In the Laboratory
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Solar Irradiation of Bilirubin: An Experiment in Photochemical Oxidation A. E. Pillay*,† Department of Chemistry, College of Science, Sultan Qaboos University, Al Khoud, 123, Sultanate of Oman; *
[email protected] F. M. Salih Department of Clinical and Biomedical Physics, College of Medicine and Health Sciences, Sultan Qaboos University, Al Khoud, 123, Sultanate of Oman
This laboratory experiment is an interesting application in biological chemistry and is beneficial to students in chemistry who are eventually branching out into the health sciences or biochemistry. The experiment deals with bilirubin, which is a well-known light-sensitive biological compound (1– 6) that is pedagogically ideal for photochemical experiments at tertiary institutes. The chemicals and reagents required to run the experiment are relatively inexpensive thus making an investigation of this nature perfect for implementation as an undergraduate laboratory experiment. An important outcome of the exercise is that it provides students with an exciting insight into the potential of sunlight and its capability of producing information on chemical kinetics. (In regions where sunlight is weak any source of visible light will do). The native compound undergoes photochemical oxidation to biliverdin when exposed to light (mechanistic considerations appear in refs 7 and 8). The reaction and the data are exponentially functional for the facile abstraction of parameters such as the rate constant and half-life. Measurements are made by spectrometric absorption at 453 nm (Figure 1) and factors that influence the reaction such as autooxidation and temperature are additional aspects of the study. A secondary feature of the experiment is monitoring the growth of the photoproduct, biliverdin, which appears at 365 nm. Biliverdin is a significant precursor in biosynthesis (5, 6), and knowledge of its preparation (and isolation) under these conditions could be useful information for students combining chemistry with biochemistry courses.
Hazards Sodium hydroxide is corrosive and should not come into contact with the skin, eyes, and clothing. Bilirubin, and its product biliverdin, can irritate the skin and eyes. Ingestion of the relevant solutions can be harmful. When preparing the solutions it is advisable for instructors or students to wear gloves. Laboratory coats are recommended in addition to mandatory safety goggles. Waste-containers should be provided for the waste generated from this experiment. Results and Discussion To gauge the length of the experiment and the timeframe to acquire sufficient data and overcome the pitfalls, we used a graduate student to conduct a dummy run. The pitfalls usually occur in the solar exposure part of the experiment. Students may not be able to reproduce the exact geometry for irradiation, and may turn the test-tube holder at different angles to the incident solar radiation. This could lead to minor anomalies in the data, but can be obviated by demarcating a fixed outline (with a permanent marker), possibly on the ground or a wall, against which the test-tube holder can rest. The rest of the procedure is straightforward. After exposure the bilirubin solution is measured spectrophotometrically and a table with the relevant data can be prepared.
Experiment A typical stock solution of bilirubin (Fluka ChemikaBiochemika, Buchs, Switzerland) is prepared by dissolving 0.50 g of bilirubin in 1 L of a 0.05 M NaOH solution, in the absence of light (small red lamp only). About 400 mL of this stock solution can be used by each student to prepare dilutions. Brown glass containers, wrapped in aluminium foil, are used to store the stock in the dark, and a fresh stock solution is prepared each time a new experiment is conducted. During irradiation the bilirubin solutions are placed in regular glass test tubes, which are exposed at a fixed geometry. The exposure times range from 1 to 10 minutes (at successive increments of 1 minute). The bilirubin levels are measured spectrophotometrically with a standard UV–vis spectrophotometer. Blank measurements are made with the appropriate solvent. †
Current address: Department of Chemistry, The Petroleum Institute, P.O. Box 2533, Abu Dhabi, UAE.
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Figure 1. Aqueous solution of bilirubin recorded before exposure to sunlight and after a 20 minute exposure. After 20 minutes most of the bilirubin has been converted to biliverdin.
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Typical results are shown in Table 1. From these data a plot of [bilirubin] versus time can be constructed (Figure 2) for abstraction of the necessary information on the reaction rate and half-life. The entire procedure, including the data reduction and plotting of graphs, should fit well into a 3-h laboratory session. Clearly, from the didactic perspective, obtaining the reaction half-life is best taught from an experimentally derived plot. However, it is necessary for students to have a background to photochemical kinetics, which utilizes the quantum yield in evolving the theory (see refs 9 and 10). Instantaneous reaction rates can be evaluated by taking tangents to the curve at designated points. For purposes of instruction (Figure 2) we have selected two points at 20.0 µM and 10.0 µM and calculated the instantaneous rates of 3.5 µM兾min and 1.7 µM兾min, respectively. From inspection it is clear that when the concentration is halved the rate is halved. Students can now compute the half-life graphically, directly from the plot.
Table 1. Photooxidation of Bilirubin with Time of Sunlight Exposure t/min
Conc/ (µmol L᎑1)
Abs
Fraction Remaining (%)
ln[bilirubin]
00
2.40
40.0
10 0
᎑10.13
01
2.02
33.6
084
᎑10.30
02
1.70
28.3
071
᎑10.47
03
1.43
23.8
060
᎑10.78
04
1.20
20.0
050
᎑10.82
05
1.01
16.8
042
᎑10.99
06
0.85
14.1
035
᎑11.16
07
0.71
11.9
030
᎑11.34
08
0.60
10.0
025
᎑11.51
09
0.50
08.4
021
᎑11.69
10
0.43
07.1
018
᎑11.86
A good routine exercise for students is to determine the half-life at different initial concentrations, as shown on Figure 2. The shaded areas on Figure 2 reflect the half-life of the depletion process, which is 4.0 minutes. The rate constant k is not obvious from Figure 2. The data have to be reconfigured linearly (see inset) to determine k, which is the negative value of the slope. We found that autooxidation can be appreciable in NaOH. The extent of autooxidation is clearly a function of time and it is expedient for students to record the absorbance of the control initially and after all the measurements have been taken. A drop in bilirubin concentration of about 10% can occur over several hours so it is vital that the solutions are prepared a short while before the experiment commences, and analyzed immediately after exposure. It is difficult to devise a correction factor for autooxidation because the bilirubin concentration can also vary with temperature (see below). However, the change in the absorbance reading of the control before and after the experiment can be used as a source of error in the investigation. Thermal effects also affect the results. This feature of the work is relatively unexplored and has only been recently reported by us (5). This part does not have to be included but is given for purposes of information. We made additional measurements at different temperatures and found that the fraction of bilirubin converted after progressive increases in temperature in intervals of 5 ⬚C up to about 40 ⬚C was under 10%. Our measured decreases in the relevant levels of the control (initially at 25 ⬚C) were as follows: 25–30 ⬚C: 3.1%; 30–35 ⬚C: 5.4%; and 35–40 ⬚C: 8.1%. This showed that the depletion in bilirubin concentration increases with increasing temperature and this treatment can be usefully employed to implement approximate corrections for heat-up effects in solar irradiation. Such corrections can be roughly applied by monitoring the temperature of the solution after each exposure, and if it falls in any of the temperature ranges above (e.g., 29 ⬚C) the true absorbance reading is approximately:
ln [Bilirubin]
true Abs = measured Abs兾(100 − 3.1)%.
40
−11 − 12 0
5
10
Time / min
30 25
7.5 mol/L
20 15
rate = 3.5
mol L min rate = 1.7
2.14 min
5.0 mol/L
[Bilirubin] / ( µmol/L)
35
slope = −k = 0.17 minⴚ1
−10
10
mol L min
2.94 min
5
From the perspective of reproducibility the absorbances of 5 individual aliquots can be recorded by students (over a few minutes to avoid possible autooxidation) and the standard deviation calculated for the 95% confidence limit. Our repeated trials (n = 5) produced reproducibility within ±2%. Further validation, in terms of the accuracy was established by comparing the experimentally determined value of the molar absorptivity of bilirubin at 453 nm, 60.05 ± 0.01 mM᎑1 cm᎑1, with the documented value (11) of 60.1 ± 0.1. A marginal relative error of 0.08% was attained, indicating that the performance of our solution and system was satisfactory. Our control solution of bilirubin had a concentration of 40.0 µM, which is recommended for optimum results. At levels above this value Beer’s law could be violated and some aberrant behavior could be encountered.
0 0
2
4
6
8
10
Time / min Figure 2. [Bilirubin] vs time of sunlight exposure. Inset: ln[bilirubin] vs time of sunlight exposure.
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Conclusion This experiment is both facile and student-friendly. It is robust and relatively inexpensive to run and would be ap-
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In the Laboratory
propriate to demonstrate the impact of the kinetics of photochemistry on biological compounds using solar radiation in a 3-h laboratory period. It is not technically demanding and students will find it enjoyable and interesting. A useful extension to this experiment would be to isolate the photoproduct (biliverdin) for possible implementation in organic synthesis. W
Supplemental Material
A student handout including procedures, reagents, and an equipment list; instructor’s notes; examples of spectra; and questions and answers are available in this issue of JCE Online. Literature Cited 1. Roth, M. Clinical Biochemistry: Principles and Methods; Walter de Gruyter: New York, 1974; pp 1372–1389.
2. Salih, F. M.; Al-Hamdi, A.; Pillay, A. E. J. Trace Microprobe Techn. 2001, 19, 409–417. 3. Pillay, A. E.; Salih, F. M.; Al-Hamdi, A. J. Trace Microprobe Techn. 2002, 20, 601–609. 4. Salih, F. M. Photodermayol. Photoimmunol. Photomed. 2001, 17, 272–277. 5. Pillay, A. E.; Salih, F. M.; Al-Hamdi, A.; Al-Kindy, S. J. Trace Microprobe Techn. 2003, 21, 295–310. 6. Ritter, S. Chem. Eng. News 2002, 80 (49), 9. 7. Knobloch, E.; Mandys, F.; Hodr, R. J. Chrom. 1988, 428, 255–263. 8. Pillay, A. E.; Salih, F. M. J. Radioanal. Nucl. Chem. 2004, 261, 211–214. 9. Logan, S. R. J. Chem. Educ. 1997, 74, 1303. 10. Willett, K. L.; Hites, R. A. J. Chem. Educ. 2000, 77, 900– 902. 11. Salih, F. M.; Pillay, A. E. J. Radioanal. Nucl. Chem. 2005, 264, 561–564.
The structures of a number of the molecules discussed in this article are available in fully manipulable Jmol and Chime format as JCE Featured Molecules in JCE Online (see page 1329).
Featured Molecules
an interactive modeling feature, Only@JCE Online
http://www.JCE.DivCHED.org/JCEWWW/Features/MonthlyMolecules/
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