Experiment #6 Introduction to the Principles of Spectrophotometry PDF

Preview

Summary

Lab Report...

Description

Laboratory Exercise #6: Introduction to the Principles of Spectrophotometry Introduction: In the identification of a given substance, there are two types of analysis that are observed: qualitative and quantitative. For qualitative analysis, the shape of the spectrum is most useful since each substance has a unique spectrum and is determined by the electronic or other energetic transitions that a sample may emit. The spectrum is determined by a spectrophotometer, an instrument that measures the wavelength of electromagnetic radiation that is absorbed by a sample and to what extent, the sample absorbs specific radiation. On the other hand, for quantitative analysis, measuring the amount of absorption in order to determine the concentration of absorbing species present is most effective. This is most effective because for a specific substance and a given wavelength, the amount of radiation absorbed is determined by the number of absorbing species in the beam of radiation. This is determined using Beer’s Law: I =Kcx Io A = absorbance ε = molar absorption coefficient c = molar concentration l = optical path length

Beer ' s Law : A=ε cl−log

Io = intensity of light going in I = intensity of light coming out c = solution concentration x = thickness of solution K = constant

The objective of this laboratory exercise is to introduce us to the use and operation of the Spectrovis Plus spectrophotometer and utilize it to determine qualitative and quantitative analysis. A spectrophotometer provides a source of continuous radiation that covers a section of the electromagnetic spectrum. The continuous stream of radiation is then required to be dispersed into a spectrum. In order to choose a portion of the spectrum that illuminates the sample given, a monochromator will be used. The narrow band of wavelengths that leave the monochromator is then directed towards the sample and through the sample onto a detector. The detector needs a way to measure the intensity of the beam when no absorbing units are present (Io) and some of the absorption by the sample (I). This becomes a ratio I/Io called transmittance (T). The Spectrovis Plus will be used, which covers the visible light portion of the electromagnetic spectrum. Experimental Section: Prior to completing the quantitative and qualitative analysis, the Spectrovis Plus needed to be calibrated. First, the Spectrovis Plus was warmed up for at least 5 minutes. Then, an empty

3 of the way with distilled water. The sides of the cuvette 4 was cleaned with Kim Wipes to insure that there were no marks left on the cuvette that would affect the results of the calibration. The cuvette was then placed into the Spectrovis Plus spectrophotometer with the clear side facing the light. Then the machine was calibrated cuvette that was supplied was filled

using the experiment menu→ calibrate→ spectrophotometer. A blank absorbance spectrum was displayed which indicated that the spectrophotometer was successfully calibrated. Qualitative Analysis There were five cuvettes that were supplied with the varying colors: Blue, Red, Yellow, Green, and Purple. Each sample was wiped with a Kim Wipe before being put into the Spectrovis Plus. The operating wavelength and the color of the area of which the absorbance band was observed was measured and recorded on the Logger Pro software. Then the data was transferred to Table 1. Quantitative Analysis A seal unknown solution was given by the TA. Then, two burettes were cleaned with distilled water. One burette was filled with the appropriate solution that corresponded to the unknown solution given. The other burette was filled with distilled water. Using Table 2, 5 specific dilutions were to be made. Each dilution was made in a beaker and transferred to test tubes and labeled 1-5. After all the dilutions have been made, each sample was poured into an empty cuvette and the absorbances were measured using the SpectroVis Plus. The results were recorded in Table 2 and then the sample concentration was determined using the equation CiVi = CfVf . The initial seal unknown sample was also measured using the SpectroVis Plus, in order to determine the operational wavelength. A graph was generated from the data collected and used to find the absorbance and concentration of the unknown sample. Results and Discussion: Qualitative Analysis

Table 1: Observation of Operating Wavelength and Absorbance Band of Sample Solutions

Solution Identification

Color of Solution

Color of the area where the absorbance band is observed

Operating Wavelength (nm)

1

Blue

Red/Orange

630.1

2

Red

Green/Blue

504.2

3

Yellow

Light Blue

485.3

4

Green

Orange

630.1

5

Purple

1st Peak: Green/Blue 2nd Peak: Red/Orange

504.2 630.8

The qualitative analysis portion of the experiment allowed for the understanding of the color wheel. It demonstrated how the colors are corresponded based on the operating wavelength of each respective color. The purple solution had two peaks because it was a mixture of the blue and red solutions. There may have been errors regarding the reading of the operating wavelengths. When trying to find the highest peak, there were other operating wavelengths that also had the highest absorbance value so the average of those wavelengths were taken. However, the recording of the absorbance spectra were done with precision. Quantitative Analysis Table 2: ppm and Absorbance of Diluted Solutions Solutions (Dilutions)

Milliliters Stock Solution (mL)

Milliliters H2O (mL)

Sample Concentration (ppm)

Absorbance

1

1.00

7.00

2.50

0.109

2

2.00

6.00

5.00

0.204

3

4.00

4.00

10.0

0.449

4

6.00

2.00

15.0

0.656

5

8.00

0.00

20.0

0.865

The quantitative analysis portion allowed for the understanding of the relationship between concentration and absorbance. It demonstrated a linear trend which can be used to determine unknown samples within the spectrum. There may have been human errors associated with the diluted samples. The proportions may not have been exactly correct, however, the procedure was done with precision. The use of spectrophotometry is seen in many different fields in the industry. In the food industry, preservatives are commonly used in numerous food products in order to broaden the time span of usability. Additives such as nitrates and sulfites can be effectively and adequately estimated using UV-VIS spectroscopy. Spectrophotometers are used to determine whether regulatory standards are met by analyzing the color saturation of food samples. Spectrophotometry is flexible to be utilized in a broad range of fields, so, there should be further experimentation to see if it can be utilized in other ways. Calculations: Concentration of Diluted Solutions: C i∗V ❑i=C ❑f ❑∗V ❑f 1) (20 ppm)(1.00 mL) = C❑ f❑ C❑ f❑ = 2.50 ppm 2) (20 ppm)(2.00 mL) = C❑ f❑ C❑ f❑ = 5.00 ppm 3) (20 ppm)(4.00 mL) = C❑ f❑ C❑ f❑ = 10.0 ppm 4) (20 ppm)(6.00 mL) = C❑ f❑ C❑ f❑ = 15.0 ppm 5) (20 ppm)(8.00 mL) = C❑ f❑

(8.00 mL) (8.00 mL) (8.00 mL) (8.00 mL) (8.00 mL)

C❑ f❑ = 20.0 ppm Concentration of Unknown Solution: Unknown Solution: Cuvette F Equation of Graph: y = 0.0437x - 0.0019 x = concentration y = absorbance Recorded Absorbance: 0.680 y = 0.0437x - 0.0019 0.680 = 0.0437x - 0.0019 0.6819 = 0.0437x x = 15.60 ppm → concentration of unknown solution Conclusion: The purpose of this laboratory exercise was to become familiar with the operation of the SpectroVis Plus spectrophotometer. Using Beer’s Law and the relationship between absorbance and concentration, it resulted in an understanding for the qualitative and quantitative analysis capabilities of the spectrophotometer. The concentration of the unknown sample was able to be determined, which was 15.60 ppm, and the absorbance of the sample was also determined, which was 0.680. The experiment and the reading of the operating wavelengths were done with precision. References: CHEM 107 Laboratory Manual, Department of Chemistry, Binghamton University, Binghamton, New York, 2019, pp. 91-96. Ramanathan, G. Color Saturation: Measuring the Concentration of Food Additives with UV-VIS Spectrophotometry. https://www.hunterlab.com/blog/color-food-industry/colorsaturation-measuring-the-concentration-of-food-additives-with-uv-vis-spectrophotometry/ (accessed Oct 21, 2019)....