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Lab report on spectophotometry
- Instelling
- Universiteit Utrecht (UU)
This document is a lab report on spectometry, the experiment was done in a first-year lab for the module 'Chemistry for Biologists'.
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Experiment 3: SpectrophotometryAnne Versluijs, CHU11B01 23/24, 12/10/23
Introduction
This experiment consists of two parts. The aim of part one of the experiment is to confirm the
Beer-Lambert law for KMnO4. The aim of the second part of the experiment is to estimate the
equilibrium constant for the formation of Fe(NSC)2+ from Fe3+ and NCS-.
Part 1: the Beer-Lambert law for KMNO4
Absorption of radiation can stimulate a transition from a low-energy state to one of higher
energy. A photo spectrometer can be used to monitor the absorption of light passing through
a sample at a certain wavelength (Atkins & De Paula, 2018).
According to the Beer-Lambert law absorption of radiation of monochromatic light is directly
proportional to the path length, the concentration of absorbing species, and the extinction
coefficient (“Handbook of UV Degradation and Stabilization,” 2015).
𝐼0
𝐴 = log10 = 𝜀𝑐𝑙
𝐼
The logarithm of the intensity of transmitted light (I) divided by the intensity of incident light
(I0) is the absorbance A. In this formula 𝜀 is the extinction coefficient, c the concentration of
the sample, and l the thickness of the sample.
Part 2: the equilibrium constant for the formation of Fe(NSC)2+
The equilibrium of a reversible chemical reaction is the state in which the forward and
reverse processes occur at equal rates (Flowers et al., 2019). The equilibrium constant for
the reaction between ferric (aq) and thiocyanate ions (aq) is given by:
[Fe(NSC)2+ ]
𝐾𝑐 =
[𝐹𝑒 3+ ][𝑁𝐶𝑆 − ]
Fe3+(aq) + NSC- (aq) FeNCS2+(aq). The reaction has very fast forward and reverse
reaction rates, meaning that an equilibrium is reached quickly (Lahti et al., 1999).
The equilibrium concentration can be determined from the measured absorbance of the
solution using a spectrophotometer (University of Colorado, n.d.). This is made possible
because Fe(NSC)2+ absorbs wavelengths that correspond with blue and green light (458
nm), making for a solution deep red in colour. Fe3+ and NCS- ions do not absorb visible light.
Experimental procedure
Part 1
The materials used were 4 test tubes, 6 cuvettes, a 10 mL and 5mL pipette, and a UV-VIS
spectrophotometer.
This method is based on the method in the ‘Junior Fresh Practical Chemistry for Life and
Health Sciences’ lab manual.
- 4 clean test tubes were prepared and labelled 1 to 4. 10mL of 4*10-4 M KMNO4 was
added into test tube 1 using a pipette.
- 5mL of test tube 1 was pipetted into test tube 2 and 5mL distilled water was added.
- 5mL of test tube 2 was pipetted into test tube 3 and 5mL distilled water was added.
- 5mL of test tube 3 was pipetted into test tube 4 and 5mL distilled water was added.
, - A baseline was run through the spectrophotometer using distilled water in a range of
500-550 nm, with the solutions in test tubes 1 to 4. The absorbances of test tubes 1
to 4 were read at 524 nm.
- Two samples with an unknown concentration of KMnO4 were run through the
spectrophotometer. The absorbance was read at 524 nm.
Part 2
The materials used were 5 test tubes, a 50mL volumetric flask, 4 25mL volumetric flasks, a
25mL, 10mL and 5mL pipette, 6 cuvettes and a UV-VIS spectrophotometer.
This method is based on the method in the ‘Junior Fresh Practical Chemistry for Life and
Health Sciences’ lab manual.
- 5 clean test tubes were labelled ‘a’ to ‘e’. Into each 5mL of a 0.0004 M NaNCS
solution was pipetted.
- To prepare solution A 25mL of a 0.8 M Fe(NO3)3 was pipetted into the 50mL
volumetric flask. 25mL of 2 M HNO3 was added afterwards. The mixture turned light
yellow-orange. 5 mL of solution A was pipetted into test tube ‘a’. The colour of the
solution turned deep red.
- Solution B was made by pipetting 10mL of solution A into a 25mL volumetric flask
and adding 15mL 1M HNO3. 5 mL of solution B was pipetted into test tube ‘b’.
- Solution C was made by pipetting 10mL of solution B into a 25mL volumetric flask
and adding 15mL 1M HNO3. 5 mL of solution C was pipetted into test tube ‘c’.
- Solution D was made by pipetting 10mL of solution C into a 25mL volumetric flask
and adding 15mL 1M HNO3. 5 mL of solution D was pipetted into test tube ‘d’.
- Solution E was made by pipetting 10mL of solution D into a 25mL volumetric flask
and adding 15mL 1M HNO3. 5 mL of solution E was pipetted into test tube ‘e’.
- A baseline was run through the spectrophotometer using distilled water in a range of
450 – 500 nm, with the solutions in test tubes ‘a’ to ‘e’. The absorbances of test tubes
‘a’ to ‘e’ were read at 458 nm.
Results
Part 1
Table 1 Spectrometer results KMnO4 at 524 nm
[KMnO4] (10^-4 mol/dm^3) Absorbance
0,5 0,121
1,0 0,247
2,0 0,494
4,0 0,998
The absorbances measured by the spectrophotometer were collected in a plot using Excel. A
trendline was calculated by the computer program. The formula was added in the lower right
corner.
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