SPECTROSCOPY MINI RESULTS

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Georgia State University *

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3810

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Chemistry

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Feb 20, 2024

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SPECTROSCOPY MINI RESULTS Spectroscopy of the Known Sample A protein sample with a known concentration of 0.125 mM was stained with the optically active chromophore Fast Green FCF. The sample was diluted at 7 different concentrations using 0.1M Tris and the OD values of each sample was measured at 625nm. Each of the 7 diluted concentrations was placed into 7 different cuvettes, and the goal was to create a standard curve using the OD readings of these 7 different concentrations in order to observe the effect of increasing concentration on Optical Density (OD). It was found that the concentration of the protein sample was directly proportional to the OD. This result can be explained by the Lambert-Beer Law; OD = εcl, where y = OD and x = concentration (c) with a slope of 0.0361 which is equal to the extinction coefficient (ε) (Table 1, Figure 1). Table 1. Fast Green FCF Known Concentrations and its Optical Density The protein sample, which was stained with Fast Green FCF, was diluted into seven different concentrations and placed into seven separate cuvettes. The optical density (OD) of each sample was then measured and recorded in Table 1 using a spectrophotometer at a wavelength of 625nm. The results were used to plot a standard curve (Figure 1), which indicated that an increase in concentration led to an increase in the OD values. Cuvette # Concentration (µM) (Independent Variable) Optical Density at 625nm (Dependent Variable) 1 0.625 0.063 2 1.25 0.080 3 2.5 0.137 4 5 0.177 5 7.5 0.257 6 10 0.391 7 12.5 0.509 Figure 1. Fast Green FCF Standard Curve 0 2 4 6 8 10 12 14 0 0.1 0.2 0.3 0.4 0.5 0.6 f(x) = 0.04 x + 0.03 Fast Green FCP Standard Curve at 625nm Concentration (µM) OD at 625nm

A scatter plot was created to show the relationship between the OD values of the seven cuvettes from Table 1 and their corresponding wavelengths. The line of best fit was plotted as a dotted line with an equation, indicating a positive relationship between OD and concentration as per the Lambert-Beer Law. Spectroscopy of Unknown Samples The spectrophotometer was used to measure the OD values at wavelength 625nm of three stained samples: A, B, and C. Using the equation of the line generated from the standard curve (y= 0.0361x + 0.0273 in Figure 1), the concentrations of these three samples were calculated and recorded in Table 2. For samples B and C, a series of serial dilutions were performed because their respective OD values were above 1.0. The dilution factor was 1:2 for sample B and 1:10 for sample C (Table 2). Another concentration was calculated for the diluted samples of B and C and recorded in Table 2. Next, a manual spectrum analysis of Sample A was conducted to determine the peak wavelength at which Sample A absorbed the most visible light. The selected wavelength range was between 400nm to 700nm with an increment of 50nm (Figure 2). Two peaks were observed for sample A, the first one at 400nm with an OD value of 0.376 and the second one at 650nm with an OD value of 0.368 (Figure 2). Table 2. Unknown Samples A, B, and C The OD values of samples A, B, and C were obtained using the spectrophotometer. Sample B and C underwent a series of serial dilutions until their OD values were below 1.0. The diluted OD values were measured and recorded in Table 2 along with their dilution factors. Sample A did not undergo any dilution because its first OD reading was below 1.0. The obtained OD values for each sample were used to calculate their concentrations using the equation of the line found in Figure 1. Unknown Samples Undiluted OD at 625nm Dilution Ratio Diluted OD at 625 nm Undiluted Concentration (µM) Diluted Concentration (µM) A 0.836 None 0.836 22.40 22.40 B 1.391 1:2 0.689 37.78 18.33 C 4.157 1:10 0.328 114.40 8.33 Calculation for Concentration of Unknown Samples A, B, and C  Sample A  Using the equation, y = 0.0361x +0.0273, y= 0.836 and x is the concentration of A in µM  Hence, 0.836 = 0.0361(x) + 0.0273  x = ( 0.836 − 0.0273 ) 0.0361  x = 22.40 µM  Sample B o Undiluted:  Using the equation, y = 0.0361x +0.0273, for undiluted OD, y= 1.391 and x is the undiluted concentration of B in µM

 Hence, 1.391 = 0.0361(x) + 0.0273  x = ( 1.391 − 0.0273 ) 0.0361 z  x = 37.78 µM o Diluted:  Using the equation, y = 0.0361x +0.0273, for diluted OD, y= 0.689 and x is the undiluted concentration of B in µM  Hence, 0.689 = 0.0361(x) + 0.0273  x = ( 0.689 − 0.0273 ) 0.0361  x = 18.33 µM  Sample C o Undiluted:  Using the equation, y = 0.0361x +0.0273, for undiluted OD, y= 4.157 and x is the undiluted concentration of C in µM  Hence, 4.157 = 0.0361(x) + 0.0273  x = ( 4.157 − 0.0273 ) 0.0361  x = 114.40 µM o Diluted:  Using the equation, y = 0.0361x +0.0273, for diluted OD, y= 0.328 and x is the undiluted concentration of C in µM  Hence, 0.328 = 0.0361(x) + 0.0273  x = ( 0.328 − 0.0273 ) 0.0361  x = 8.33 µM

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Figure 2. Sample A Spectrum Analysis A spectrum analysis was conducted on sample A to determine the wavelength at which it absorbs the most visible light. The analysis was performed using a range of wavelengths from 400nm to 700nm with a 50nm increment. The dependent variable was the OD, while the independent variable was the wavelength. The results showed that the peak wavelength at which sample A absorbed the most light was 400nm and 650nm. However, the lowest wavelength for visible light absorption for sample A was observed to be around 550nm and 700nm, as depicted in Figure 2. Concentration and Purity of Samples D and E Two colorless samples, D and E, were subjected to a spectrum analysis. Sample D was a DNA solution while sample E was a protein solution. The spectrophotometer was set to a specific range of wavelengths, from 220nm to 360nm. This was done to determine the concentration of DNA in sample D, calculate its purity level, and identify the peak positions of both samples D and E. Sample D had OD values of 2.872 and 2.142 at wavelengths 260 nm and 280nm, respectively. Using the formula: DNA concentration = 50ug/mL x # OD units, the DNA concentrations of sample D at 260nm and 280nm were calculated and recorded in Table 3. The purity for sample D was calculated using the formula: Ratio = OD 260 OD 280 , and it was found to be 1.34, which indicates protein contamination in sample D since it falls below 1.8. The peak of sample D was observed at 260nm while the peak of sample E was observed at 280nm. (Table 3, Figure 3) Table 3: Calculation of Sample D’s DNA concentration and purity ratio The OD readings of sample D at both 260nm and 280nm were recorded and compared. With the use of the formula, DNA concentration = 50ug/mL x # OD units, the DNA concentrations of sample D were calculated at both 260nm and 280nm. To obtain the purity level of sample 400 450 500 550 600 650 700 750 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Sample A Spectrum Analysis Wavelength (nm) OD

D, the ratio of the OD readings 260nm and 280nm was calculated; = OD 260 OD 280 . The purity value was 1.34 which indicates protein contamination of sample D. Sample D Data Results OD at 260nm 2.872 OD at 280 nm 2.142 DNA Concentration (ug/mL) at 260nm Using equation, DNA concentration = 50ug/mL x # OD units = 50 x 2.872 = 143.6 DNA Concentration (ug/mL) at 280nm Using equation, DNA concentration = 50ug/mL x # OD units = 50 x 2.142 = 107.1 Purity Ratio = OD 260 OD 280 = 2.872 2.142 = 1.34 Figure 3: Sample D and E Spectrum Analysis From Figure 3 above, the OD values of samples D and E were plotted against a selected range of wavelengths of 220nm-360nm. The dependent variable in Figure 3 is the Optical Density (OD) while the independent variable is the wavelength in nm. Sample D is represented by the graph in green with a peak at 260nm with an OD reading of 2.872 hence, it contained DNA samples. The blue plot represents sample E, which shows a peak at 220nm. Despite containing protein, the peak for this sample was detected at 220nm instead of the expected 280nm. This could be due to an error of omission as we forgot to dilute samples with OD's above 1.0. After the peaks for each sample was attended, the general trend was a decrease in OD values. 220 240 260 280 300 320 340 360 380 -0.5 0 0.5 1 1.5 2 2.5 3 Sample D and E Spectrum Analysis D E Wavelength (nm)