Precision Measurements Using UV/Vis Spectroscopy And Quantitative Determination Of Salicylate Via Reaction With Fe(III)

Introduction to Precision Measurements and the Science of Measurement

Part 1: Precision Measurements using UV/Vis Spectroscopy

This lab work was meant to foster understanding in:

1. Use of an air-displacement piston pipette
2. Use of an analytical balance
3. Calibration of glassware

4.To demonstrate the influence of volume measuring equipment on precision

Introduce the concept of propagation of uncertainty 

The science of measurement is known as metrology. Mass, length, area, volume, time, pressure, and other weights and measurements are all part of physical metrology. The CSIRO National Measurement Laboratory, NATA (National Association of Testing Authorities), ISO9000, and other regulating agencies such as the FDA (Federal Drug Administration) in the United States are the bodies that supervise weights and measures in Australia (Jorio and Dresselhaus, 2016).

 The link between a sequence of measurements, especially whether they show the same or comparable results, is referred to as precision. Precision is sometimes used interchangeably with accuracy, which characterizes the connection between a measurement and a predetermined ‘actual’ value, such as a standard reference material.

Because no two measurements are ever precisely the same, analysts often refer to measured values as having a degree of uncertainty, which refers to the range of possible values on each side of the observed value that the measurement is most likely to fall into.

In a data collection, precision is expressed as the standard deviation, range, or variance. The width of the curve in a Gaussian distribution plot represents the standard deviation, which is a measure of how tightly a collection of results are grouped around the mean.

Part 2: Quantitative determination of Salicylate via Reaction with Fe(III)

This practical exercise reinforces knowledge in:

1. Preparation of calibration curves
2. Quantitative analysis using a complexometric reaction
3. Determining reaction stoichiometry using a Job plot

Spectroscopic analysis is an important method for identifying and quantifying various compounds. This experiment teaches you how to determine salicylate using electronic absorption spectroscopy in the visible portion of the spectrum.

Salicylate has a variety of applications, hence it’s found in a variety of items. Salicylic acid is the most prevalent metabolite of aspirin, and it’s used to treat acne, warts, and other skin conditions (https://www.facebook.com/Drugscom, 2020).

Salicylic acid reduces the loss of skin cells from hair follicles, which helps to cure acne. These cells are usually the ones that obstruct pores and cause pimples. Salicylic acid also has a keratolytic (peeling) action, which promotes the shedding of dead cells. This makes it easier to remove a thin layer of skin and helps to unclog pores. Salicylic acid at higher concentrations is used in wart therapy to soften the wart and induce an immunological response against the human papillomavirus, which causes wart production.

Salicylic acid’s diverse medicinal uses need the development of analytical procedures for its measurement. Gas-liquid chromatography (GLC), ultraviolet spectroscopy, and fluorescence spectroscopy were among the techniques used. Colorimetric or visible spectrophotometry, on the other hand, is the most extensively used approach in clinical labs. To measure salicylate in a commercial product, a variation of this approach will be used throughout the trial (face wash). The second stage of the technique employs spectrophotometry to look into the chemical composition of the reaction that results in the colored product under investigation.

 Beer’s Law says that a compound’s absorbance is proportionate to its concentration (A=εbc). We may use this linear connection to create a calibration curve by collecting absorbance values for samples of known concentration at a certain wavelength, ideally the max, which is the wavelength where maximum absorption occurs. The linear regression equation that results allows us to calculate the concentration of an unknown material by measuring its absorbance at the same wavelength. The inability of salicylate and salicylic acid to absorb visible light poses an experimental obstacle. However, when iron (III) ions are added, a brightly colored species emerges:

Understanding Precision and its Relationship with Accuracy

With a basic spectrophotometer, you can readily identify the complex, allowing you to measure salicylate in unknown materials. All salicylate will be protonated under the acidic experimental circumstances, as illustrated in the chemical equation above. The coefficients and subscripts x and y are indicated in the chemical equation above. The method of continuous variation (also known as Job’s method) will be used in the second part of this experiment to determine these numbers for the dominating complex. Several solutions containing various amounts of salicylate and Fe3+ will be created for this technique. The total moles of both reagents will stay constant while the quantity of each reactant is altered. The solution with the highest absorbance at max is the one with the most stoichiometry.

The method was adapted as described in the Analytical Chemistry 1 Practical Manual. Briefly, for the first part of the experiment, the air-displacement piston pipette and 10.0 mL volumetric flask to be used for the experiment were firstly calibrated. For the pipette, it was done by pipetting 1.00mL(1000uL) of water into a 20.0mL vial and recording the weight displayed on the balance. This was repeated by adjusting the scale on pipette until the balanced displayed 1.00g (which was at 1.009uL on the pipette and then 4 more times after that to ensure the correct volume was being dispensed. The volumetric flask was then calibrated by placing it on the balance and filling it to the line with water to record the weight displayed. This was repeated 4 times.

After the calibration of the pipette and the volumetric flask, the 1000uL of the stock solution that was provided (methyl orange in water) was transferred to the 10.0 mL volumetric flask using an air-displacement piston pipette and then diluted to volume with distilled water. The absorbance of this solution was measured at 473nm on the UV/Vis spectrometer and the value recorded. This was repeated four times using the same pipette and volumetric flask so that there were 5 absorbance values recorded in total at the end.

For the second part of the experiment, at first, for the spectrophotometric determination of salicylate in acne medication, five standard solutions of sodium salicylate in deionised water were prepared as following- 1. A 100ml volumetric flask was used to prepare an initial stock solution of 100mM (0.100 M) of sodium salicylate. The mass of sodium salicylate needed for this was calculated to be 1.60g and accurately weighed out on the balance.

By dilution in 10mL volumetric flasks, standards of 20.0mM, 30.0mM, 40.0mM and 50.0mM were made and another 100mL of a 10mM standard in water was also made.

A sample of the acne face wash solution was collected and the suggested quantity of the salicylate on the label is noted that is 2.00%.

In 5 separate test tubes, 20.0uL of each standard and of the acne face wash was transferred using an air-displacement pipette. Then 10.0mL of acidic 10.0mM ferric nitrate solution was added to each test tube and mixed well.

Quantitative Determination of Salicylate via Reaction with Fe(III)

After the spectrophotometer was blanked using the ferric nitrate solution, λ_max was determined from an absorbance spectrum (400-700nm) collected with the most concentrated standard and then the absorbance values were recorded for each of the standards at the determined λmax.

Within the second part of the experiment, to examine the reaction stoichiometry of the reaction between Fe (III) and salicylate, 20.0mL of 10.0mM acidic ferric nitrate, 50.0mL of dilute nitric acid (60.0mM) and the 10.0mM sodium salicylate solution made earlier were used to prepare solutions for spectrophotometric analysis by pipetting the correct amount of each solution into a vial as suggested in the table below. After that, the vials were labelled and 4.00mL of dilute nitric acid was added to each vial to make a total of 5mL. Using the dilute nitric acid to blank the spectrophotometer, the absorbance values were recorded at λmax for each solution. 

A table of Calibration of Pipette and Volumetric Flask

A standard curve of Iron Salicyclate solutions. 

A job plot for data from part 2

A table showing statistical analysis of the unknown sample.

A comparative data analysis table for Part 1 data sets.

When the quantified value of salicylate is converted to g/L, a value of 3.9% is noted. This is contrary to the literature value of 2%. This is due to errors both in measurements carried out and the inherent uncertainty of the apparatus.

In the comparison of two data sets in part 3, it is found that the data indeed come from the same population but vary widely and their standard deviations are quite different. This can be attributed to random errors.

From your data, which dilution method offers the best precision? Discuss the possible reasons with reference to the statistical results obtained and sources of error and uncertainty.

Precision from standard deviations show pipettes to possess a lower standard deviation (0.003725) compared to that of volumetric flaks (0.02838). Precision can also be deduced from the uncertainty results. The pipette offers an experimental uncertainty (0.001665) lower than that of the volumetric flask (0.01269). Pipetting methods are thus considered more precise in producing more reproducible results. These uncertainties can either be grouped as systemic or random errors.

Compare and contrast the experimental and the theoretical uncertainty associated with the air-displacement pipette. Comment on potential differences.

The theoretical uncertainty is observed to be higher when compared to the experimental uncertainty. This can be inferred to experimental uncertainty not only accommodating for systemic errors but also random errors.

Why is it important to use the same pipette and glassware when completing the 5 repeat measurements for each solution?

This helps to keep systemic errors due to apparatus changes to a minimum. Every apparatus will give a different uncertainty, for success in the experiment the value of uncertainty by apparatus should remain constant.

In your Discussion, answer the following questions;

a) Were there any outliers in the data sets? Show the values that identify if there are or aren’t outliers.

The dataset did not contain any outliers as shown by no value going below G_critical in the Grub’s test.

Was your standard deviation similar to your lab partners? Show the values that identify if the standard deviations are or aren’t the same.

F-test gives a good discerning platform as it compares the variances in the data. It is hypothesised that the standard deviations are similar. It can thus be observed that; F>F_critical. leading to rejection of the hypothesis.

Was your data from the same population as your lab partners? Show the values that identify if they are from the same population.

The null hypothesis is that the samples are from the same population. This is evaluated by using a t-test. It can be observed that t_sample<t_critical   thus the null hypothesis is upheld.

Briefly comment on a possible source of error that might provide outliers or differences in the data.

Random errors caused by human error are mainly associated with the errors observed from the comparison of the two data sets.

Conclusion

The effect of errors can be clearly observed in the measurement and quantification od salicylate with propagation of uncertainty. The quantified concentration value of salicylate is observed to be 24.31⁺_–0.6830.

The job plot reveals that the best stoichiometric ratio is 0.4. this gives the mole ratio of ferric nitrate to salicylate at 1:1.50.

Errors would have been mitigated by being more attentive and keen to reduce the impact of random errors, which has been seen to have a big impact on the variance of samples in the same population.

References

https://www.facebook.com/Drugscom (2020). Salicylates. [online] Drugs.com. Available at: https://www.drugs.com/drug-class/salicylates.html.

Jorio, A. and Dresselhaus, M.S. (2016). Nanostructured Materials: Metrology. [online] ScienceDirect. Available at: https://www.sciencedirect.com/science/article/pii/B9780128035818025911 [Accessed 7 Apr. 2022]. 

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