Sample lab report
Introduction to the Spectrophotometer:
Wavelength, Absorbance, and Concentration
In Methylene Blue
Kevin Donnelly
2 March 2006
Cell Biology
TA: Alex Trachtenberg
Lab partners: Tamara Jette and Wayne Thornton
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Sample lab report
Introduction to the Spectrophotometer:
Wavelength, Absorbance, and Concentration
In Methylene Blue
Kevin Donnelly
2 March 2006
Cell Biology
TA: Alex Trachtenberg
Lab partners: Tamara Jette and Wayne Thornton
INTRODUCTION
The spectrophotometer is an essential tool for biologists and chemists in analyzing chemical and biological samples. Gaining familiarity with its operating protocols and understanding what its outputs mean are very important in the development of lab technique for students of cell biology. This experiment will help laboratory students gain experience in using the spectrophotometer. This instrument takes advantage of the regular light absorption and scattering patterns of chemical structures (Lab Manual, p.19). Specifically, it detects compounds absorbing light at selected wavelengths and produces a number corresponding to its absorption (Alberts, 2004). Each compound absorbs and scatters this light more than others at specific wavelengths. Years of research have yielded information on a myriad of compounds, permitting one to use spectrophotometry as a way of identifying unknown compounds and determining the concentration of a substance. Specifically, the spectrophotometer measures quantitatively the amount of light passing through a compound in solution as a fraction of the light emitted by the machine (“Spectrophotometry”). A monochromator is used to produce light in very small ranges of wavelength. A photodetector detects how much of the light emitted by the machine actually transmits through the solution. A specific wavelength is input, and a display indicating percent transmittance (and its inverse, absorption or optical density) communicates to the user the absorption of a compound at this wavelength. This experiment includes two pieces related to the spectrophotometer. First, a determination of the wavelength at which a compound (in this case, methylene blue) absorbs light best is made. Second, using this wavelength, solutions of varied concentrations are measured for their absorptions; this measurement relies on the idea that a greater number of molecules in a given volume will absorb more light than one that has fewer (Lab Manual, p. 23). Two hypotheses may be made prior to performing this experiment. First, the absorption spectrum obtained from methylene blue should peak at an intermediate range approximately equal to 668 nm (“Optical Absorption of Methylene Blue”). In addition, the absorptions of serial dilutions of methylene
by 25 nm, zero the machine, measure the OD of the stock solution at the new wavelength) was repeated 21 times, until readings for the same stock solution across a wide spectrum of wavelengths was recorded. The only extra adjustment needed during this process occurred at the 600 nm reading, when the machine’s filter lever needed be flipped to its second position; this was forgotten at first, and resulted in initial readings at 600nm, 625nm, and 650nm inconsistent with the hypothetical trend. The final readings recorded, however, reflect the proper adjustment of the lever as required by the spec-20. The results of this part of the experiment were recorded in a table, and then plotted using Microsoft Excel. A peak wavelength was recorded for use in the next step in the experiment. To determine the standard curve of absorbance versus concentration, several steps were necessary. First, serial dilutions of the stock solution were made. Using the pipettes from step 1, 5 ml of stock solution was placed in one vial without any water. A second vial containing 4.5 ml and 0.5 ml of stock solution and water, respectively, was made. This process was continued for a total of 9 vials (each totaling 5 ml, with remaining ratios of stock:water::4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5, 2.0:3.0, 1.5:3.5, 1.0:4.0). The wavelength was held constant through this experiment. The wavelength used was the peak wavelength from the previous portion of this experiment: 625nm.^1 The machine was zeroed for this wavelength using the steps outlined earlier. Each dilution tube was placed individually in the sample chamber and the resulting OD was recorded. Alongside this data, the molarity of each sample was calculated and their corresponding g/L concentrations were, as well. This data was plotted using Microsoft Excel in an X-Y scatter plot. A best fit line (standard curve) was calculated and plotted using Excel, and the equation for this line included. A tube of the stock solution with unknown concentration labeled “B” was obtained from the instructor for evaluation. This tube was placed in the sample chamber of the spec-20 without adjustment from the previous step in the experiment; its OD was recorded at a wavelength of 625 nm and its OD was
(^1) 625 nm is not the actual peak wavelength recorded for the stock solution used. Two higher results were excluded because there was instruction to exclude wavelengths with correlated OD values greater than 0.8. See Table 1 in theresults section for these values.
recorded. Having established the standard curve for this wavelength, the sample of unknown concentration was plotted on the best fit line using OD. Its correlating concentration was then found. RESULTS The first experiment yielded wavelength and absorbance (OD) readings as shown in Table 1. These results were plotted in Figure 1. Figure 1 shows the absorbance increasing as the wavelength is increased until approximately 650 nm is reached; from this point, the absorbance pattern decreases rapidly. Therefore the peak wavelength for Methylene Blue is approximately 650 nm. Table 1
Absorbance vs. Wavelength^ Figure 1:
(^0 100 200 300 400) Wavelength (nm) 500 600 700 800 900 1000
Absorbance (Optical Density)
Wavelength (nm) Absorbance (OD) Wavelength(nm) Absorbance(OD) 400425 0.0180.010 675700 0.8000. 450 0.025 725 0. 475500 0.0380.055 750775 0.0100. 525 0.068 800 0. 550575 0.1800.300 825850 0.0090. 600 0.560 875 0. 625 0.725 900 0. 650 0.
DISCUSSION
Two hypotheses were evaluated in the course of this experiment. The first hypothesis, that methylene blue would absorb light at 668 nm, was not able to be falsified. The peak wavelength occurred at 650 nm. The next highest occurred at 675 nm, and the third at 625 nm. Given that the OD measured at 675 nm was slightly higher than that at 625 nm, it is likely that the peak wavelength actually exists somewhere between the measures evaluated at 650 nm and 675 nm. The second hypothesis, that a linear relationship between absorbance and concentration would be found, was also not falsified based on the experiment. The R^2 value for the best fit line was 0.987; in statistics, the R^2 value can be anywhere from 0 to 1, with 0 being least likelihood of correlation, and 1 indicating a perfect correlation between events. As the R^2 value was very close to 1, a high degree of correlation was found. Given the results, little error appears likely in the experimental procedures. However, any doubts regarding the results may be traced to a few elements of the experiment that lend themselves to possible error. The need to zero the machine between each of the readings in obtaining the absorption spectrum, and resulting peak wavelength, leaves room for error between each reset procedure. Though the student performing the zeroing was supported in his procedure by two lab partners, the precision with which a person can accurately adjust the needle on the spectrophotometer to zero is limited. Another place where error is likely is in the serial dilution of methylene blue. Though there is a presumption that the pipetting was done accurately, similar room for human error in measurement is possible as in the repeated zeroing of the instrument. Finally, reading the OD outputs became less precise the higher the OD reads; the machine measures more precisely smaller increments, with larger increments closer together on the dial. A lack of precision in all three of the stated procedures above is possible. However, error appears to have been minimized in the experiment, assuming the hypotheses made were not misguided. One way of determining the accuracy of these results would be to repeat this experiment several times, finding a mean between the data obtained.
REFERENCES
Alberts, Bruce. Dennis Bray, et al. Essential Cell Biology, ed 2. New York: Garland Science, 2004. p. 103 Lab Manual, Cell Biology. Atrium Graphics, 2006. “Monochromator.” http://en.wikipedia.org/wiki/Monochromator Prahl, Scott. “Optical Absorption of Methylene Blue,” http://omlc.ogi.edu/spectra/mb/index.html. “Spectrophotometry.” http://en.wikipedia.org/wiki/Spectrophotometry