Spectroscopy. n fascinating field of chemistry and physics, has bccn used in an array of diverse applications from the analysis of chemicals in foods and pharmaceuticals to the detertnination of’ age nnd composition of distant stars and entire galaxies. The fundamental idea behind spectroscopy involves the interaction bctwccn matter (molcculcs and atoms) and radiation
(e.g. infrared radiation). For exatnple. thc dctailcd images of thc human body obtained from MRI (magnetic resonance inmging) scans arc the product of the interactions of hydrogen atoms with radio waves (under certain controlled conditions). We use these interactions of matter with radiation to obtain information about substances, specifically, the structure and behavior of molecules.
Not all molecules interact with radiation in the same way. The nature of the interaction and the region of the electromagnetic spectrum where these interactions occur depend on the structure
Figure I: diluted solution from a
color (Figure 1). You or someone you know may have
commercial drink oxytops. wordp
used this technique, called colorimetry (measure of the
of the molecules (or atoms). Color results from the interaction of substances with a specific portion of the electromagnetic field that we call “visible”. As expected, the amount of substance influences the extent of that interaction. You can easily tell which of the solutions is more concentrated just by looking at the intensity of their
intensity of color). For instance, you can find fast, cheap and easy to use medical assays to assess blood glucose levels that use colorimetric principles. They use strips of paper impregnated with chemicals that change color with the amount of glucose present in a small drop of blood. The final pattern of colors on the strip is the “reading” that indicates the glucose levels.
In this project, your team will apply the principles of spectrophotometry for chemical analysis and you will use your chemistry knowledge and understanding to explain the interaction of UV-Visible radiation and molecules in commercial products.
2. PROJECT DESCRIPTION
Figure 2: Spectrophototneter from Ocean Optics Company
molecules and how these interactions can be used determine their concentration in solutions. Food dyes
Food dyes, widely used in t11C food industry as additives, may possess potential negative health efTccts related to their use and have recently sparkcd somc controversy. To help the ptiblic make inli)rtned decisions. n non-profit orgnni?lltion called Clean Eating Advocates (CEA) is creating an online datnbnse With the food dye content in various commercial drinks. They propose using spectrophotomctry for the analyscg. measuring the absorption of tJV-Visible radiation by a solution and using it to find the concentration of dyes in thc drinks. Ijnaginc that you have been asked to assist CEA in this projcct. Sincc their project rcccntly started, the database iB “till small. Ilowcvcr, the CEA provided us with n list or target dyes, Your team will investigate the interaction of WV-visible radiation with food dye to
absorb electromagnetic radiation in the visible light spectrum, and the absorption of light and the concentration of dyes are related. Thus, your team will use a spectrophotometer (Figure 2) to determine the unknown dye concentrations in solutions.
Standard Curve vith In•nginary Data You will determine the concentration of color dyes in commercial products. Your team will have to identify the wavelength at which the absorbance is greatest (lambda maxima, Imax) per color. Then, create a calibration curve per color which must comply with industrial standards (linearity of R2 = 0.95 and a minimum offive data points). Each calibration curve can be used to calculate the unknown concentration of that
CorEentration per cent (VIV)
Figure 3: This is an example of a standard curve. A best fit line has been generated and the resulting equation
specific dye in the commercial product (Figure 3).
It is important to use the linear equation from the calibration curve and correlate it with Beer’s law (A =
and r-squared value are shown below tlc) to calculate the unknown concentration. The the X-axis label
spectrophotometer that you will be using gives more
accurate readings if the absorbance is between the range of 0.1 and l, then you will have to adjust
the concentrations of the dye solutions to obtain values within this range. To start, your TA will assign three of the dyes available in the stockroom to your team. Specific instructions for the use of the spectrophotometer will be provided to you during class. Create a calibration curve for each assigned color (same as mini-project 2) and get the linear equation (Beer’s law). Use Microsoft excel or similar software to create your calibration curve (absorbance vs concentration). Create the calibration curve during class as you perform the readings on the spectrometer. Both tasks should be completed at the stitne tinte. Discord nnd repeat samples that deviate from the linearity of the graph so your team can have n five point graph with and R2 of 0.95 or higher. Answer the following questions before you stnrt your experiment:
t. What is the goul Cor the entire project?
- What are the chemical formulas and the maximum absorbance of the dyes assigned to your team?
- How are color and wavelength of maximum absorbance related?
- What will the concentration of your initial stock solution be?
- What measurements will you collect and how will you organize them in your notebook?
- CONCEPTS AND TECHNIOUES
Topics you may need to review depending on your experimental decisions are: electromagnetic spectrum, light wavelength and color, UV-Visible absorption spectroscopy, spectrophotometer, concentration of solutions, dilution calculations, Beer’s law, calibration curves, lambda maxima (Xmxx), accuracy and precision of measurements, interpretation of data using Excel. Reviewing the following techniques before coming to the lab will make your work easier and more productive: measuring volumes at different levels of accuracy (use of burets and pipets), serial and parallel dilution, and graphing using Excel.
- SUPPLEMENTAL INFORMATION AND ONLINE RESOURCES
These links provide information about: concepts, research connection and laboratory techniques. Do not limit your search of information to these resources only.
- USF Laboratory Toolbox: Canvas Course Homepage
- Principle of Spectrophotometry:
http://www.chm.davidson.edWvcc Spectrophotomctry Spectrophotometry.html
http://www.chm.davidson.cdu/vcc Spectrophotometry/Absorbance.Spectrum.html Beer’s Law: BeersLaw.htrnl
- Spectroscopy information (Michigan State University, Department of Chemistry):
http://www2.chetnistry.tnstl.edt1/ract11ty/rcusgh/VirC11xtJrnl/SpectrPY=-IYV—t——!J! Spectrophotometer, how does it work?
http://www2.chen1istry.msu.edu/raculty reusch/Virt•rxtJlnl/Spectrpy/UVVis/uvspec.httnl/uv I
- Preparation of standard solutions: http:/ASAvw.youtube.com/watch?v=XMtm4hVCGWg How to dilute a solution?
5. SAFETY NOTES
You may be asked to leave the laboratory if you do not abide by safety norms:
- Laboratory coat, splash proof goggles and gloves MUST be worn during this experiment.
- Wash your hands thoroughly at the end of the experiment.
- Water is not a chemical hazard; however, other chemicals you may propose to use in your experiments may present hazards. Consult the SDS (Safety Data Sheet) for each chemical; some chemicals may irritate/burn eyes and skin.
- Do not pour any of your solutions or samples down the drain. Use the proper waste containers (solid, liquid waste).
6. BASIC AVAILABLE MATERIALS
The following will be available in the laboratory. You will need to ask your TA for authorization before using any substance or equipment not listed here:
*All dyes are in solid form unless otherwise stated by the TA or the stockroom staff
- General laboratory glassware (volumetric flask, beaker)
- Red Dye Red Dye
- Yellow Dye #5,6
- Green Dye #3
- Blue Dye #2
- Other dye colors may be available
- Graduated pipettes
- Pipette pumps
- Ocean Optics Spectrophotometer Instrument (the “operation instructions” for this instrument will bc shared with you before the experiment)
7. GUIDING IDEAS AND PLANNING OUESTIONS FOR THE PROJECT
- What safety protocols need to be followed for this project? What are the specific safety concerns for each chemical used? Consult the SDS for each.
- Mix two of your dyes, predict what the spectrum will look like.
- Why is finding the wavelength of maximum absorbance necessary?
- What glassware will you need to use? What equipment will you need?
- Suggest the concentration of food dyes in a few specific products.
- You will need to prepare a calibration curve. What is a calibration curve? What is your independent variable? What is your dependent variable? What are you measuring? How are you going to measure it? How do all these components fit together to allow you to obtain useful data from a calibration curve?
- When performing serial and parallel dilutions, accuracy is extremely important. A small error in an initial dilution propagates throughout the experiment becoming a large error by the end. How will you minimize error when measuring solutions?
- The absorbance values for your solutions must be in the range of 0.1-1.0. If the absorbance value is lower than 0.1 or higher than 1.0 what will you have to do to get a value between those numbers?
- How many different concentrations are needed to produce a calibration curve?
- You will need to find a line of best fit. What is the point of finding the line of best fit? What does the variance show? Do you want a high or low R2 value?
- Analyze your data and prepare a graph of absorbance vs. concentration using Excel.
Determine the “line of best fit” or “trend line” from the graph. How can you relate this line of best fit to the Beer-Lambert Law?
- How can you determine if the correlation for the line-of-best-fit works?
- Use your graph to calculate the molar absorptivity (c) for the food dye. What is the physical meaning of the molar absorptivity?
- Does your molar absorptivity match the reported value in the literature? What error sources could affect the accuracy of your measurement?
- What type ol’ sample preparations arc necessary? For example, how would you prepare a solid sample? What happens if your sample is too concentrated? Too dilute?
- What other factors are ilnportant to consider when analyzing commercial products?
- If a samples absorbance value falls outside the range of the calibration curve? How would you address this?
Check your planning with your TA. If there is enough time left, your TA may allow you to start working on the analysis of one or more samples. This will allow you to advance some work or simply get acquainted with the glassware and the preparation of your sample. If any new concepts or techniques came up during your planning, you need to review them before coming to the lab next week.
8. PROJECT SUMMARY
I. What is the goal of the entire project? Relate your results to the goal of the overall project.
- Prepare a table of wavelength of maximum absorbance vs. solution color using your data and observations from the initial activity.
- Compare the spectra for three dyes. Does the wavelength of maximum absorbance for each color solution match color of the solution?
- How are your spectra similar? How are your spectra different? What causes these similarities and differences? You have an idea of how the absorbance spectrum for a single substance looks. How do you think the absorbance spectrum will look if instead of a single dye in a solution, you have multiple food dyes? What do you notice? What does
this tell you about the relationship between and the dyes? Can you differentiate between multiple dyes?
- Analyze your data and determine the concentration of food dye in your food products. Label concentrations with proper units.
- What is the concentration of food dye reported in the commercial product you studied?
- List possible sources of error in this analysis. How did the errors affect your measurements? Label them as random or systematic.
- Several dyes have been banned. Why have they been banned?
- Are dye concentrations safe in the products you studied?
- Fill out the table below and compare your results to the results of other teams using the same color dyes. Is there any difference? Why?
Table 1: Data collection from color samples.
(M) sample 1
(M) sample 2
9. THINGS YOU MAY WANT TO CONSIDER FOR YOUR PROJECT
Make sure you follow the lab report or oral/poster presentation guidelines provided by your instructor. Present your experimental data in an easy-to-read format. Sometimes it is better to use graphs instead of tables for specific purposes; this helps the reader understand your findings and your claims. Always use and show the appropriate units for measurements and significant figures. Consider describing general aspects and findings from your study of color and wavelength. Was there anything that initially seemed counterintuitive? Do not overemphasize procedures that you know other teams performed as well. Stress steps that you think are new to other teams, for example details of food sample preparation and analysis.
Remember that we want you to connect your experimental work to research. What kind of similarities do you find between your lab work and what you understand for research?
10. RESEARCH CONNECTION
Many researchers in the natural and medical sciences at USF routinely use spectroscopic techniques. Dr. Dean F. Martin, Distinguished University Professor Emeritus at the University of South Florida researches aspects of environmental chemistry motivated by his interest in fast and cheap removal of toxic chemicals from the environment. One method he is interested in uses substances that act as ”molecule traps”. They encapsulate the toxic molecules within their structure, something like a fishing net catching fish of specific sizes and letting others free. One of these molecule-trapping substances, Ortogil@, has been widely studied by undergraduate and graduate students working under the supervision of Dr, Martin. Ortogil@) shows particular attraction to certain food dyes and pharmaceutics present in watcr samples. Professor Martin uses spectrophotometry to determine how clean the water sample is after treatment with Ortogil@.
The picture shown illustrates an experimental setup used by Dr. Martin’s students. The yellow solution in the bottle is a sample contaminated by the organic molecule 4-nitrophenol. Dr. Martin and his students quantify the concentration of 4-nitrophenol using the spectrophotometer on the right. The solution is pushed through a glass column filled with a packing material covered with Ortogil@. As you can see, the liquid eluting from the column in the graduated cylinder is clear. Dr. Martin’s students quantify the content of 4-nitrophenol by subtracting the initial concentration from the final concentration measured by the spectrophotometer and calculate Photo taken in Dr. Martin’s Laboratory at USF how much contaminant was removed.
Medical research findings suggest that the presence of food dyes is correlated with development of attention deficit hyperactivity disorder (ADHD) in children. You can imagine the impact of Dr. Martin’s development of cheap and easy ways to remove harmful materials from our
drinking water sources.
provided Raw Data for Result Section
-Red Dye #40 dilution preparation, calculate the V1 for each dilution and complete the table. Include this in your result section of the lab report.
M1 (stock ,M)
-Data for calibration plot :Red Dye # 40. Lambda max at 490 nm.
-Data for unknown drink. Use the calibration plot and equation you created to solve for the concentration of Red due #40 in the two unknown drinks:
Sample containing red dye#40
Mystery red drink #1
Mystery red drink #2
UV-visible spectrophotometry is an analytical technique to determine the concentration of a compound in solution. It is based on the fact that molecules absorb electromagnetic radiation and at the same time the amount of light absorbed depends on the concentration in a linear way (Skoog, 2016). Visible UV spectroscopy is an instrumental technique to determine the concentration of a sample by measuring the absorption of visible light. This technique is based on Beer Lambert’s law, which linearly relates the absorbance of a sample to its concentration (Skoog, 2016).
A = εbc
Where: c is expressed in mol/L, b is the length of the optical path (width of the cell containing the substance’s solution) and is expressed in cm, and ε is the molar absorbance.
In order to be able to quantify the concentration of a solution it is necessary to make a linear adjustment that relates the concentrations of the absorbent species and the absorbance of the same, this relationship is known as a calibration curve and is made to ensure the accuracy in the results obtained.
On the other hand, the color is one of the first impressions you have of a food; it tends to modify subjectively other sensations such as taste and smell, and can define the success or failure of a product in the market. Colorants are the group of food additives that are responsible for providing the desired and expected color of each food. According to their origin they can be classified as synthetic and natural colorants. There is the belief that natural colors are harmless, however, the addition of all of them is regulated according to national and international standards and have a maximum accepted concentration value, so it is necessary to control what kind of colors were added to commercial products and in what quantity. When they are added in higher concentrations than those accepted may present risks to human health. During the last three decades, several studies have shown that moderate concentrations of synthetic dyes in foods can cause hyperactivity and other behavioral disorders in children. Also in people with special organic characteristics can occur intolerance, intensification of the symptoms of asthma and allergies (Stevens, 2013).
The Red #40 is a synthetic red dye that is an azoderivative compound. It is a disodium salt that comes in the form of a dark reddish powder that is very soluble in water and is usually used in the food industry as a food colorant. Red 40 is used as an additive / colorant in sweets, dairy products, cookies, gelatin, condiments, drinks, desserts, cake mixes and fruit flavored fillings. It is also used in medicines and cosmetics (Stevens, 2013).
Figure 1. Red 40 (molecular formula).
It has been reported that Red 40 may accelerate the appearance of tumors in the immune system of mice. It also causes hypersensitivity (allergy-like) reactions in some consumers and may cause hyperactivity in children (Stevens, 2013). For this reason, this practice is focused on determine the concentration of red dye #40 in beverages by means of the UV-vis spectrometry technique in order to know if these beverages comply with the limits allowed to be consumed.
In UV-Vis spectroscopy, light passes through a sample at a certain wavelength in the ultraviolet or visible spectrum. If the sample absorbs some of the light, not all of the light will be passed through or transmitted. Transmission is the ratio of the intensity of the transmitted light to the incident light and is related to absorbance. The absorbance can be used in a quantitative way, to obtain the concentration of a sample. It can also be used in a qualitative way, to identify a compound by combining the absorbance measured in a range of wavelengths, called the absorbance spectrum, to the published data. This video will introduce UV-Vis spectroscopy and demonstrate its use in the laboratory in the kinetic determination of sample reaction and concentration (Skoog, 2016).
When a photon hits a molecule and is absorbed, the molecule is promoted from its state into a higher energy state. The difference in energy between the two is the gap in the bandage. The energy of the photon must coincide exactly with the gap of the bandage in order for the photon to be absorbed. The chemical structure determines the gap of the bandage; therefore the molecules each have unique absorption spectra (Skoog, 2016).
Absorbance follows Beer’s law, which shows the states is equal to the molar attenuation coefficient times the path length and concentration. The molar attenuation coefficient is related to the individual capacity of compounds to absorb light of a specific wavelength. Path length refers to the distance traveled by light through the sample, which is typically 1 cm for standard cuvettes. Beer’s Law can be used to calculate the concentration of the sample, if the absorptivity is known, or a calibration curve can be used (Skoog, 2016).
UV-Vis is called a general technique, as most molecules absorb light in the UVvisible wavelength range. The UV range extends from 100 – 400 nm and the visible spectrum ranges from 400-700 nm. However, other spectrophotometers do not operate in the deep ultraviolet range of 100 – 200 nm, as light sources in this range are expensive. Most UV-Vis spectrophotometers use a deuterium lamp for the UV range, which produces light from 170 – 375 nm and a tungsten filament lamp for the visible range, which produces light from 350 – 2,500 nm (Skoog, 2016).
Since the light source is generally a lamp with wide wavelengths, the specific absorbance wavelength is selected by means of filters or a monochromator. A monochromator is a device that spatially separates wavelengths of light and then places an output slit where it is the desired wavelength of light. The monochromator can be analyzed in a wavelength range to provide an entire absorbance spectrum. This makes the technique useful for quantifying and identifying a wide range of molecules (Skoog, 2016).
Among the applications it has:
- Determination of functional groups in organic molecules
- Analysis of biochemical samples
- Determination of metals in coordination compounds
- Semiconductor Analysis
- Color measurements
- Quantitative determination
- Monitoring the kinetics of chemical and biochemical processes
The steps of the methodology are listed below in the scheme.
Initial solution of 0.01 M of colorant red #40 (MW=
Take aliquots of 2.5, 5, 7.5 and 18 mL to a final volume of 50 mL
Measure of the absorbance with the UV
vis spectrometer at
Make the calibration curve
Measuring the absorbence of two drinks at
Calculate the final concentration of the drinks
Table 1 shows the absorbance values for the different measured standards. It can be seen that as the concentration of the dye increases, the absorbance.
Table 1. Concentration and absorbance data.
Diluted Solution Concentration (M)
y = 129.93x
R² = 0.9858
Figure 2. Absorbance – Concentration fitting curve
Figure 2, represents the linear adjustment between the concentration of Red 40 solutions and the absorbance, it can see that the equation of the line is y=129.9x and R2=0.9513. It can relate the adjustment of the curve with the equation of Beer Lambert, in that case the equation that relates the absorbance with the concentration is:
The values of the concentration of red 40 in the analyzed drinks are shown in Table 2. In this table, it can be seen that drink 1 has a higher concentration of red 40 than drink 2.
Table 2. Colorant concentration in drinks
Mystery red drink 1
Mystery red drink 2
In order to know if the concentrations of the drinks comply with those allowed for daily consumption, it was decided to compare the results obtained with those reported in a literature work. The values calculated for the analyzed samples and those reported by this work are detailed in Table 3. In it, it is appreciated that the concentrations of drink 1 is allowed for the infantile consumers but not for the adult.
While drink 2 has concentrations of red 40 that are allowed for all consumers. Table 3. ADI values for daily consumption of Red 40 colorant
ADI (children) (mg/kg/d)
Mystery red 1
Mystery red 2
The calculated values of ADI were made considering that the commercial beverage has a volume of 250.0 mL, and that the weights of children and adults are 30 and
80.7 kg, respectively.
- Sample concentration (mystery red drink #1)
𝐶 = 𝑠𝑙𝑜𝑝𝑒 = !” = 0.0063 𝑀
Where: C is concentration of the problem sample (M), A is absorbance and slope is the slope of the fitting curve (M-1).
- Calculated ADI (mystery red drink #1 for adults)
𝑚 = 𝑛 ∗ 𝑀 = 0.0016 𝑚𝑜𝑙 𝑚𝑔
𝑚 783 𝑚𝑔 𝑚𝑔
Where: ADI stands for acceptable daily intake (mg/kg), m is the mass of dye in a portion of the commercial product (mg), W is the weight of the person drinking the commercial product (kg), V is the volume of the product (L) and M is the molar mass of the dye (g/mol)
The adjustment obtained by the calibration curve is adjusted to a straight line, complying with the estimates of Beer Lambert’s Law. The adjustment of the straight line gave as equation: 𝐴 𝐶. The value of R2 was close to 1, which meant that the experimental data fit the equation of the line with a probability of
0.9513. In this experience there are many sources of error because there is no experience preparing solutions, nor handling the volumetric material, so this could be the cause of the R2 value being less than 1. In order to minimize errors in other work, one must ensure that the aliquots of solutions are taken correctly, the entire volume is transferred to the volumetric flasks, and the volumetric materials are correctly wrapped. The results of the final concentrations of red 40 in the drinks showed that both are allowed for children, but drink 1 cannot be ingested by adults as its value is above the allowed ADI value.
- The relationship between red 40 standards concentrations and absorbance was linear with an R2 equal to 0.9513, the equation of the line was 𝐴 =
- The concentrations of the red colorant in drinks 1 and 2 were: 0.0063 and
0.0010 M, respectively.
- Drink 1 can be ingested by all types of consumers, since its ADI values are below the permitted ones. Drink 2 can only be consumed by children, since its ADI value is above the permitted.
- Skoog, D. A., Holler, F. J., & Crouch, S. R. (2016). Principles of instrumental analysis.Thomson Brooks/Cole.
- Stevens, LJ; Burgess, JR; Stochelski, MA & Kuczek, T (2013) Amounts of Artificial Food Colors in Commonly Consumed Beverages and Potential Behavioral Implications for Consumption in Children. Clinical Pediatrics XX (X) 1 – 8.