2.2: Lighting Up – Smokers, Solutions, and Spectroscopy

Important: Complete the prelab questions as you read through the text before coming to lab!

Purpose

• Develop a process by which thiocyanate concentration can be quantified spectroscopically.
• Determine whether a lung disease patient could be a smoker by analyzing their saliva for thiocyanate.

Skills

• Familiarize yourself with various types of laboratory glassware: beakers, graduated cylinders, pipettes, and volumetric flasks.
• Dilute a stock solution.
• Measure absorbance with a spectrometer.

Chemistry Learning Objectives

• Explain and interpret complexation reactions at the microscopic view. • Calculate concentration from mass of solute and volume of solution. • Calculate concentration using the dilution equation, \(C_1 V_1 = C_2 V_2\).
  Calculate concentration algebraically from a best-fit line on a Beer’s Law plot (graph of absorbance vs. concentration).

Analytical chemistry – A branch of chemistry that focuses on identifying and quantifying the components in a substance.

Spectroscopy – The scientific study of how matter interacts with light.

Cardiopulmonologist – A medical professional who specializes in the heart and lungs.

Biomarker – A measurable substance in an organism whose presence is indicative of some phenomenon such as disease, infection, or environmental exposure.

An important branch of chemistry is analytical chemistry, in which instruments are used to determine the composition of a substance. Applications of analytical chemistry occur every day in doctor’s offices when patient samples are tested to diagnose diseases and keep us healthy!

A common analytical technique is spectroscopy, where light is used to determine the composition or properties of a substance. Your eyes perform spectroscopy when you decide what to do at a stoplight, or which snow is safe to eat.

In this lab, you will take on the role of a cardiopulmonologist trying to diagnose the cause of two patients’ lung disease. You will develop a test for thiocyanate, a biomarker of smoking, and use the results to determine whether the patients are smokers or not.

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Spectroscopy – A laboratory technique which quantifies the amount of light that passes through a substance. A spectrometer is the instrument used for this technique (Figure 3-1).

A.Background

Burning plant matter contains many harmful compounds that are inhaled when smoking, including cyanide (CN–). Cigarettes contain nicotine, an incredibly addictive chemical, and many people find it difficult to quit smoking even though they know it is bad for them. This can lead to embarrassment, and as a result some patients are reluctant to tell their doctors about their smoking history.

The detoxification of cyanide occurs in the liver and produces thiocyanate (SCN–), which appears in saliva.1 There are many sources of cyanide, including almonds, some fruits and vegetables, vitamin B12 supplements, and inhaling burning plant matter.2

Imagine you are a doctor trying to diagnose a patient’s lung disease. The patient claims that they don’t smoke, but you have your doubts. You’d like to test the patient’s saliva for thiocyanate so that you can provide the patient with the correct treatment. Everyone has some level of thiocyanate in their saliva, but smokers tend to have more than non smokers.1 You heard of a technique called spectroscopy, where the composition of a solution can be determined from its color.

Figure 3-1. Diagram of a spectrometer, courtesy of JASCO, 2024.

In this lab, you will use spectrometer to determine the

concentration of thiocyanate in a patient’s saliva. But first,

you need to discover how spectroscopy is used to measure

concentration.

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Spectrometer – An instrument that measures the absorbance of a substance at a specific wavelength.

Absorbance – How much light is blocked by the sample. Darker colors indicate high absorbance.

Wavelength – Basically, a specific color of light. FeSCN2+ has a maximum absorbance at 492 nm.

Calibration curve – A series of measurements of samples with known concentration, used to standardize a technique so it can be used to measure concentration in unknown samples.

Analyte – A chemical substance that is the subject of chemical analysis. In this case, the analyte is thiocyanate.

Beer’s law plot – A graph of absorbance vs. concentration at a specific

wavelength. Absorbance generally increases with increasing concentration.

�������� = ��������

Eqn 3-1. Dilution equation, where

C1 is the initial concentration,

V1 is the initial volume,

C2 is the final concentration, and V2 is the final volume of the solution.

Aliquot – A small, carefully measured volume.

Experimental

Theory

You will use a spectrometer to determine the concentration of thiocyanate in the saliva of a lung disease patient. Thiocyanate is colorless, but when it forms a complex with Fe3+ it turns deep red (Rxn. 3-1).

����3+(����) + ������−(����) → ����������2+(����)

Yellow Colorless Deep red

Rxn 3-1. The complexation of iron(III) with thiocyanate changes its color.

You will first need to determine the relationship between absorbance and concentration. This is commonly done by the preparation of a calibration curve. A Beer’s law plot is a specific type of calibration curve, where absorbance is plotted as a function of concentration.

The class will create a series of standard solutions with increasing concentration of thiocyanate to determine how absorbance depends on thiocyanate concentration. You will be responsible for preparing one standard solution, then the class’s measured absorbance data will be combined and graphed in a shared Google Sheet.

A stock solution with a high concentration of thiocyanate has been prepared. You will dilute a small amount of the stock solution by adding it to an iron(III) solution; this will make the concentration of thiocyanate lower according to the dilution equation (Eqn. 3-1). Your instructor has assigned your group an aliquot volume to use during your dilution. Write your group’s aliquot volume below; this will be the initial volume of stock solution for your dilutions.

Aliquot volume:

__________ mL

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Precision – A measure of how close a set of measurements are to each other.

Molar (M) – A unit of concentration equal to ������ ������������

�� ����������������. Note: Molar (M) is not the same thing as moles (mol).

You have one additional task: determine the appropriateness of various types of glassware for solution preparation and dilution. Your lab manager needs to order new glassware and is trying to decide between beakers or volumetric glassware. To assist, each group member will prepare the diluted solution twice – once using each type of glassware – then compare the results, and report your findings back to your lab manager.

Beaker Volumetric glassware

Volumetric flask Volumetric pipette

Although you and your group member(s) will prepare solutions of the same concentration, your samples may not have the same absorbance, due to experimental uncertainties and potential errors in solution preparation and measurement. You will compare your measured absorbance to your partner(s)’ to assess the precision of each preparation technique.

Part 0 Procedure: Preparation

This has already been done for you, but you will use the information to answer the prelab questions before class. Show your work on the next page.

1. The lab specialist has prepared 3.00 L of iron(III) stock solution, which contains 121.7 g FeCl3 (162.2 g/mol) and 250. mL 12.0 M HCl.

Prelab Q1 – Calculate the concentrations of the iron(III) stock solution in molar (M).

a. What is the concentration of FeCl3 in M?

b. What is the concentration of HCl in M?

c. What is the total concentration of Cl–in M?

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Millimolar (mM) – A unit of concentration equal to 10-3 M (1 ����

1000 ��), or �������� ������������

�� ����������������.

Saliva sample collection and analysis procedure3

1. The patient abstains from eating or drinking for at least 60 minutes.

2. The patient rinses their mouth with water, then waits 10 minutes.

3. At least 2 mL of saliva are collected from the patient. The saliva is filtered and centrifuged to remove any solids.

4. A 0.72-mL sample of saliva is combined with 1.60 mL of

0.25-M FeCl3 solution and 1.68 mL of pure water.

5. The resultant solution is light sensitive and should be stored in the dark.

Shown work for pre-lab questions

2. The lab specialist has prepared 500. mL of thiocyanate stock solution, which contains 0.1317 g NaSCN (81.07 g/mol).

Prelab Q2 – Consider the thiocyanate stock solution.

a. What is the concentration of the NaSCN in

millimolar (mM)?

b. When NaSCN dissolves in solution, what two ions are formed?

3. Saliva samples have been collected from two patients and prepared as described to the left. In Part 3, you will measure the absorbance of both samples and use your results from lab to determine the concentration of thiocyanate in each patient’s saliva.

Prelab Q3. – Consider the Saliva sample collection and analysis procedure to the left.

a. Why is the concentration of thiocyanate in the cuvette less than the concentration of

thiocyanate in the patient’s saliva?

b. Once you have the concentration of thiocyanate in the cuvette, explain how can you calculate

the concentration of thiocyanate in the patient's

saliva. No calculations needed.

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Beaker

Removing Sharpie from glass – Spray ethanol on a paper towel and wipe the Sharpie marks until they are removed.

Safety note – The Fe3+ solution is prepared in 1.0 M hydrochloric acid, which can burn your skin. If any acid gets on your skin, rinse it off immediately in the sink and inform your instructor!

Graduated

cylinder

Note – If your DI (deionized water) squirt bottle ever runs out, refill it from the white plastic tap at the center sink.

Glass stir rod

Part 1 Procedure: Beaker Dilution

1. Obtain two 100-mL beakers. Use a Sharpie to label one ‘Stock SCN–’ and the other ‘Stock Fe3+’. Bring both beakers to the fume hood. Pour at least 15 mL of the

sodium thiocyanate (NaSCN) solution per group member into its beaker, and pour at least 20 mL of the iron(III) chloride (FeCl3) solution per group member into its beaker.

2. Obtain one large (400-600-mL) beaker. This will be your waste beaker, a place to dispose of any unused reagents.

Take turns following steps 3 through 9. While one group member prepares their dilution, the other member(s) will assess their technique on the laminated sheet.

3. Get a 20-mL beaker. Pour Stock SCN–solution into the 20-mL beaker until the volume approximately equals your assigned aliquot volume (pg 3-4). You may need to estimate between the lines. Pour this into a 50-mL beaker, and label the beaker with your initials.

4. Use a graduated cylinder to measure 10.0 mL of the Stock Fe3+ solution and add it to the 50-mL beaker labeled with your initials.

5. Carefully add deionized water to your initialed beaker until the final volume is 25 mL. (Use the dropper when you get close.) If you make a mistake, start over from the beginning of the dilution with a clean 50-mL beaker.

6. Stir thoroughly with a clean, dry glass stir rod until it is evenly mixed.

7. Make observations about the preparation process and solution color change, and write them in Table 3-1 of the worksheet.

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Volumetric pipette

Note – When dispensed correctly, some liquid will remain in the pipette tip after draining; do not expel the last bit of solution from the pipette!

Volumetric flask

8. Set the 50-mL beaker aside. You will use it in the spectroscopy portion of the lab (Part 3).

9. Your partner can reuse the 20-mL beaker and the graduated cylinder without cleaning them, since they are used to measure the same solutions.

Part 2 Procedure: Volumetric Dilution

1. Obtain a volumetric pipette from your instructor; the volume should match your assigned aliquot volume.

Take turns following steps 2 through 7. While one group member prepares their dilution, the other member(s) will assess their technique on the laminated sheet.

2. Practice using your volumetric pipette with a clean beaker of deionized water. Draw up liquid until it reaches the line, then allow the liquid to drain out – this will deliver the exact volume of liquid into the container. Make sure not to get any liquid into the bulb.

3. Use your volumetric pipette to measure one aliquot of the Stock SCN–solution and add it to a 25-mL volumetric flask. Label the flask with your initials.

4. Use a graduated cylinder to measure 10.0 mL of the stock Fe3+ solution and add it to your initialed flask.

5. Carefully add deionized water to the flask until it reaches the line. (Use the dropper when you get close.) If you make a mistake, start over from the beginning of the dilution with a clean 25-mL volumetric flask.

6. Put the cap on the flask and invert it several times to mix the solutions together.

7. Make observations about the preparation process and solution color change. Write them in Table 3-1.

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Blank – A sample which contains everything except the analyte.

Safety note – Gloves should not be worn while interacting with instruments such as the LabQuest.

Cuvette

Part 3 Procedure: Spectroscopic measurement of absorbance

1. Before you measure the absorbance of your sample, you need to calibrate your spectrometer with a blank. This shows the spectrometer what it ‘looks’ like when a sample contains no analyte (thiocyanate).

2. To calibrate your spectrometer, press the red box in the middle of the LabQuest screen and press Calibrate… a. While you wait 90 seconds for the lamp to warm up, look at the Fe cuvette, which contains the Fe3+ solution. Note that two of the sides are

transparent, and two are cloudy or ridged. The transparent sides often have triangular markings at the top.

b. Wipe off the transparent sides of the cuvette with a Kimwipe. Make sure to only handle the cuvette from the cloudy/ridged sides so you don’t get fingerprints where the light needs to pass.

c. Ensuring that the beam path passes through the transparent sides of the cuvette, place the cuvette into the spectrometer and firmly press down until it is fully inserted.

d. When the lamp has finished warming up, press Finish Calibration, then press OK. Remove the Fe cuvette from the instrument and set it aside.

e. Press Collect, , at the bottom left of the screen. If the LabQuest asks about unsaved data, press Discard.

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3. All group members will repeat steps 3a through 3c for both of their dilutions.

a. Carefully fill a cuvette with one of your solutions until it is around ¾ full.

b. Use a Kimwipe to ensure the transparent sides are clean, then insert the cuvette into the

spectrometer with the correct orientation.

c. To measure the absorbance of the sample, press Keep, . When prompted to Enter value for event, enter your initials and either ‘1’, for the beaker dilution, or ‘2’ for the volumetric dilution.

d. You do not need to write down the absorbance values right now; you will first measure the

absorbance of all samples, then record all values in your worksheet pages (Step 6).

4. Now that you have measured the absorbance of your standard solutions, you’ll measure the absorbance of both unknowns: Patient A and Patient B.

a. From your instructor, obtain the cuvettes

containing the patients’ saliva samples mixed

with Fe3+ solution. They are labelled A and B.

b. Use a Kimwipe to clean the sides of one patient’s cuvette and insert it into the spectrometer with the correct orientation.

c. Press Keep, . Name the sample A (for Patient A) or B (for Patient B).

d. Repeat the above steps for the other patient.

5. Once the absorbance of all samples has been measured, press Stop, , to stop data collection, then press the ‘XY’ tab at the top of the screen. This will display your data.

6. Write down your absorbance values in Table 3-2, along with the absorbance values of your group member(s) and Patients A and B. Then, clear your LabQuest data by pressing Table → Clear All Data → Meter Screen, .

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Removing Sharpie from glass – Spray ethanol on a paper towel and wipe the Sharpie marks until they are removed.

Linear regression line (aka line-of-best fit) – A straight line applied to a scatterplot which passes as close to as many points as mathematically possible. The line’s equation relates the x and y values mathematically.

R2 value – A measure of how close a regression line is to its data. An R2 of 1.00 indicates a perfect match to the data. An R2 of 0.00 indicates no correlation between the data.

Part 4: Clean-up

1. Return cuvettes A and B to your instructor. Set aside cuvette Fe, it will be used by the next lab section.

2. Pour all the other solutions into your waste beaker (but don’t let it overfill). If the waste beaker gets close to full, or you are done cleaning up, transfer the contents to the waste jug in the fume hood.

3. Clean all Sharpie marks off the glassware using ethanol and a paper towel. Refill the DI squirt bottle(s).

4. To clean your volumetric pipette, pour deionized water into a clean beaker, then suction up the water with the pipette and release it into the waste beaker. Do this at least three times.

5. To clean the other glassware, rinse them with warm tap water in the sink. If any residue remains, use the soap and brush near the sink to scrub the residue away.

6. Bring the glassware back to your station. Set the beakers upside-down on the tray to dry.

Part 5: Enter data into class spreadsheet

1. Open the Google Sheet link emailed to you by your instructor.

2. In an empty row, write your name, aliquot volume, and the absorbance of both dilute solutions. (You will not enter Patient A or B data into the class spreadsheet.)

3. Once everyone has entered their data, sketch each graph in Figure 3-4 of the worksheet. Also write down the equation for the linear regression line and its R2 value.

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Worksheet

Note: You will complete these activities as part of your instructor’s explanation of the lab procedure.

����3+

������−

+→

����������2+

Figure 3-2. Microscopic view of the complexation reaction between iron(III) and thiocyanate (water molecules omitted for clarity). You may use different shapes to represent Fe3+ and SCN–.

Low absorbance sample: Light source Cuvette Detector

High absorbance sample: Light source Cuvette Detector

Figure 3-3. Schematic diagrams of a spectrometer measuring a sample with a low absorbance (top) and a sample with high absorbance (bottom).

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Data tables and observations

Table 3-1. Aliquot volume and observations for your solutions. Make sure to calculate your final SCN– concentration using the dilution equation, ��1��1 = ��2��2.

Concentration of stock SCN–: 3.25 mM Final dilution volume: 25.0 mL
Your group’s SCN– aliquot volume (mL):
Final [SCN–] in your samples (mM):
Solution	Procedural observations (solution colors, glassware use, measurement process, etc.)
Beaker dilution
Volumetric dilution

Shown work for calculation of final [SCN–]

Table 3-2. Absorbance at 492 nm for your group’s standard solutions and each patient sample.

	Sample	Absorbance (Group member #1)	Absorbance (Group member #2)	Absorbance (Group member #3)
Standard solutions	Beaker dilution
	Volumetric dilution
Patient samples	Patient A cuvette
	Patient B cuvette

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Beakers Volumetric glassware Absorbance

Absorbance

[SCN—] (mM) [SCN—] (mM)

�� =

��2 =

�� =

��2 =

Figure 3-4. Sketches of the scatterplots and regression lines from the class’s Google Sheet data. You do not need to add values to the axes, nor do you need to include all points, just enough to represent the general trend.

Questions

1. You and your group member(s) each prepared solutions with the same aliquot volume, meaning they should have the same thiocyanate concentration and absorbance. How close were your absorbance values to those of your group member(s)? Did the type of glassware affect how close the absorbance values were? What does this say about the precision (page 3-4) of the glassware?

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2. Discuss the following types of glassware with your group member(s), then list at least one advantage and one disadvantage for each. Provide as much detail as possible.

Beaker	Advantages:
	Disadvantages:
Graduated cylinder	Advantages:
	Disadvantages:
Volumetric pipette	Advantages:
	Disadvantages:
Volumetric flask	Advantages:
	Disadvantages:

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3. Each group was given a different aliquot volume. What happens to the appearance of the solution as the aliquot volume increases? Justify your observation mathematically using Eqn. 3-1 (pg 3-3).

4. Consider both graphs from the Google Sheet, which you sketched in Figure 3-4. a. What is one way that the regression lines are similar?

b. What is one way the graphs are different?

c. Comment on how well each graph tells you about the relationship between absorbance and concentration. Briefly explain, making sure to mention reference plot’s R2value in your explanation.

5. Your lab manager needs to buy new glassware and is trying to decide between beakers and volumetric glassware. They want to order beakers, since beakers are the least expensive. Do you think this is a good idea? Imagine a conversation between you and your lab manager. Have one group member act as the lab manager and the other(s) act as lab technician(s). Which glassware did you decide to purchase? Give at least three reasons for your choice.

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6. You are trying to develop a process to measure the concentration of thiocyanate in saliva to determine whether a lung disease patient is a smoker or not.

a. How can you use the plots of absorbance vs. concentration to determine the thiocyanate concentration in a patient sample?

b. Figure 3-4 shows the plots, equations, and R2values for the standard series of thiocyanate for each type of glassware. Which type of glassware is more precise (pg 3-4)? Circle your choice in Table 3-3 and explain your logic.

c. In Table 3-3, input the equation from the plot corresponding to the type of glassware you chose in the last question, rewrite the absorbance values for Patient A and Patient B cuvettes, then calculate the thiocyanate concentration in Patient A’s cuvette and Patient B’s cuvette. Show your calculations and input the values into Table 3-3.

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d. Calculate the thiocyanate concentration in each patient’s saliva (consult the Saliva sample collection and analysis procedure). Show your calculations and input the values in Table 3-3.

Saliva sample collection and analysis procedure3

1. The patient abstains from eating or drinking for at least 60 minutes.

2. The patient rinses their mouth with water, then waits 10 minutes.

3. At least 2 mL of saliva are collected from the patient. The saliva is filtered and centrifuged to remove any solids.

4. A 0.72-mL sample of saliva is combined with 1.60 mL of

0.25-M FeCl3 solution and 1.68 mL of pure water.

5. The resultant solution is light-sensitive and should be stored in the dark.

Table 3-3. Absorbance at 492 nm for your group’s standard solutions and each patient sample, as well as calculated thiocyanate concentrations in patient cuvettes and saliva.

Glassware chosen (circle one): Beakers Volumetric glassware
Equation relating Absorbance to [SCN–]:
	Patient A	Patient B
Absorbance (cuvette) From Table 3-2.
[SCN–] (cuvette), mM
[SCN–] (saliva), mM

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7. Typical nonsmokers have saliva thiocyanate concentrations of 0.5 – 2 mM.1 Some smokers may have saliva thiocyanate concentrations as high as 6 mM.1 Recall that there are other dietary sources of cyanide (pg 3-2).

a. Do you believe that Patient A is a smoker? Explain your logic.

b. Do you believe that Patient B is a smoker? Explain your logic.

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8. Beer’s Law relates a solution’s absorbance at a specific

wavelength to the

concentration of the absorbing species, according to Eqn 3-2.

Eqn. 3-2. Beer’s law ������ = �� ∗ �� ∗ ��
	Description	Value for Exp 3
Abs	Absorbance – Measured spectroscopically. Unitless.	See Table 3-3
a	Molar absorptivity – A constant representing how strongly the species absorbs light at a specific wavelength.	2.567 mM-1 cm-1
b	Path length – The distance the light travels through the solution.	1.00 cm
C	Concentration of the absorbing species.	Calculated below

a. Calculate the concentration of thiocyanate in cuvettes A and B using the Beer’s Law equation, along with the given values for molar absorptivity (a), path length (b), and your measured absorbances (Table 3-3). The results should be considered as the true values for thiocyanate concentration, [SCN–]true. Add your values to Table 3-4 and show your work for patient A below.

b. Compare the calculated [SCN–]true for patients A and B to the experimental values of [SCN–] you got using the calibration curve equation of your chosen glassware (Table 3-3, cuvette row) by calculating the percent error. Add your values to Table 3-4 and show your work for patient A below.

Table 3-4. Calculation of percent error.

	Patient A	Patient B
Measured absorbance From Table 3-3
[SCN–]true
[SCN–]experimental From Table 3-3, cuvette row
% ���������� =������������������������ −�������� ��������∗ ������%

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References

1. Tenovuo, J. In Human Saliva: Clinical Chemistry and Microbiology, Vol II; Tenovuo, J., Ed.; CRC: Boca Raton, FL, 1989; pp 55–91

2. The Facts about Cyanides, New York State Department of Health; Albany, NY, 2024

3. Beltzer, E.K., Fortunato, C.K., Guaderrama, M.M., et al. Salivary flow and alpha-amylase: Collection technique, duration, and oral fluid type. Physiol Behav, 101(2), 2010; pp 289-96

Acknowledgements

Conceptualization of this lab was a joint effort of Dr. MacManus-Spencer, Dr. Soliman, and Dr. Schabes of the Union College Department of Chemistry. Critical testing and input was performed by students Ferneidy Dotel (Biology) and Shamil Williams (Neuroscience).

Development was funded by NSF grants #2337027 and #2337028. We are grateful to St. Mary’s College of Maryland for the use of their materials and lab space, as well as pedagogical guidance through the Chemistry Laboratory Curriculum Innovators Program.

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