Overview

In this experiment we introduce you to luminescence spectroscopy. You’ll also explore some of the advantages and disadvantages of this technique compared to absorption spectroscopy, and gain practice measuring and evaluating the detection limits of an analyte using luminescence versus absorbance spectroscopy.

To cite this lab manual: “A Spectrophotometric Study of Fluorescein”. A Manual of Experiments for Analytical Chemistry. Department of Chemistry at UW- Madison, Summer 2024.

Visual Abstract

Background

Fluorescein is an organic compound used as a fluorescent tracer in a variety of products, ranging from the standard yellow highlighter pen, to the antifreeze for the radiator in your car, to medical and research applications that use fluorescence as a way to tag biological molecules for detection. You have used a structurally similar compound to detect the colorimetric endpoint of the titration between NaOH and potassium hydrogen phthalate. Phenolphthalein is different from fluorescein only by one oxygen atom, which bridges two of the aromatic rings in the latter as seen in Figure 1.

This small structural difference changes the spectral behavior of fluorescein significantly: the result allows fluorescein to be used as an agent in fluorescence spectroscopy, whereas phenolphthalein does not fluoresce.

Luminescence is observed when light is released from a molecule as electrons relax from an excited to a ground state. Phosphorescence occurs when the photon emitted results from a transition between different spin states, while fluorescence occurs when the transition happens within the same spin state (for example, between S₁→S₀). When a molecule fluoresces, emission occurs at longer wavelengths (or lower energy), compared to the excitation wavelength, since some of the excitation energy lost is due to vibrational relaxation.

More about the spectrometer

Your textbook covers how luminescence is used in analytical chemistry. In general, quantitative analysis is possible under the conditions where the intensity of the luminescence (I) is proportional to concentration (c). The intensity of the incident radiation (P₀), also impacts I and should remain constant when collecting standard and sample data.

To perform a quantitative study, a set of standards of known concentration must first be made and measured. The resulting linear standard plot can then be used to determine the concentration of the sample. Figure 2 illustrates the major internal components of the SpectroVis Plus Vernier instrument, which can be used for both fluorescence and absorption spectroscopy applications.

Notice that the excitation sources for fluorescence spectroscopy are offset 90° from the detector. This orientation minimizes the detection of scattered incident radiation, which is deflected by particles, the cuvette surface, and large molecules in solution. Similar to absorption measurements, fluorescence is measured on a relative scale of 0 – 1.

Just like Beer’s Law has limiting conditions, there are processes that limit the linear relationship between luminescence intensity and concentration. At high concentrations, for example, neighboring molecules in a sample can absorb light from an excited molecule before the light can leave the cuvette. A sample is said to be “quenched” when the analyte is so concentrated it absorbs its own fluorescence.

In this experiment you will compare and contrast spectra of fluorescein collected by absorption versus fluorescence spectroscopy. Note from the structure shown in Figure 1 that fluorescein is also a weak acid. As a result, the fluorescence of this molecule exhibits a small pH dependence, within the range from pH 5 to 11. For this reason, as you make your samples from the stock solutions provided, be sure to use 0.1 M NaOH when making dilutions.

Using the methodology you learned in previous experiments, your group will design an experiment to measure the method detection limit of fluorescein using fluorescence versus absorbance spectroscopy. A more complete explanation for determining a method detection limit is provided in the Spike Recovery and Method Detection Limit lab. Read it carefully and be sure you understand the procedure before coming to lab. Finally, you will measure the concentration of fluorescein present in antifreeze samples using a set of standards you make, as well as the spectroscopy your of choice.

Performing serial dilutions

Using serial dilutions to prepare a set of solutions (either for preparing samples or standards) is standard practice in the analytical lab. In using this method, you will perform successive dilutions of a stock standard (or sample).

Consider Figure 3, for example, in preparing standards for this experiment using the stock solution provided. In this case we start with a solution that is 7.5 × 10^-5 M fluorescein. To achieve a set of standards using a serial dilution scheme using a 100 mL flask for the dilutions, one solution is to take 50 mL of the stock and dilute to 100 (a 50:100 or 1:2 dilution), resulting in 7.5 × 10^-5 M/2 = 3.7₅ × 10^-5 M fluorescein solution. Next, you can do the same thing with this second solution (again a 1:2 dilution) resulting in a 3.7₅ × 10^-5 M/2 = 1.8₇ × 10^-5 M solution.

As you work to design your experiment for this lab, keep in mind the volumes of volumetric pipets available to you, which include 50, 20, 5, 2, and 1 mL volumes. Other pieces of glassware can be used to perform the volume transfer of the previous solution, including a 1 mL Mohr pipet for small volumes, your Eppendorf pipets (potentially not as accurate as good old glassware!), or a buret.

Lab Concept Video

Write your observations or notes from the video in your lab notebook.

Pre-lab Work

Lab Skills

Review these lab skills videos prior to lab.

Extra Resources:

• EPA Method Detection Limit Determination Procedure
• Using the LabQuest to Collect Spectrophotometry Data
• Making Scientific Plots

Prelaboratory Exercises

1. The stockroom will provide a stock solution of 7.5 × 10^-5 M fluorescein for preparing standard solutions. You will need to create a set of standards to calibrate the instrument in preparation for an absorption study of fluorescein. Design a table in your notebook describing how you would make four 100 mL solutions of fluorescein from this stock solution ranging in concentration from 1.0 to 75 µM fluorescein. Make sure you consider what pieces of glassware you have in the lab!
2. Using your lowest standard concentration from the previous question, create a new table for an additional four standards, where the concentrations range from 0.01 to 0.5 µM fluorescein. This will ROUGHLY be the concentration range where fluorescence measurements will be made. It will be up to you to decide where the cutoffs exist for when absorption versus fluorescence spectroscopies are best suited.
3. Consider the following intensity spectrum of fluorescein and phenolphthalein:
   
   Assuming a linear relationship is preserved between signal intensity and concentration, estimate in molarity units a detection limit for fluorescein (MW = 332.31) at 515 nm, using fluorescence spectroscopy. Remember your estimate should fall within a signal to noise range of 2.5 – 5.
4. What is the difference between the units of absorbance and relative intensity?

Before You Take The Quiz on Canvas

• Review the EPA Procedure for Method Detection Limit handout and understand how to determine a suitable, estimated detection limit.
• Have confidence in how to perform serial dilutions, and understand the process for performing this important technique.
• Understand how to calculate the concentration of an unknown sample given the luminescence of an unknown sample and a calibration curve of luminescing standards.
• Know the difference between absorption and luminescence, and how these differences impact the instrumentation used for quantitative study.

Experimental

Part 1: Determine the method detection limit of fluorescein using fluorescence versus absorption spectroscopy

Determining the MDL for absorption spectroscopy

1. Using the stock solution of 7.5 × 10^-5 M fluorescein, prepare four 100 mL solutions by serial dilution ranging from 1 × 10^-6 to 7.5 × 10^-5 M fluorescein. Be sure to use 0.1 M NaOH when diluting your solution to the mark. (The calculations for making these solutions should already be completed in your notebooks. You captured the procedure in Prelaboratory Question 1.)
2. Connect a SpectroVis Plus spectrophotometer to the USB port of a LabQuest. Calibrate the spectrophotometer using 0.1 M NaOH.
3. Collect and save full wavelength spectra of your solutions, noting the optimal wavelength for analysis. Comment in your notebook whether these data support that Beer’s Law works for the system. Be sure you also capture the spectrum of the blank (you should save a total of 5 spectra).
4. Using the lowest concentration of the solutions made in (1), follow the EPA procedure for determining the MDL for fluorescein. Use the signal values obtained at the optimal wavelength determined in (3) in evaluating whether your estimated detection limit concentration value corresponds to an instrument signal/noise in the range of 2.5 to 5. If the solutions are too concentrated, dilute by a factor of 5 to 10 and retake the measurements. Repeat this step until you have found the MDL using absorption spectroscopy.

Determining the MDL for fluorescence spectroscopy

5. Prepare four 100 mL solutions by serial dilution ranging from 0.5 × 10^-6 M to 1 × 10^-8 M fluorescein. Be sure to use 0.1 M NaOH when diluting your solution to the mark. (The calculations for making these solutions should already be completed in your notebooks. You captured the procedure in Prelaboratory Question 2.)
6. Prepare the SpectroVis Plus for measuring fluorescence.
   • From the “Sensors” menu, choose Change Units → USB: Spectrometer → Fluorescence 500 nm.
   • Set up initial conditions for the instrument by tapping the “Mode: Full Spectrum” box on the Meter screen. Begin by entering 150 ms for the Sample time, Wavelength smoothing 0, Samples to Average 1. P₀ can be adjusted by changing the sample time. If the peak intensity is greater than 1, reduce the sample time in intervals of 10 ms. If it is too small, increase by units of 10 ms. Remember you want to keep P₀ constant for all measurements, so take care to calibrate your measurements to a median concentration of your standards before getting too far into the procedure.
7. Do NOT blank the instrument when the SpectroVis Plus is in fluorescence mode. Note the placement of the cuvette faces as you insert the cuvette into the sample holder. Be sure to place the cuvette in the spectrophotometer using the same orientation for all measurements.
8. Collect full wavelength fluorescence spectra of your prepared solutions, noting the optimal wavelength for analysis. Measure the two lowest standards prepared for the absorption experiment, and then continue with the solutions prepared for the fluorescence experiment (The calculations for making these solutions should already be completed in your notebooks. You captured the procedure in Prelaboratory Question 2.) Comment in your notebook whether these data support that luminescence intensity is proportional to concentration for the range of solutions prepared. Be sure you also capture the spectrum of the blank (so you should save a total of 7 spectra)!
9. Using the lowest concentration of the solutions made in (5), follow the EPA procedure for determining the MDL for fluorescein using fluorescence spectroscopy. Use the signal values obtained at the optimal wavelength determined in (8) in evaluating whether your estimated detection limit concentration value corresponds to an instrument signal/noise in the range of 2.5 to 5. If the solutions are too concentrated, dilute with 0.1 M NaOH by a factor of 2 and retake the measurements. Repeat this step until you have found the MDL using fluorescence spectroscopy.

Part 2: Determine the concentration of fluorescein in commercial antifreeze

Obtain fluorescence intensity and/or absorbance measurements of a sample of commercial antifreeze.*

To prepare your sample, begin by diluting 25 mL of sample to 100 mL using 0.1 M NaOH. Visually compare the resulting sample to your standards as a first guess as to whether you need to make more dilutions or not. Perform dilutions as needed until your sample concentration falls within the range of standards you prepared. Report the concentration of fluorescein in the ORIGINAL SAMPLE.

*Reblanking the instrument and rechecking that your standards still provide a similar absorbance or intensity value is good laboratory practice. We suggest you take a few minutes and do that before finalizing the analysis. If your group chooses to do this by fluorescence, be sure to keep the orientation of the cuvette the same for each measurement and remember, don’t blank the instrument when taking a fluorescence measurement.

Post-Lab Work Up

Results/Calculations

Fill out the answer sheet for this experiment completely. Answer the following post-lab questions.

1. Provide samples of absorbance and intensity spectra for fluorescein. Write a few sentences comparing and contrasting key features of each, including differences in sensitivities and wavelength maxima/minima.
2. Show calibration plots for each method. Is the concentration linearly proportional to the signal for both methods? Suggest improvements to the procedure to optimize the calibration curves presented.
3. Report the MDL for each method. List at least one advantage and disadvantage to using absorption or fluorescence spectroscopy for quantitative study.
4. Write a data-driven reflective summary describing your procedure for determining the concentration of fluorescein in radiator fluid. Why is fluorescein added to antifreeze?

Challenge Questions

Challenge questions are designed to make you think deeper about the concepts you learned in this lab. There may be multiple answers to these questions! Any honest effort at answering the questions will be rewarded.

Beer’s law is useful in many additional contexts besides determining concentrations of analytes in solution. For example, Beer’s law governs light absorption in solid crystals, which in turn is relevant to many techniques that study the photophysical properties of materials. In lieu of molar concentration and absorptivity, absorbance of crystals depends instead on a material- and wavelength-dependent property called the absorption coefficient (α, in units of cm^-1) as well as the path length through the crystal (L), such that log(I₀/I) = αL.

Based on this information, answer the following questions to your best ability. Note that there are multiple ways of approaching these questions and that you are not expected to follow a particular method. As long as you show your reasoning, any honest attempt to answer each of these questions will be awarded full points.

1. Consider a monochromatic beam of violet light passing through a μm-sized, nm-thin red crystal of thickness L = 100 nm. Assume no light is reflected by the surface of the crystal, and let the absorption coefficient of the crystal for the wavelength of interest be α = 1.0 × 10⁵ cm^-1.
   
   1. Which of the plots, A-D, above best describes the evolution of the light intensity across the path length in the crystal? Based on the characteristics of the graph you chose (such as the slope or intercept), describe in detail how the intensity of light changes through the crystal.
   2. Draw a picture to show how you consider the light to interact with the crystal. Use the picture to provide a physical explanation for why the variation of light intensity with path length follows the plot you selected.
2. Replicate the plot you chose in (1) in your lab notebook. Label this shape as “RED 1”.
   1. Sketch the intensity of light through a blue crystal (of a different material), whose absorption coefficient at the same wavelength is α = 5.0 × 10⁴ cm^-1. Label this shape as “BLUE”. Explain the reasoning behind your drawing, providing:
      • A mathematical reason based on “Beer’s Law” for this crystal, and
      • A physical explanation in terms of the behavior of atoms in the crystal. How does the picture you drew in (1)b change when light interacts with the blue crystal? How does this lead to the plot you drew?
3. Consider a third red crystal of the same material as the first one (α = 1.0 × 10⁵ cm^-1), but half the thickness (L = 50 nm). Let’s call this crystal “RED 2”.
   1. Draw the intensity profile of the same beam of purple light through the thinner red crystal on the same graph you used for the questions above. Label this shape as “RED 2.”
   2. Explain the reasoning behind your drawing.
   3. It turns out that the red crystal can also fluoresce at a glowing green color – How exciting! Curious to examine its emission, you grab a glass microscope slide (you’re lucky – there was only one slide left!), place the precious and delicate RED 2 crystal on it, and prepare to measure its fluorescence spectrum as shown in the diagram below. In this case, you place a filter before your detector to block the excitation wavelength and measure the fluorescence.
      

      Just as you are about to start the experiment, your lab partner interrupts you and asks where you found the glass slide. “From the bench, next to the SpectroVis spectrophotometer,” you reply, hesitantly, “Why?”

      You look at the bench space next to the spectrophotometer, which your lab partner was using during the Fluorescein Lab. After an awkward silence, you and your lab partner realize, in horror, what has happened. Some fluorescein has dripped onto the bench! What’s worse, it made its way to the bottom of your glass slide, before you placed the crystal on it. Of course, you fear that the emission from the fluorescein will interfere with your measurement of the crystal’s fluorescence, especially because they both emit light of similar wavelength. However, you also remember that your crystal’s absorption coefficient α is quite large. This may solve your problem, since most of the excitation light could get absorbed by the red crystal before it has a chance to reach the fluorescein and cause it to interfere. You want to carry out a calculation to determine if that’s the case. After consulting your TA, you learn that the standard measure of “how far” light travels into objects is the penetration depth, which is defined as the depth by which the light intensity drops to a factor of 1/e = 36.8% of its original value. Based on this information, will the fluorescein spilled on the bottom of the glass slide interfere with your measurement of the fluorescence of the crystal?
      In support of any calculations you perform to solve this problem, please address the following questions:
      1. What question should you ask to determine whether the excitation light will “reach” the contaminated glass slide?
      2. Given the information you have about the RED 2 crystal, perform a calculation to determine whether the excitation light will reach the contaminated glass slide.
      3. Based on your answer to (ii), is it possible to measure the fluorescence of RED 2 without interference from the fluorescein droplets? What assumption did you make to answer this question?

Lab Report Submission Details

Submit your lab report on Canvas as 1 combined PDF file.  This submission should include:

1. The completed answer sheet.
2. Your lab notebook pages associated with this lab, which should include answers to the post-lab questions and challenge questions.

The grading rubric can be found on Canvas.

References:

1. Harris, D. C. & Lucy, C. A. Quantitative Chemical Analysis, 10^th ed.; W. H. Freeman: New York, NY, 2020