985716 0.0584772

3 3.25 1.5460361 0.2210995

4 4.88 1.2814844 0.5415226

5 6.33 0.4236569 1.4367165

6 7.74 0.2452599 2.7667481

7 9.12 0.1644434 2.5847152

8 10.54 0.2230504 2.6182042

9 11.99 0.3021874 3.0599169

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

2 2.01 0.622783 0.7088602

3 3.43 1.0907634 1.2865518

4 4.86 0.6084439 0.6911659

5 6.29 0.2033223 0.1326611

6 7.72 0.1284271 0.0368175

7 9.13 0.1552028 0.0570897

8 10.57 0.1282185 0.0358453

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.

Wavelength is the distance between corresponding points in two consecutive

waves. In the field of spectroscopy, the component colors of the visible portion
can be separated by passing sunlight through a prism which acts to bend the light
in diferent degrees according to the wavelength. When white light is reflected by
a colored substance, a portion of the mixed wavelength is absorbed, and the
remaining light will then assume the complementary color to the wavelengths
absorbed. The relationship of the wavelength and the color of the substance can
be determined by the image shown. The image displays a ROYGBIV pattern which
includes a wavelength in increasing order from left to right.
2. What is the significance of pKa?
pKa is a property of a compound that measures the strength of an acid in a
solution. The lower the pKa, the more acidic the compound, and the more it
dissociates in water. It is essential in the fields of water treatment and drug
development.
3. Which is the favored species of the indicator (Hln o ln-) in a neutral
environment?
An indicator is a solution that changes color at a specific pH. One kind of
indicator is the acid-base indicator which is a complex organic substance that
is a weak acid and an example of this kind of indicator is the methyl red
indicator. Methyl red is the yellow weak acid and when combined water it
dissociates thus forming red colored solution in a neutral environment. Under
acidic conditions, the equilibrium of the solution is to the left and the
concentration is too low for the red color to be observed. However, under
neutral and basic/alkaline solutions, the equilibrium is shifted to the right
wherein the concentration is enough for the red color to be observed thus
making the methyl red favor the alkaline conditions in a neutral environment.
4. What are the other regions of spectroscopy aside from the visible region?
The other regions for of spectroscopy, in order of decreasing hazard, are
gamma, x-ray, UV, IR, microwave and radio. Gamma, x-ray and high UV
exposure can cause chemical reactions with their high energy that are
enough to ionize atoms and cause damage to one’s health by radiation
sickness, DNA damage, or cancer. The rest cannot cause such efects.
REFERENCES
• L. (2016, July 21). Spectrophotometry. Retrieved May 13, 2018, from
https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/
Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectr
ophotometry
• Libertin, C. R., Panozzo, J., Groh, K. R., Chang-Liu, C. M., Schreck, S., &
• Woloschak, G. E. (1994). Efects of gamma rays, ultraviolet radiation,
sunlight, microwaves and electromagnetic fields on gene expression
mediated by human immunodeficiency virus promoter. Retrieved May
13, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/7938460
• Visible and Ultraviolet Spectroscopy. Retrieved May 13, 2018 from
https://www2.chemistry.msu.edu
• Manallack, D. T. (2007). The pKa Distribution of Drugs: Application to
Drug Discovery. Perspectives in Medicinal Chemistry, 1, 25–38.
• Acid-Base indicators. Retrieved May 13,2018 from
https://schools.ednet.ca
Thank You For Listening