Spectrophotometry and Calibration Curves PDF

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Title: Spectrophotometry and Calibration Curves Day and Date: Sept 12 2019, Thursday Name: Nikhila Sampath Partner: Harrison Park Course: Chem 223 Section: AB 5 TA: Sanja Pudar

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Abstract The purpose of this experiment was to determine the unknown concentration of methylene blue using Beer’s Law. This was done using calibration curves and linear regression. The unknown concentration was determined to be 19.9 ± 1.48. Possible sources of error include improper wiping of the cuvette and background noise.

Introduction The main goal of this lab was to determine the unknown concentration of methylene blue in a mixture using calibration curves and linear regression. This was accomplished using Beer’s Law, which states that the amount of energy absorbed or transmitted by a solution is proportional to its molar absorptivity and the concentration of solute1 . In other words, A= εLC

(1)

where A is absorbance, ε is molar absorptivity in cm-1  M-1, L is path length in cm and C is the concentration in M. In this lab, a red tide spectrophotometer was used to measure absorbance. In a spectrophotometer, a white light is shone at a sample and the light returning from the sample is measured. A spectrophotometer measures the intensity of light relative to wavelength. Electromagnetic energy collected from the sample enters the device and is separated into component wavelengths by holographic grating. The grating separates each color from the white light which is then focused onto a CCD array detector where the intensity of each wavelength is then measured The CCD is then read-off to a computer and the result is a spectrum which displays the intensity of each wavelength of light. Spectrophotometers are used in a variety of

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fields. Some of its uses include quality control of clothing and LEDs2 . Spectrophotometers are the preferred means of measuring absorbance because of its flexibility and graphical representation of data which makes it easier to interpret. Another advantage is that the instrument is capable of measuring small volumes and highly concentrated samples3 . However, like everything else, using spectrophotometers has its own disadvantages. The equipment can be expensive. Moreover, the instruments don’t work well if left on for a long period of time (personally experienced in the lab).

Chemical Information Chemical Methanol4

Hazards ➔ Flammable liquid and vapor ➔ May be fatal or cause blindness if swallowed. ➔ Harmful if inhaled or absorbed through skin. ➔ Causes irritation to skin, eyes, and respiratory tract ➔ High vapor concentrations may cause drowsiness

Routine safety and handling procedures Inhalation: Remove to fresh air. If breathing is difficult, administer oxygen. If the victim is not breathing, provide artificial respiration. Get medical attention. Ingestion:Do not induce vomiting unless directed to do so by medical personnel. If vomiting occurs, keep head low so that vomit does not enter the lungs. Never give anything by mouth to an unconscious person. GET MEDICAL ATTENTION IMMEDIATELY. Skin Contact: Wash affected area with plenty of water for at least 15 minutes. Remove contaminated clothing and shoes. Wash clothing before

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reuse. Get medical attention if symptoms occur. Eye Contact:Check for and remove contact lenses. Immediately flush eyes with gentle but large stream of water for at least 15 minutes, lifting lower and upper eyelids occasionally. Get medical attention. Methylene blue5

➔ Harmful if swallowed ➔ Toxic to aquatic life

Inhalation: Remove the victim into fresh air. Ingestion:Rinse mouth with water. Immediately after ingestion, give lots of water to drink. If the victim is fully conscious, immediately induce vomiting. Call Poison Information Centre. Skin Contact: Rinse with water. Soap may be used. Take victim to a doctor if irritation persists. Eye Contact:Rinse with water. Remove contact lenses, if present and easy to do so. Continue rinsing. Do not apply neutralizing agents. Take victim to an ophthalmologist if irritation persists.

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Procedure The first part of the lab was about setting up the spectrophotometer and handling the equipment. A cuvette was obtained and a mark was made on one side of the frosted area to ensure consistent placement in the spectrophotometer. Extra caution was taken to touch only the top and frosted area while handling the cuvettes to minimize error. Gloves were worn for the experiment. The cuvette was wiped with a Kimwipe before being inserted into the cuvette holder. The spectrophotometer was calibrated following the instructions provided. In the second part of the lab, the spectrophotometer was blanked using water and the absorbance of water at 500 nm was measured ten times. The cuvette was then replaced with 30% methanol . The absorbance of 30% methanol at 500 nm was measured. The spectrophotometer was the renblanked with 30% methanol and the cuvette was removed from the cuvette holder. The cuvette was then replaced back inside the cuvette holder and absorbance at 500 nm was measured ten more times. This process was repeated again, but the cuvette was taken out and replaced in between readings. The mean and standard deviation was calculated for both sets of data. The third part of the lab dealt with determining the unknown concentration of methylene blue in a mixture. The spectrophotometer was blanked using the 30% methanol and the unknown was chosen. Based on the color of the unknown, five standard solutions of known concentrations were picked. The cuvette was emptied and filled with the standard of lowest concentration and an absorbance spectrum was obtained. In this manner, three spectra were obtained for each of the standard solutions, in ascending order of concentration. Using the average absorbance between the three spectra, absorbance vs concentration calibration curves were plotted for 648 nm, 656

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nm, and 664 nm. This data was then used to determine the best wavelength at which the absorbance spectrum for the unknown should be acquired. Three spectra were obtained for the unknown at the picked wavelength.

Data and observations TABLE 1- Absorbance of water 1

2

3

4

0.01

0.01

0.01

0.01

Trial # Absorbance at 500 nm

5

6

7

0.01 0.01

8

9

0.01 0.01 0.01

10 0.01

TABLE 2- Absorbance of 30% methanol Trial # Absorbance at 500 nm

1

2

3

4

5

6

7

8

9

10

0.012

0.012

0.012

0.012

0.012

0.012

0.012

0.012

0.012

0.012

TABLE 3- Absorbance of 30% methanol after reblanking Trial # Absorbance at 500 nm

1

2

3

4

5

6

7

8

9

10

0.009

0.009

0.1

0.009

0.1

0.009

0.009

0.009

0.1

0.009

TABLE 4- Absorbance of 30% methanol when its taken out in between trials Trial # Absorbance at 500 nm

1

2

3

4

5

6

7

8

9

10

0.005

0.009

0.034

0.01

0.017

0.034

0.031

0.041

0.045

1

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TABLE 5- Statistical analysis of values in table 3 and 4 Data Set

Mean

Standard Deviation

Table 3

0.0363

0.0440

Table 4

0.123

0.309

GRAPH 1- Absorbance vs. Concentration calibration curve at 648 nm

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GRAPH 2- Absorbance vs. Concentration calibration curve at 656 nm

GRAPH 3- Absorbance vs. Concentration calibration curve at 664 nm

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TABLE 6- Absorbance of unknown at chosen wavelength (664 nm) Trial #

Absorbance

1

0.407

2

0.408

3

0.408

TABLE 7- Details of unknown Detail

Value

Unknown #

7

Mean Absorbance at 664 nm

0.4077

Standard Deviation for absorbance

0.0005774

ε at 664 nm in cm-1M-1

2.05294E-08

Standard Deviation for ε

1.52408E-09

Concentration in μ M

19.9

Propagation of error in concentration

± 1.48

Calculations Mean= X=

T rial 1+ T rial 2+ T rial 3+ T rial 4+ T rial 5+ T rial 6+ T rial 7+ T rial 8+ T rial 9+ T rial 10 = 10

0.009+0.009+0.1+0.009+0.1+0.009+0.009+0.009+0.1+0.009 0.363 = 10 = 10

Standard Deviation=

√

n

∑ (xi−X )2 i=1

n−1

= 0.0440

0.0363

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ε= C=

A C×L

=

0.07633333 4×10 6M ×1cm

= 1.90833 × 10−8 cm-1M-1

A 0.4077 = ε×L 1.90833×10−8 cm−1M −1×1 cm

× 10−6 = 19.8593 μ M

 1.52408E−09 )2 ] ½ SC= C × [( SA )2 +( Sεε )2 ] ½ = 19.9 × [( 0.0005774 )2 +( 2.05294  = ± 1.47762 A 0.4077 E −08

Discussion The first part of the lab involved calibrating the spectrophotometer with 30% methanol since the methylene blue standards were in a solution of 30% methanol. It was observed that the 30% methanol did not have a significant absorbance at 500 nm. Its absorbance was accurate when compared to that of water. When the spectrophotometer was reblanked, the absorbance reading changed since it was measuring the amount of 30% methanol in solution as opposed to water. When the cuvette was taken out in between trials, there was a slight change in absorbance readings (both mean and standard deviations changed). This could be due to the disturbance caused when the cuvette was taken out and placed back in. The spectrophotometer was highly sensitive, meaning that even the slightest amount of noise and light change could affect the absorbance reading. In simpler words, removing and reseating the cuvette in between trials affected the accuracy and precision of the data. The extent of change can be measured by performing the t-test. A t-test is a statistic used to determine if there is a significant difference in the mean of two sets of data which are related to each other. The best range of wavelengths to measure absorbance would be between 350 to 700 nm. Below 350 nm, the absorbance reading reflects the absorbance of the cuvette and not the chemical inside it. In the second part of the lab, an unknown was obtained and five standards were chosen based on it. Some standards were darker in color and some lighter. This was done to ensure that

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the unknown concentration fit the range of concentrations chosen. The same cuvette was used throughout the experiment to ensure accuracy and precision since the spectrophotometer was calibrated to the exact cuvette. As a result the standards had to be tested in ascending order to prevent residue left in the cuvette from contaminating the sample. If there was residue left over from a lower concentration when testing a higher concentration of sample, it would not have a huge affect. However, if residue from a high concentration mixes with a lower concentration sample, it would skew the data. After obtaining three spectra for each standard and graphing mean absorbance for each standard at 648 nm, 656 nm and 664 nm, it was determined that 664 nm was the best wavelength to measure the absorbance of the unknown. 664 nm was chosen because it had the highest R2 value, making it the most accurate. In other words, the best wavelength to assay methylene blue was 664 nm. The concentration of the unknown was determined to be 19.9 μ M with an error of ± 1.48. This result seemed consistent with the data obtained for the various standards. According to the graphs, the unknown concentration should be around 20 μ M. The curvature seen in the working curve is due to the instrument since there is always noise when taking measurements using the spectrophotometer. The slight curves are due to the signal to noise ratio. The lowest concentration at which absorbance can be accurately measured is the limit of detection and should be about three times the standard deviation of the noise from the blank measurement. The highest concentration that can be measured is known as dynamic range. Having relatively low absorptivity constants leads to ideal methylene blue behavior while having a high absorptivity constant or one with high deviations does not lead to ideal methylene blue behavior. The wavelengths used were the ones that had constant absorbance readings. If

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another compound was absorbed and caused interference, this would lead to an overestimation in the concentration of the unknown since the absorbance would be higher than it ideally should be. The closer the R2 of the linear curve is to 1, the more linear it is. While choosing the ideal wavelength to measure the absorbance of the unknown, 664 nm was chosen since its R2 value was the closest to 1. R2 is a better measure of linearity than the standard deviation of the slope. A source of error could have been that the cuvette was not wiped properly and this could have either clouded the cuvette sides or possibly scratched it, causing the data to be skewed. One way the experiment can be improved in the future would be to use a more sensitive material to clean the cuvette so that it would not get scratched. Another way would be to measure absorbance in a quiet room so that no other noise interferes with the absorbance readings. Using spectrophotometers that have a shield or cover around them could also help reduce the noise.

Conclusion The absorbance of water and 30% methanol was determined. The spectrophotometer was then blanked with 30% methanol and the absorbance of 4 μ M, 8 μ M, 12 μ M, 20 μ M and 30 μ M methylene blue was measured at 648 nm, 6565 nm and 664 nm. Based on the R2 values, 664 nm was chosen as the best wavelength to measure absorbance. The absorbance of the unknown was measured at 664 nm and the unknown concentration was determined to be 19.9± 1.48 using Beer’s Law. This agreed with the absorbance readings collected for the standards.

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Reference 1. Illustrated Glossary of Organic Chemistry - Beer's Law (Beer-Lambert Law), http://www.chem.ucla.edu/~harding/IGOC/B/beers_law.html. 2. Martin, Paul. What Is a Spectrophotometer?, http://www.microspectra.com/support/learn/what-is-a-spectrophotometer. 3. Luftman, Marcelo. “Using Spectrophotometer To Determine Concentration (UV/VIS).” PROAnalytics, LLC, 19 Dec. 2018, https://pro-analytics.net/using-spectrophotometer-to-determine-concentration/. 4. Sds.chemtel.net. (2019). [online] Available at: http://sds.chemtel.net/webclients/safariland/finished_goods/Pioneer%20Forensics%20-% 20PF032%20-%20PF033%20-%20PF034%20-%20Methanol.pdf [Accessed 18 Sep. 2019]. 5. Labchem.com. (2019). [online] Available at: http://www.labchem.com/tools/msds/msds/LC16850.pdf [Accessed 18 Sep. 2019].

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Appendix GRAPH 4- Absorbance vs. Wavelength for 4 μ M 4.1- Trial 1

4.2- Trial 2

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4.3- Trial 3

GRAPH 5- Absorbance vs. Wavelength for 8 μ M 5.1- Trial 1

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5.1- Trial 2

5.3- Trial 3

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GRAPH 6- Absorbance vs. Wavelength for 12 μ M 6.1- Trial 1

6.2- Trial 2

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6.3- Trial 3

GRAPH 7- Absorbance vs. Wavelength for 20 μ M 7.1- Trial 1

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7.2- Trial 2

7.3- Trial 3

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GRAPH 8- Absorbance vs. Wavelength for 30 μ M 8.1- Trial 1

8.2- Trial 2

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8.3- Trial 3...