# Theory of Photometry Essay

1408 Words Jun 5th, 2014 6 Pages
Biochem 300 Lab
Fall 2012 Lab #1
Theory of Photometry
In this lab we are conducting a series of test to verify the theory of photometry. A spectrophotometer is used to measure the absorbance of light of a sample. The absorption of light mechanism consist of a light bulb, a compartment to place the test tube, a wavelength selector, a slit a light detecting photocell and a meter to show the measurement of the sample. The compound can absorb a photon of light, if the energy of the photon, is equal to the amount of energy needed to emit electrons of the sample to a higher energy orbital. The mechanism works by turning a light bulb, which permits light to go through a prism that is set in a specific wavelength, then this light is
Experimental results:
The wavelength of the absorbance peak found was at 400nm which is fairly close to the standard λ that was used to conduct the experiment of 405nm. It was also noted the PNP solution changed form a clear solute at pH of 4 to a yellow solute at pH of
10.
Data Collected:
Worksheet #1
Test
tube
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

Volume of
0.12
μmole/ml
PNP (mls)
0.0
0.5
0.5
1.0
1.0
1.5
1.5
2.0
2.0
2.5
2.5
xxx xxx xxx xxx xxx xxx xxx xxx Volume of
Unknown
#85 (mls) xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx 0.5
0.5
1.0
1.0
1.5
1.5
2.0
2.0

Volume of pH
4 Buffer B
(mls)
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0

Volume of 0.1M, pH 11 Na
Carbonate (mls)
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0

Calculations:
For test tubes 2-11
Concentration
of PNP
X
(µmoles/ml)
Equation

Volume Of
(ml)
Results

=

Amount of
PNP in Tube
(µmoles)

Absorbance
(at 405nm)
0.000
0.210
0.210
0.420
0.425
0.660
0.665
0.840
0.840
1.060
1.060
0.055
0.055
0.110
0.115
0.160
0.170
0.220
0.220

Amount of
PNP in tube
(μmoles)
0.000
0.060
0.060
0.120
0.120
0.180
0.180