Lab 2 post-lab (spectrophotometry) PDF

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Completed lab report for Lab 2 (assigned by Prof. Wentz-Hunter)...

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Spectrophotometry LAB 2

Carolyn Straub & Morgan Youskevtch (lab partner) PROFESSOR WENTZ-HUNTER | PERFORMED 6/3/15, DUE 6/17/15

Straub, 1

Introduction In spectrophotometry, a crucial process to many biological assays, the pigments of a solution placed in the machine are quantified by the amounts of light absorbed and transmitted. During measurement, a specific wavelength on the visible spectrum is chosen. When that specific wavelength, which corresponds to a specific amount of energy, is absorbed by the sample, the absorbance and percent transmittance readings change accordingly. The more light absorbed, the higher the absorbance reading and the lower the transmittance reading. The relationship between absorbance and transmittance is explained by the BeerLambert Law, which states A=2−log10 %T , where A is absorbance and %T is percent transmittance. When combined with Lambert’s law, which states that “the proportion of any incident of light absorbed by a substance is independent of the intensity of the light” (WentzHunter 2015), we get the following relationship: A=Eλcl . In this equation, A is absorbance, Eλ (Epsilon sub-lambda) is molar extinction coefficient for the absorbing substance at wavelength λ , c is concentration of the absorbing solution, and l is length of light path in the absorbing substance. These equations will be necessary for interpreting the data later on. In this lab, my partner and I used spectrophotometry to measure the absorbance of numerous solutions. This was done by the method of photometry, which compares the intensity of a beam of light (Io) to the intensity of the light transmitted (I). The objectives were to create a standard curve of the protein concentration and then use that curve to determine the unknown protein concentration.

Methods My partner and I followed the lab protocol “Lab 2: Spectrophotometry” on Blackboard (Wentz-Hunter, 2015). The one difference was that our data was not actually used to create the standard curve, nor was our own unknown data used. This is because the dye me and my partner used was ineffective.

Results The concentration of BSA protein in a sample affects its ability to absorb light. My partner and I prepared six protein standards from a 10 mg/ml BSA stock solution. These were mixed with a dye reagent, placed in the spectrophotometer, and used to create a standard curve (in reality, we used another group’s data). When collecting our data, we read the transmittance and then converted it to absorbance using the equation A=2−log10 %T . Morgan and I chose to do this because in the higher absorbance ranges, it allows us to read more significant figures. These results can be found in table 1 and figure 1.

Straub, 2

Standard Curve Absorbance vs [BSA]

Absorbance at 595 nm

1.8 1.6

f(x) = 0.06 x + 0.16

1.4 1.2 1 0.8 0.6 0.4 0.2 0

0

5

10

15

20

25

[BSA] (mg/μl)

Figure 1. The standard curve for BSA protein at wavelength 595 nm with line of best fit. These results are from my classmates, not Morgan and me.

Standard Curve (Absorbance vs [BSA]) Own Data [BSA] (mg/μl)

Absorbance at 595 nm Try 1

0 5 10 15 20 25

Try 2

%Transmittance Try 1

Blank 0.359 0.387 0.010 0.392 0.389

Try 2 Blank

0.344 0.377 0.010 0.403 0.416

43.8 41.0 97.7 40.6 40.8

45.3 42.0 97.8 39.5 38.4

Table 1. Standard curve data with my own data. We discarded this data because it was acquired with faulty dye.

Next, we prepared four test tubes from the protein sample of unknown concentration. Again, we discarded our own data (found in table 2). The data we used from our classmates is in table 3. Our classmates’ 0.01 ml solution was chosen as the unknown to find the concentration of because it best fit on the standard curve.

Unknown Protein Concentration (Absorbance vs [BSA])

Straub, 3

Own Data Protein (ml)

Absorbance at 595 nm Try 1

0 0.01 0.02 0.1

Try 2

%Transmittance Try 1

Blank 2 2 2.05

Try 2 Blank

2 2 2.05

1 1 0.9

1 1 0.9

Table 2. Unknown protein concentration with my own data. We discarded this data because it was acquired with faulty dye.

Discussion After collecting our quantitative data, I moved focused attention on the purpose of the lab: determining the concentration of the unknown BSA sample. To do this, I used the equation of the linear standard curve, which was y=0.0628 x +0.159 . I took the absorbance found, which was 0.54, and multiplied it by the dilution factor (120), then plugged it in for y and solved for x . The answer came out to be 1030 mg/μl of BSA in the unknown sample. One source of error could have occurred during pipetting. I felt like we could have been more careful about our accuracy, especially at the end when Morgan and I were trying to finish up. The main source of error, however, was the faulty dye used in the experiment, which was prepared by the TA.

Conclusion With the help of my partner, I was able to determine that the BSA concentration was 6.07 mg/μl for the unknown in this experiment. I came to this conclusion by comparing the absorbance of the unknown solution to the standard curve made by the data from my classmates.

Straub, 4

References Wentz-Hunter, K. (2015). Lab 2: Spectrophotometry. Blackboard.com accessed on May 26, 2015....