Lab Bio – 103
Experiment: September 5, 2018
Report due: September 17, 2018
Lab Report 1: Spectrophotometry
Determination of Concentration by Light Absorbance
This experiment introduced the direct measurement approach to absorption spectrophotometry.
Purpose: Recognize the proper functioning of the spectrophotometer, acquire knowledge of the
absorption of light in some compounds, understand quantitative relationships between
transmittance, absorption and concentration, and to use spectrophotometric data to quantify
copper (II) ion concentration is an unknown sample.
Introduction: Visible light spectrum extends between 400 to 700 nm wavelength, contains all the
colors of the rainbow and is the part of the electromagnetic radiation that can be perceived by the
human eye. What we perceived as color is the ability of a substance to absorb and reflect light in
a certain part of the visible spectrum. The amount of light absorbed by a substance is determined
with the use of a spectrophotometer, which is a laboratory equipment that measures the amount
of light intensity absorbed, after passing a specific wavelength through a sample solution.
Spectrophotometer measurements yield light absorbance numerical values, which are used to
calculate the concentration of a solution in accordance to the Beer-Lambert Law, which
expresses the relationship between light absorbance and concentration in a solution A= εcl. That
is, the amount of light absorbed by a solution is directly proportional to its concentration,
therefore; the concentration of a solution can be determined by measuring its absorption of light
A
at a specific wavelength and rearranging the equation: C = εI . In addition, the concentration
of test samples can also be determined by means of a standard curve, which is a graph that
relates known concentrations of substances with the absorbance of each of them at the maximum
wavelength.
Methods: Prepare six test tubes, place only distilled water in test tube one which will blank the
spectrophotometer, place standard copper sulfate (II) and water in test tubes two to five and
unknown copper sulfate (II) in test tube six, following the table below:
Test Tube # 1 2 3 4 5 6
Water (mL) 10.0 8.0 6.0 4.0 2.0 0
Standard copper (II) sulfate (mL) 0 2.0 4.0 6.0 8.0 -
Unknown copper (II) sulfate - - - - - 10.0
(mL)
Once all six test tubes are ready, the next step is to calibrate the spectrophotometer by adjusting
the absorbance to zero, which means that the colorimeter is left blank. First, connect and turn on
the unit, a colorimeter display should appear on the screen with transmittance values (% value)
and absorbance values of the colors, red (660 nm), green (565 nm), blue (468 nm) and orange
(610 nm). Then, pour the water from test tube one into the colorimeter bottle, place it into the
1
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, Lab Bio – 103
Experiment: September 5, 2018
Report due: September 17, 2018
holder of the unit, close the top and press the green button only once to set the absorbance to zero
and blank the first solution, which means that the absorbance value in the orange color should
show 0.000. After this, remove the bottle from the holder, pour the contents back into tube 1,
pour the contents of tube 2 into the colorimeter bottle, place it in the unit, do not press the green
button, the screen will show the absorbance reading and record it for the orange wavelength 610
nm in the data sheet. Next, pour the contents back into tube 2, clean the colorimeter bottle, pour
the contents of tube 3, place it in the unit and record readings in the data sheet. Repeat this
procedure for the remaining test tubes and record readings.
Results:
1. Report Sheet
Volume, mL Concentration, mM Absorbance (at 610 nm)
0 0 0
2.0 0.40 0.25
4.0 0.80 0.50
6.0 1.20 0.75
8.0 1.60 1.00
10.0 UNKNOWN 3.00
2. Calculations of Potassium Dichromate concentration in each of the samples test-
tubes 1-5
Test-tube # 1: Only water
Test-tube # 2: C1V1 = C2V2
2
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Experiment: September 5, 2018
Report due: September 17, 2018
Lab Report 1: Spectrophotometry
Determination of Concentration by Light Absorbance
This experiment introduced the direct measurement approach to absorption spectrophotometry.
Purpose: Recognize the proper functioning of the spectrophotometer, acquire knowledge of the
absorption of light in some compounds, understand quantitative relationships between
transmittance, absorption and concentration, and to use spectrophotometric data to quantify
copper (II) ion concentration is an unknown sample.
Introduction: Visible light spectrum extends between 400 to 700 nm wavelength, contains all the
colors of the rainbow and is the part of the electromagnetic radiation that can be perceived by the
human eye. What we perceived as color is the ability of a substance to absorb and reflect light in
a certain part of the visible spectrum. The amount of light absorbed by a substance is determined
with the use of a spectrophotometer, which is a laboratory equipment that measures the amount
of light intensity absorbed, after passing a specific wavelength through a sample solution.
Spectrophotometer measurements yield light absorbance numerical values, which are used to
calculate the concentration of a solution in accordance to the Beer-Lambert Law, which
expresses the relationship between light absorbance and concentration in a solution A= εcl. That
is, the amount of light absorbed by a solution is directly proportional to its concentration,
therefore; the concentration of a solution can be determined by measuring its absorption of light
A
at a specific wavelength and rearranging the equation: C = εI . In addition, the concentration
of test samples can also be determined by means of a standard curve, which is a graph that
relates known concentrations of substances with the absorbance of each of them at the maximum
wavelength.
Methods: Prepare six test tubes, place only distilled water in test tube one which will blank the
spectrophotometer, place standard copper sulfate (II) and water in test tubes two to five and
unknown copper sulfate (II) in test tube six, following the table below:
Test Tube # 1 2 3 4 5 6
Water (mL) 10.0 8.0 6.0 4.0 2.0 0
Standard copper (II) sulfate (mL) 0 2.0 4.0 6.0 8.0 -
Unknown copper (II) sulfate - - - - - 10.0
(mL)
Once all six test tubes are ready, the next step is to calibrate the spectrophotometer by adjusting
the absorbance to zero, which means that the colorimeter is left blank. First, connect and turn on
the unit, a colorimeter display should appear on the screen with transmittance values (% value)
and absorbance values of the colors, red (660 nm), green (565 nm), blue (468 nm) and orange
(610 nm). Then, pour the water from test tube one into the colorimeter bottle, place it into the
1
This study source was downloaded by 100000830604181 from CourseHero.com on 04-19-2022 03:38:23 GMT -05:00
https://www.coursehero.com/file/34648446/Lab-Report-1-Spectrophotometrydocx/
, Lab Bio – 103
Experiment: September 5, 2018
Report due: September 17, 2018
holder of the unit, close the top and press the green button only once to set the absorbance to zero
and blank the first solution, which means that the absorbance value in the orange color should
show 0.000. After this, remove the bottle from the holder, pour the contents back into tube 1,
pour the contents of tube 2 into the colorimeter bottle, place it in the unit, do not press the green
button, the screen will show the absorbance reading and record it for the orange wavelength 610
nm in the data sheet. Next, pour the contents back into tube 2, clean the colorimeter bottle, pour
the contents of tube 3, place it in the unit and record readings in the data sheet. Repeat this
procedure for the remaining test tubes and record readings.
Results:
1. Report Sheet
Volume, mL Concentration, mM Absorbance (at 610 nm)
0 0 0
2.0 0.40 0.25
4.0 0.80 0.50
6.0 1.20 0.75
8.0 1.60 1.00
10.0 UNKNOWN 3.00
2. Calculations of Potassium Dichromate concentration in each of the samples test-
tubes 1-5
Test-tube # 1: Only water
Test-tube # 2: C1V1 = C2V2
2
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