Lab #7 Report

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University Of Arizona *

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447

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Mechanical Engineering

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Jan 9, 2024

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McKenna Nichols BME 447 Loeffler 25 October 2023 Chapter 8 Lab Report: Spectrophotometry Results Task 1: Hemoglobin Quantification with a Spectrophotometer Intensities Measured At Wavelength: 530 Concentration (mg/mL) Intensity Absorbance 0 12258.46 0 0.125 3389.73 0.55827 0.25 2701.61 0.65681 0.5 2683.69 0.06597 1 679.41 1.25630

Task 2: Hemoglobin Quantification with LED/PD Circuit Concentration (mg/mL) 𝑉 𝑜?? Absorbance 0 11.1 0 0.125 10.3 0.03249 0.25 9.6 0.0630 0.5 9 0.09108 1 8.1 0.13684 Task 3: Unknown Sample 𝑉 𝑜?? Absorbance Assumed Concentration (mg/mL) 9.9 0.04969 0.25

Discussion In the first portion of this lab, we began by making five solutions which each have different concentrations of hemoglobin. The concentrations we made are 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0.0 mg/mL. We then used the spectrophotometer to measure the intensities of these concentrations. We then used these intensity values to calculate the absorbance for each solution. The equation we used for absorbance is , where is 𝐴 = 𝑙𝑜𝑔( 𝐼 0 𝐼 ) 𝐼 0 the light intensity from the light source and is the light intensity passing through the material. 𝐼 Now that we have the absorbance, we graphed that in comparison to the concentrations. This graph can be seen in the results section above. Our results showed that there is a direct relationship between the absorbance of hemoglobin and the concentration of hemoglobin. This makes sense because as the hemoglobin concentration increases, the solution becomes less transparent. This means that more of the light is absorbed by the hemoglobin and less of the light passes through the solution. In the second portion of this lab, we used the same five solutions that we made for the previous section. To begin we had to wire the circuit that is shown to the right. From here, we measured the value of when 𝑉 𝑜?? the cuvette was filled with different solutions of different concentrations. We did this for the concentrations of 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0.0 mg/mL. We then used these values to find the absorbance for each solution. We used the 𝑉 𝑜?? equation where is the voltage output of the 0 mg/mL solution and is 𝐴 = 𝑙𝑜𝑔( 𝑉 𝑜??,0 𝑉 𝑜?? ) 𝑉 𝑜??,0 𝑉 𝑜??

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the voltage output of the solution in the cuvette. Finally, we took this absorbance and graphed it against the concentration. This graph can be seen in the results section above. Our results showed the same direct relationship between concentration and absorbance that was seen in the first part of the lab. We also observed that as the value of decreased, the absorbance increased. This 𝑉 𝑜?? makes sense because the highest absorbance reflects the highest concentration. The highest concentration would mean the lowest value because the concentration blocks light from 𝑉 𝑜?? being picked up by the photodiode. The final portion of this lab involved the same circuit and solutions that were used in the previous section. However, we were given a random solution from another group and we had to measure the and calculate the absorbance. From here we had to make an assumption about 𝑉 𝑜?? the concentration of the solution we were given. For this section, we measured a value of 𝑉 𝑜?? 9.8 V which corresponds to an absorbance of 0.05409. Based on this value and our line of best fit from the graph from part 2, we would assume that the concentration of this solution is 0.25 mg/mL. After verifying with the group who gave us the solution, this was correct. Review Questions 8.1 Compare your spectrum to the ones shown in Fig. 8.5. Is your hemoglobin oxy-, deoxy-, or methemoglobin? Explain why your hemoglobin is in that state. Based on our spectrum compared to the one shown in Fig. 8.5, I would say that our hemoglobin is deoxyhemoglobin. This is because the graph we were given only has one distinct peak which is in the area of green wavelength (495-570 nm), there is a slight increase in the red wavelength (625-740 nm) and the NIR is very close to zero. This follows the same pathway that is shown in Fig. 8.5 which is why I am concluding that our hemoglobin is deoxyhemoglobin.

8.2 Compare the absorbance readings (zero-adjusted) of Task 1 and Task 2. The absorbance readings of Task 2 are likely smaller than a half of those of Task 1. What factors are responsible for this deviation? The factors that are responsible for the Task 2 absorbance readings being smaller than a half of those of task 1 are the color of the LED. Only some of these wavelengths may have been able to be read and contributed to the value. The spectrophotometer can produce and pick up all 𝑉 𝑜?? wavelengths including ones that the LED could not. The stock solution had a lower absorbance value because it was unable to pick up all of the wavelengths. 8.3 Does the absorbance (1) increase, (2) decrease, or (3) stay the same if the gain is decreased to 1000? The absorbance would decrease if the gain is decreased to 1000.