AC47386 BeersLawReport 030713 FINAL

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Ariel Chen (ac47386)

VDS Spring 2013

Beers Law Lab Report - Final

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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 pickit 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 dilutions

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 H 2O,


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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 thecurve, 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)

(by Beer's

Law)

Con.

(known)

0.5 0.05 0.025 0.012

5

0.0062

5

0.0031

3

0.00156

3

0.00078

1

0.00039

1

0.008047

2

0.00768065

3

Absorbance

Trial 1

2.363 2.201 1.779 1.020 0.528 0.264 0.134 0.068 0.035 0.663

AbsorbanceTrial 2

2.383 2.214 1.800 1.022 0.529 0.268 0.135 0.071 0.041 0.662

Absorbance

Trial 3

2.381 2.166 1.794 1.029 0.525 0.267 0.138 0.065 0.034 0.669

Average

Abs

2.376 2.194 1.791 1.024 0.527 0.266 0.136 0.068 0.037 0.665

Standard

Dev.

0.011 0.025 0.011 0.005 0.002 0.002 0.002 0.003 0.004 0.004

Molar

Absorptivit

y

4.751 43.87

3

71.64

0

81.89

3

84.373 85.227 86.827 87.040 93.867

Avg of Molar Absorptivity 86.53778


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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.


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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, andthe 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 adifferent 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

y = 82.596x

R = 0.9995

0.000

0.200

0.400

0.600

0.800

1.000

1.200

0 0.002 0.004 0.006 0.008 0.01 0.012 0.014

Absorbanc

e

Concentration (mM)

Standard Curve

Unknown

Linear (Standard Curve)


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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 ornot 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/chem-ed/spec/beerslaw.html (accessed February 12, 2013).

2. Allen, D., Cooksey, C., Tsai, B. Spectrophotometry . http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm (accessed March 06, 2013)

3. Greis, K. D.Mass spectrometry for enzyme assays and inhibitor screening: an emerging application inpharmaceutical research.Mass Spectrom Rev 2007, 26 (3), 324-39.

AC47386 BeersLawReport 030713 FINAL

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