Spectrophotometer

Objectives:
1. To determine the wavelength of maximum absorption, Amax, of bromophenol blue.
2. To construct a standard concentration curve of bromophenol blue.
3. To determine the concentration of the unknown bromophenol blue solutions.
4. To determine the concentration of two different solutes, bromophenol blue and methyl orange in a mixture.
Materials and Methods:
Refer to Biological Science practical manual page 5-11.
Results:
Part 1: Determination of Amax of bromophenol blue

The wavelength with maximum absorbance reading form the graph is 590nm.
Part 2: The effect of concentration on absorbance of bromophenol blue solution

Tube
1
2
3
4
5
6
Distilled water (ml)
2.5
2.0
1.5
1.0
0.5
0.0
Bromophenol blue 10 mg/L (ml)
0.0
0.5
1.0
1.5
2.0
2.5
Concentration of bromophenol blue (mg/L)
0.0
2.0
4.0
6.0
8.0
10.0
Absorbance at 590 nm (Amax of bromophenol blue)
0.000
0.135
0.199
0.404
0.596
0.724

From Figure 2 above, the molar absorptivity of bromophenol blue at 590nm is the gradient of the linear regression line which is 0.0744 L mg-1 cm-1.
Part 3: Determination of the concentration of the bromophenol blue solutions of unknown concentration By plotting on the standard concentration curve from Part 2, the concentration of bromophenol blue are as follows:

By using the formula of Beer-Lambert Law,

Part 4: The effect of concentration on absorbance of methyl orange solutions

From Figure 4 above, the molar absorptivity coefficient of methyl orange is the gradient of linear regression line at 460nm which is 0.0722 L mg-1 cm-1.

Part 5: Determination of the concentrations of two different solutes, bromophenol blue and methyl orange, in mixture C

The molar absorptivity coefficient of bromophenol blue at 460nm is 0.0036 L mg-1 cm-1.

The molar absorptivity coefficient of methyl orange at 590nm is 0.0003 L mg-1 cm-1.

According to Beer-Lambert law,

0.375 = 0.0744 (CBB) + 0.0003 (CMO) CBB =

References: 1. Hanna Instruments. Technical advice. http://www.hannacan.com/conseils_colorimetres_en.htm (accessed 6 March 2015). 2. Sausen Silmi. Determining the Concentration of a Solution: Beer 's Law. http://www.scienceteacherprogram.org/chemistry/sausen.html (accessed 6 March 2015). 3. Oliver Seely. Determination of the Mn Content of Steel. http://www.csudh.edu/oliver/che230/labmanual/mangnese.htm (accessed 7 March 2015).