Absorption Spectra

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Introduction

At any particular wavelength the absorption of light when visible light passes through a solution depends on two factors: * The length of the light path
* The concentration of the colored path
The connection of these two variables is known as the Beer-Lambert Law:

Absorbance (A) = ε c l

Where ε is the absorption coefficient
C is the concentration of the compound
And l is the length of light usually 1cm

When I is constant, this proves a linear relationship between absorbance and concentration. This means it can be rearranged to find the find the optimum concentration for maximum absorbance.

C= A/ ε

Methods:

The two reagents used are Bromophenol blue and Methyly orange.

Experiment 1:

1 Set up the colorimeter at 400nm, measure the absorbance of both reagents against distilled water, this will be used as a blank (make sure to zero the colorimeter after each interval)

2 Repeat this procedure at 20nm from 420nm to 700nm

3 After getting a range of results make further readings in smaller intervals to precisely locate the wavelength for λmax

Experiment 2:

1 Using the protocol provided prepare a series of dilutions in testubes for each compound for the range of concentrations from 1 to 10mg/l.

2 Using the λmax for each reagent from Experiement 1 set the colorimeter against distilled water and measure the absorption

3 Repeat for other dilution series

Results:
Experiment 1:
Bromophenol Blue|  | Further readings to determine λ max|  | Wavelength (nm)| Absorbance| Wavelength (nm)| Absorbance| 400| 0.093| 595| 0.98|
420| 0.113| 590| 0.942|
440| 0.035| 610| 0.672|
460| 0.048| | |
480| 0.069| | |
500| 0.111| | |
520| 0.154| | |
540| 0.301| | |
560| 0.451| | |
580| 0.728| | |
600| 0.976| | |
620| 0.347| | |
640| 0.066| | |
660| 0.011| | |
680| 0.002| | |
700| 0| | |

Methyl orange|  | Further readings to determine λ max|  | Wavelength (nm)| Absorbance| Wavelength (nm)| Absorbance| 400| 0.377| 470| 0.723|
420| 0.51| 465| 0.731|
440| 0.619| | |
460| 0.687| | |
480| 0.686| | |
500| 0.515| | |
520| 0.513| | |
540| 0.119| | |
560| 0.033| | |
580| 0.006| | |
600| 0.001| | |
620| 0| | |
640| 0| | |
660| 0| | |
680| 0| | |
700| 0| | |

Absorbance
Absorbance

Wavelength
(Nm)
Wavelength
(Nm)

Experiment 2:
Tube No.| Stock solution (10mg/l) ml| Distilled water (ml)| Concentration (mg/L)| Concentration%| 1| 0.3| 2.7| 1| 0.0001%|
2| 0.6| 2.4| 2| 0.0002%|
3| 0.9| 2.1| 3| 0.0003%|
4| 1.2| 1.8| 4| 0.0004%|
5| 1.5| 1.5| 5| 0.0005%|
6| 1.8| 1.2| 6| 0.0006%|
7| 2.1| 0.9| 7| 0.0007%|
8| 2.4| 0.6| 8| 0.0008%|
9| 2.7| 0.3| 9| 0.0009%|
10| 3| 0| 10| 0.001%|

 | bromophenol blue| methyl orange|
Tube number| Absorbance at λ max| Absorbance at λ max| 1| 0.102| 0.139|
2| 0.207| 0.181|
3| 0.324| 0.22|
4| 0.403| 0.297|
5| 0.504| 0.365|
6| 0.673| 0.444|
7| 0.699| 0.563|
8| 0.839| 0.613|
9| 0.934| 0.68|
10| 1.028| 0.733|

Concentration%| Bromophenol blue| Methyl orange|
Concentration%| Absorbance at λ max| Absorbance at λ max| 0.0001%| 0.102| 0.139|
0.0002%| 0.207| 0.181|
0.0003%| 0.324| 0.22|
0.0004%| 0.403| 0.297|
0.0005%| 0.504| 0.365|
0.0006%| 0.673| 0.444|
0.0007%| 0.699| 0.563|
0.0008%| 0.839| 0.613|
0.0009%| 0.934| 0.68|
0.001%| 1.028| 0.733|

Absorbance
Absorbance

Concentration (Mg/l)
Concentration (Mg/l)

Calculations:

Concentration is derived from the formula C1V1=C2V2
C1= 10 mg/l
V1= stock solution so for tube 1 its 0.3
C2= unknown concentration
V2= 3 ml stock solution + distilled water = 3
To find unknown concentration its [(C1xC1)/v2] so for the Tube...
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