Part 1: PhET/RSC Beer’s (Beer-Lambert) Law Simulation
Part 1 of today’s virtual practical is an interactive simulation to investigate the effect on absorption of different wavelengths of light as they pass through solutions of differing concentrations.
Introductory video
Beer’s Law simulation link
Step 1: create solutions of different concentrations
Create a solution with 0.5 L of solvent and one solute of your choice (found in the top right hand side drop down menu) until the solution is saturated and record that concentration (move the concentration probe over the solution). Repeat this with different solutes.
Q1: Are the concentrations of saturated solutions the same for different solutes? If not, why not?
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Starting again with one solute, create a solution with 0.5 L of solvent that is at its maximum (saturated) concentration with the solute of your choice, but do not use excess solute so care needs to be taken with the shaker!
Now dilute this saturated solution by adding 0.5 L of solvent. Measure the concentration of this solution. Now drain 0.5 L off of the solution. Repeat this dilution procedure 4 times. Note down the concentration after each dilution.
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Q2: What is the dilution factor for the above dilutions?
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Step 2: Factors affecting absorbance
Now switch to the Beer’s Law tab on the bottom of the screen within the PhET simulation.
Start this simulation by turning on the light with the large red button on the left-hand side. This will be preset to a particular wavelength depending on the sample analysed.
Q3: Why has the wavelength been preset to a particular value for the sample you are investigating?
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Move the ruler on the bottom left hand corner over the width of the sample and measure the distance the light will pass through of that sample and make a note.
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Q4: Why are we measuring the distance the light passes through the sample?
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Using the concentration slide bar at the bottom, change the concentration three times. Make a note of the path length, concentration and absorbance values.
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Q5: What effect does increasing concentration have on absorbance, and why?
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Now either increase or decrease the pathlength of the sample by moving the double-headed yellow arrow, making a note of the length.
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Q6: How does changing the path length of the sample affect absorbance?
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Underneath the light source, toggle to the ‘variable’ wavelength option. Move the slide bar three times to different wavelengths of light. At each wavelength make a note of the absorbance value.
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Q7: How does changing the wavelength of light affect the absorbance value and why? What is lmax?
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Q8: With the above information you have just gathered in mind, what relationship can be observed between absorbance, path length and concentration?
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Part 2: Exactly how red are tomatoes?
Lycopene is an unsaturated hydrocarbon natural product that is abundant in tomatoes. While many small-molecule organic compounds are colourless, lycopene is strikingly red.
Figure 1: The (skeletal) molecular structure of lycopene.
The molecule’s region of conjugated (connected in a row) carbon-carbon double bonds (C=C) is the extended structural feature that gives rise to the absorption of light that causes lycopene to appear coloured to our eyes.
Q9: In lycopene’s structure:
a) How many conjugated C=C double bonds are there? ………………………….
b) How many methyl groups (CH3) are there? …………………………………………
Figure 2: The ultraviolet-visible absorption spectrum of lycopene.
Like most dyes, lycopene absorbs some wavelengths (l) of light more than others, and these can be determined by a UV-vis spectrum.
Q10: a) Using Figure 2, determine the approximate lmax (nm) of lycopene.
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b) Which part (colours) of the visible spectrum is lycopene absorbing?
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c) What colour bias has the remaining light that lycopene reflects and transmits?
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The Beer-Lambert Law indicates that the absorbance of any substance is proportional to its concentration as follows:
Assuming the Beer-Lambert law is obeyed, the concentration of a lycopene solution can be quantified by measuring its absorbance. For lycopene this is most conveniently monitored at 503 nm.
Q11: Circle and label lycopene’s 503 nm absorption peak on Figure 2.
Every dye has its own unique absorption coefficient e, and the greater this value the ‘darker’ and more intensely absorbing it is.
Your goal is to determine lycopene’s e value using real lab data.
Note that the units of e are cm-1 M-1, i.e. absorption per centimetre of path length, per molarity unit of concentration. It is for this reason that UV-vis machines analyse samples in a special glassware of exactly 1 cm cross-section (pictured with lycopene solution).
Q12: What is the name of the small glass vessels used in UV-vis analysis? …………………………………………
Procedure
Lycopene samples of known concentration will be prepared by diluting a stock solution. The solvent for the stock and all dilutions is 20% v/v ethanol-water mixture.
1. Weigh out 0.100 g of lycopene into a weighing boat.
2. Wash the powder thoroughly into a 5L volumetric flask and top up to the line.
3. Shake the mixture to ensure homogeneity.
4. Set out 10 volumetric flasks (250 mL), labelling them A to J.
5. To each add the appropriate volume of lycopene stock solution, as shown in the table below, and shake each to mix thoroughly.
6. In the spectrophotometer, run a blank sample of pure solvent to compensate for any absorbances from the solvent or background.
7. Now proceed to measure the absorbance at 503 nm for of each of solutions A to J.
Q13: Why can pure water not be used for the solvent?
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Q14: What kind of pipette would be best for measuring different volumes of stock?
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Results
Q15: Calculate the concentration of each of your diluted solutions in mg/L and enter these into the blank column of the table.
Q16: On the graph paper, (using sharp pencil) plot absorbance (A503) against concentration (mg/L). Choose a large but sensibly convenient scale for your two axes and use fine crosses to mark your data points.
Q17: Using a ruler if relevant, draw a best fit line that encompasses the apparent trend and extend this beyond the data set in both directions.
Q18: Circle any outlying data points. What is the best way to handle these?
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Q19: Why are some data points not exactly on the line? What type of uncertainty (error) causes this scatter? ………………………………………………………………..
Q20: By drawing a large triangle at your trendline (but away from any individual data points), measure the gradient of the trendline (Dy/Dx).
Gradient of my line (with units): ………………………………………………….
Q21: Given that lycopene has a molar mass of 536.9 g/mol, calculate lycopene’s molar extinction coefficient (e):
e = …………………… M-1
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