MODULE 8.
VISIBLE SPECTROPHOTOMETRY
8.1 Introduction
Spectrophotometer is an analytical apparatus that can be used for quantitative or qualitative
measurement, by passing through the light (specifically generated by light source and was
selected by monochromator inside the spectrophotometer) from a clear solution. The
spectrum of monochromatic light compelled colour solution, when it is passed throught the
solution, some will be absorbed and some will be transmitted. The one that get absorbed is
quantitatively equivalent with the amount of species activity inside the cuvet and solution. In
order to comply the purpose of analysis, a blanko is applied thus the influence of cuvet system
and solvent matrix can be used as baseline of analysis. The deviation between intensity signal
of light spectrum after passing through blanko sample is a signal that was generate by analyte
activity. Spectronic-20 is one one visible-spectrophotometer that can be used on a range of
wavelength between 400-600 nm.
The first step to analysis the compound using spectronic-20 is by determine the maximum
wavelength (λ) and create the correlation spectrum between absorbance (A) in y-axis and
wavelength in x-axis. This correlation can be seen on Figure 12.
The peak point of curve show an optimum
wavelength in a maximum absorbance
Wavelength (nm)
Figure 12. Absorbance spectrum on various wavelengths
The maximum wavelength is a wavelength which shows maximum absorbance. This maximum
absorbance shows that monochromatic spectrum passed through sample is a spectrum that
complement with the colour of sample solution. The measurement of analyte absorbance on a
maximum wavelength (λ-max) is a important to produce data with good stability. In this λ-max,
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measurement noise because of spectrum shifting or electrical noise can be surpressed.
Manually, maximum wavelength can be obtained from the measurement absorbance spectrum
of transparent colour solution with precise concentration, scanning can be done on available
wavelength based on monochromator filter.
In the analysis using visible spectrophotometer, the absorption of light spectrum on λ-max
(specific for each compound/solution), is equal with the concentration of compound in the
solution. This statement was described on the correlation between concentration (C) and
absorbance (A), which is known as Beer-Lambert Law.
A = -log T = ε b C
where :
A : absorbance
T : transmittant
b : cuvet width (cm)
C : concentration (Molar)
ε : coefficient of molar absorptivity
The correlation between A and C (coefficient of molar absorptivity and cuvet width, are known
as constant, K) will be linier on exact range of concentration. The liniearity of this correlation
curve will determine the size of spectrophotometry analysis detectable limit (limit of detection,
LoD) on a good measurement range. On this linier area, error measurement were negligible
and this is in accordance with the justification of error factor on Beer-Lambert equation
conIn order to get the range and liniearity of the measurement, the analysis should be done on
various absorbance measurement (A) from a series of concentration. Next, the correlation of
absorbance and concentration can be plotted as follows (Figure 13).
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Figure 13. Standard correlation curve between absorbance and concentration
8.2 Objectives
1. Students are able to make a correlation spectrum between absorbance of various
wavelengths with the concentration of the solution.
2. Students are able to determine optimum wavelength for each solution.
3. Students understand the basic concept of Beer-Lambert Law
4. Students are able to make standard curve and select the concentration which meet Beer-
Lambert Law, i.e., the point which in linier area between concentration (C) and
absorbance (A)
8.3 Apparatus and Materials
1. Apparatus:
a. Volumetric pipettes
b. Pipette dropper
c. Wash bottle
d. Burette
e. Beaker glass
f. Erlenmeyer flask 250 mL
g. Volumetric flask 100 mL
h. Cuvet
i. Spectrophotometer (Spectronic-20).
2. Materials:
a. KMnO4 10-3 M solution
b. K2Cr2O7 10-3 M solution
c. Aquadest
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8.4 Experimental Procedures
8.4.1 Experiment I (Determination of Maximum Wavelength on a Measurement)
1. Turn on the spectrophotometer (Spectronic-20) and let it stable for 30 min.
2. Turn the wavelength into 380 nm
3. Turn the transmittant (%T) to “0’” (without cuvet)
4. Put the cuvet filled with aquadest inside the spectrophotometer, and turn the
transmittan (%T) to 100.
5. Take out the cuvet filled with aquadest, and replace with cuvet filled with KMnO4 10-4
M or K2Cr2O7 10-4 M, then record the transmittant (%T) of the solution at 380 nm
wavelength
6. Take out the cuvet, then turn the wavelength to 400 nm
7. Repeat procedure 3 to 5
8. Repeat the procedure on other wavelength up to 620 nm, with a range of 20 nm for each
wavelength. However, at the nearest maximum wavelength, procedure can be
conducted every ±5 nm
9. Convert %T to A with this equation: A = -logT
10. Draw the absorbance spectrum for each solution
11. Determine the maximum wavelength for each solution
12. Compare both spectrum and define the answer.
8.4.2 Experiment II (Determination of Measurement Range and Detectable Limit on a
Linier Measurement)
1. Prepare various concentration of KMnO4 solution, i.e., 1x10-4 M, 2x10-4 M, 4x10-4 M,
6x10-4 M, 8x10-4 M, and 1x10-3M
2. Measure %T from each concentration using maximum wavelength obtained from
experiment I
3. Convert %T into absorbance value (A)
4. Draw correlation graph between concentration (C)on x-axis and abosorbance (A) on
y-axis
5. If the range of those concentrations shows a linier graph, add bigger concentration
on the line.
6. Repeat the procedure 1 to 5 using K2Cr2O7
7. Compare and describe the results
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8.5 In-depth knowledge
1. Illustrate the scheme of instrument analysis and describe the concept of
spectrophotometry.
2. Define the optimum/maximum wavelength!
3. Why measurements have to be made on the range of linier measurement?
4. Why visible-spectrophotometer can only be used for the analysis of clear/
transparent coloured solution?
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