Ariel Chen (ac47386) VDS Spring 2013 Beers Law Lab Report - Final Effect of Varying Concentration on Absorbance

Introduction The Beer-Lambert Law, also often called Beers Law, is the linear relationship between absorbance and concentration of some substance that absorbs electromagnetic radiation. The law is written as A = bc, where A is the absorbance, the molar absorptivity of the substance, b the path length, and c the concentration of the substance [1]. This shows a direct relationship between concentration and absorbance. Absorbance of a solution can be found using a spectrophotometer, an instrument that transmits light through a vessel and reads the absorbance of the solution within the vessel [2]. The concentration of the solution can be determined with this instrument and Beers Law, making the process a useful tool in research areas such as drug screening. When a compound successfully inhibits an enzyme, the absorbance reading and concentration can reveal whether or not the inhibition was successful [3]. The objective of this lab is to find the correlation between concentration and absorbance of a substance and find this standard curve. The standard curve will be used to determine the concentration of a solution with unknown concentration. Our hypothesis states that as the concentration of the solutions increase (as they are less and less dilute), the absorbance reading from the spectrophotometer will increase; if the concentration is too low, the spectrometer will not be able to pick it up amidst the noise and when the concentration is too high, the spectrometer might become too saturated and the absorbance values plateau. Materials and Methods Materials needed for this investigation included a stock solution of a chemical compound of known concentration, either in red or blue (red was used, in this instance), several reusable, non-sterile plastic test tubes with a volume of more than 10 ml, a test tube rack, a beaker of deionized H2O, Parafilm, a plastic cuvette, a spectrophotometer that can read the visible spectrum, a pipettor and pipette tips, and a 10ml long pipette and a rubber pipette bulb. Due to the possibility of the dye from the stock solution staining clothes or skin, lab coats, goggles, gloves, closed toed shoes and long pants were worn in this experiment. A sample of the stock solution was obtained from the mentor overseeing the experiment: Red Dye Stock #3. Nine plastic test tubes were labeled with labeling tape and markers with Stock, Dil. 1, Dil. 2, Dil. 3, and so on. Serial dilution s of the stock solution were performed. From the stock solution to Dilution 1, the dilution performed was a tenfold dilution (1ml of stock solution and 9ml of dH2O were added to Dil. 1). Taking up 1ml of the substance was done with a micropipettor and for amounts larger than 1ml, with the 10ml long pipette and rubber pipette bulb. Before setting up the next dilution, the solution in Dilution 1 was mixed thoroughly by covering the top of the tube with a square of Parafilm and a thumb and inverting it around five times. The same tenfold dilution was performed from Dilution 1 to Dilution 2. However, from Dilution 2 to 3, a twofold dilution was performed (5ml from Dil. 2 and 5ml dH2O went into the tube labeled Dil. 3). Twofold dilutions were performed for Dilution 3 to 4 and for the rest of tubes. Between each dilution, it was ensured that the solutions were mixed thoroughly by inverting it with Parafilm. Next, a spectrophotometer was obtained: a Vernier (Ocean Optics, Dunedin, FL) RedTide VIS-NIR spectrometer (labeled Rugsby) as well as a clean plastic cuvette and a computer with LoggerPro 3.5.3 software (Vernier, Beaverton, OR) installed on it. The spectrometer was calibrated by going under the Experiment menu in LoggerPro and selecting Calibrate and Spectrometer:1 and following the instructions on screen. The plastic cuvette was loaded with 1ml H2O, 1

Ariel Chen (ac47386) VDS Spring 2013 Beers Law Lab Report - Final serving as the blank, and inserting the blank into the spectrometer so the light passed through the 1cm length. The cuvette was carefully wiped down prior to ensure that there were no smudges that could offset the readings. The cuvette was removed after the calibration process was completed and rinsed out. 1ml of the solution in Dilution 2 was loaded into the cuvette and into the spectrometer in the same fashion the blank was placed in it (with the light passing through the 1 cm length) so that the max can be found. Data was collected after Dilution 2 was loaded and the peak of the curve, the maximum absorbance, was found and the wavelength at that point was recorded. The cuvette was removed and properly rinsed and dried. 1ml from Dilution 9 was loaded and the absorbance at the determined max was found and recorded. Three trials from Dilution 9 were performed, cleaning out and loading a new sample into the cuvette between trials. After Dilution 9, Dilution 8 was tested, then Dilution 7, and so forth until Dilution 1 had readings taken of it. All data collected was recorded. Finally, the process was repeated with a solution with unknown concentration. The three absorbance values were also collected and recorded. Results and Discussion max: 523 nm

Data Table: Experimental absorbances for each trial and dilution with calculated averages, standard deviation, molar absorptivity, and concentration of unknown solution. Dil 1 Dil 2 Dil 3 Dil 4 Dil 5 Dil 6 Dil 7 Dil 8 Dil 9 Unknown (by slope) 0.008047 2 0.663 0.662 0.669 0.665 0.004 (by Beer's Law) 0.00768065 3

Con. 0.5 0.05 (known) Absorbance 2.363 2.201 Trial 1 Absorbance 2.383 2.214 Trial 2 Absorbance 2.381 2.166 Trial 3 Average 2.376 2.194 Abs Standard 0.011 0.025 Dev. Molar 4.751 43.87 Absorptivit 3 y Avg of Molar Absorptivity

0.025 1.779 1.800 1.794 1.791 0.011 71.64 0

0.012 5 1.020 1.022 1.029 1.024 0.005 81.89 3

0.0062 5 0.528 0.529 0.525 0.527 0.002 84.373

0.0031 3 0.264 0.268 0.267 0.266 0.002 85.227

0.00156 3 0.134 0.135 0.138 0.136 0.002 86.827

0.00078 1 0.068 0.071 0.065 0.068 0.003 87.040

0.00039 1 0.035 0.041 0.034 0.037 0.004 93.867

86.53778

Ariel Chen (ac47386) VDS Spring 2013 Beers Law Lab Report - Final

Fig. 1: Spectrum with maximum absorbance, wavelength and statistics box found from Dilution 2.

Fig. 2: Spectrum with absorbance of each trial at maximum wavelength.

Ariel Chen (ac47386) VDS Spring 2013 Beers Law Lab Report - Final
1.200 1.000 0.800 0.600 0.400 0.200 0.000 0 0.002 0.004 0.006 0.008 0.01 Concentration (mM) 0.012 0.014 Standard Curve Unknown Linear (Standard Curve) y = 82.596x R = 0.9995

Fig. 3: The relationship between solution concentration and absorbance with linear best fit. Unknown with Beers Law calculated concentration. Dilutions 4 through 9 used.

When the absorbance reading from the spectrophotometer was below 1.5, the concentration and absorbance had a direct relationship. The average absorbance of Dilutions 4 through 9 wer used to find the standard curve seen in Fig. 3., and these five data points were selected because Dilutions 1, 2, and 3 had absorbance values greater than 1.5, when concentration and absorbance no longer had a linear relationship. After linear fit was used on the data points to find a slope, the slope was used to find the concentration of the unknown solution. Beers Law (A = bc, where A is the absorbance, the molar absorptivity of the substance, b the path length, and c the concentration of the substance ) was also applied to find the concentration of the unknown solution in different way, and the result was 0.007680653 millimolar. The molar absorptivity was calculated using Beers Law on known solutions with known concentrations and averaging the molar absorptivity. See Data Table. The data collected from the spectrophotometer was precise through trials with small standard deviation values. The data was generally accurate as well, with the R2 value on the linear fit graph closely approaching 1. Our hypothesis was proven correct because the data collected revealed a direct relationship between absorbance and concentration, which is also in line with Beers Law. Possibilities of error still exist, and some factors that could skew of offset results include human errors when measuring volumes of solution or solvent during the dilutions, resulting in concentrations slightly different than the calculated concentrations. The dilutions could also be inadequately mixed at certain points, with parts of the solution having a different concentration than others. The covering of the tubes with Parafilm and inverting several times is meant to try to minimize the possibility of this error but it might still occur momentarily. The spectrophotometer itself may fluctuate slightly in its readings even with it is stabilized, which could be another source of slight error. Conclusion

Absorbance

Ariel Chen (ac47386) VDS Spring 2013 Beers Law Lab Report - Final An initial stock solution was diluted several times to a range of known concentrations. The solutions were loaded into a cuvette and spectrophotometer and the absorbance was found. Using these values, a standard curve was found and the unknown concentration of a solution was calculated with both the standard curve and Beers Law. In future research, the skills of performing serial dilutions and using a spectrometer will likely be utilized, and they are important skills to have when doing procedures such as enzyme assays, since absorbance is used to determine whether or not a compound is effective in inhibiting an enzyme. References 1. Tissue, B. M. The Chemistry Hypermedia Project: Beer-Lambert Law. http://www.files.chem.vt.edu/chemed/spec/beerslaw.html (accessed February 12, 2013). 2. Allen, D., Cooksey, C., Tsai, B. Spectrophotometry. http://www.nist.gov/pml/div685/grp03/spectrophotometry.cf m (accessed March 06, 2013) 3. Greis, K. D. Mass spectrometry for enzyme assays and inhibitor screening: an emerging application in pharmaceutical research. Mass Spectrom Rev 2007, 26 (3), 324-39.