In the Laboratory
A Colorimetric Analysis Experiment Not Requiring a Spectrophotometer: Quantitative Determination of Albumin in Powdered Egg White
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A High School or General Science Experiment Amanda K. Charlton, Richard S. Sevcik, Dorie A. Tucker, and Linda D. Schultz* Department of Chemistry, Geosciences, and Environmental Science, Tarleton State University, Stephenville, TX 76402; *
[email protected] Albumin is the most abundant protein constituent in human plasma and is the major constituent in egg white, also known as albumen. Albumin aids in the dissolution of many substances that are sparingly soluble in water by directly binding them to the protein. These substances include simple dyes, and this dye-binding capability has been used as the basis for measurement of albumin in biological fluids. Two examples of these techniques are the use of bromocresol green to measure serum albumin (1) and of bromophenol blue in urine dipsticks (2). Either of these dyes can be used to measure the albumin content in a solution of powdered egg white in deionized water, but in our laboratory, the color produced by the bromophenol blue was judged to be easier to evaluate visually. The human eye, with good sample illumination, is very sensitive to small differences in radiant power transmitted through two colored solutions observed side by side, but is very poor as a quantitative basis for measuring the transmission through either solution alone. Therefore, the transmittance of radiant power is generally measured using a spectrophotometer, or some similar instrument, and converted to absorbance (A = ᎑log T ). Solution concentrations are determined using the Beer–Lambert law, (1)
A = abc
where A is the absorbance of the solution, a is a constant dependent upon sample identity and wavelength of analysis, b is cell path length, and c is solution concentration. Therefore, when a solution of known concentration (c) and a solution of unknown concentration (c´) are chemically treated in the same manner and their absorbances (A and A´) measured at the same wavelength in sample cells of the same path length, c´ can be determined from the relationship, c A = c ′ A ′
(3)
b c = b ′ c ′
Journal of Chemical Education
c ′ =
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cb b ′
(4)
Procedure A standard solution containing 0.10 g of albumin in 50.0 mL of deionized water and an unknown solution containing 1.00 g of powdered egg white in 500.0 mL of deionized water are prepared. A pH 4.0 acetic acid兾sodium acetate buffer is prepared by combining 20.5 mL of 0.2 M acetic acid and 4.5 mL of 0.2 M sodium acetate and diluting to 250.00 mL. A 25.0-mL aliquot of the albumin solution is added to 100.0 mL of the pH 4.0 buffer in a 150-mL beaker. This is the standard. A 25.0-mL aliquot of the egg white solution is added to a separate 150-mL beaker also containing 100.0 mL of the buffer. After stirring, 1.25 mL (25 drops) of bromophenol blue indicator is added to each solution, and the solutions are stirred periodically for 10 minutes. Since color development depends on time and temperature, care should be taken to treat both solutions in an identical manner. After the color has developed, previously marked flat-bottom culture tubes are filled to heights of 1.0 cm, 2.0 cm, or 3.0 cm with the standard solution. The tubes are placed above a diffuse light source, and unmarked tubes are filled with the unknown solution until the color intensity matches that of the standard solution as viewed from the open end of the tube. The height of the unknown solution in the unmarked tubes is measured and the percent albumin in the unknown is calculated by the relationship
b albumin fraction = b ′ × 100%
(2)
The concentration of an unknown solution can also be determined from a relationship derived from the Beer–Lambert law and used as the basis for the Duboscq comparator (3): If two solutions with concentrations c and c´, as described above, are placed in sample cells and the solution path lengths are adjusted until A = A´,
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Therefore, if light is passed through the two solutions from below and the heights of the solutions are adjusted until the color intensities are visually the same, the heights of the solutions can be measured and
(5)
where b is the height of the standard solution and b´ is the height of the unknown solution. This experiment is appropriate for a high school chemistry or general science lab and is designed for a class size of 30 students working in pairs. If the experiment is performed during 45-minute lab periods, it can be done over three days: day 1 involves introduction, organization, and preparation of solutions; day 2 includes actually performing the experi-
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In the Laboratory
ment and gathering data; and day 3 is used for calculation, analysis, and discussion of results. Equipment All equipment should be available in a high school chemistry lab. Glassware needed includes two 250-mL volumetric flasks, one 100-mL volumetric flask, three 150-mL beakers, one 600-mL beaker, one 50-mL graduated cylinder, two 25mL graduated cylinders or pipets, one 10-mL graduated cylinder, eyedroppers, and 15 flat-bottom disposable culture tubes (Ward’s Scientific). Two magnetic stirrers are helpful, but solutions can be mixed with stirring rods. An overhead projector covered with typing or tissue paper makes an excellent light source.
Table 1. Height of Unknown Albumin Solution Compared to Standard Run
1 cm
Std (%)
2 cm
Std (%)
3 cm
1
1.40
71.4
2.40
83.3
3.80
Std (%) 78.9
2
1.20
83.3
2.30
87.0
3.60
83.3
3
1.25
80.0
2.75
72.7
3.65
82.2
4
1.25
80.0
2.40
83.3
3.80
78.9
5
1.20
83.3
2.20
90.9
3.70
81.1
6
1.35
74.1
2.80
71.4
4.20
71.4
Avg
78.7
81.4
Overall Avg
79.8
Standard Deviation
5.47
79.3
Chemicals
Results
The following chemicals are required. All are readily available and inexpensive. Bromophenol blue and glacial acetic acid were obtained from Aldrich Chemical Company, Bovine serum albumin was obtained from Sigma Chemical Company, and sodium acetate was obtained from Alfa Aesar. Powdered egg whites can be obtained from a local grocery store.
The data obtained from six different runs and statistical analysis of the calculated results are summarized in Table 1. The results of the colorimetric analysis, 0.798 ± 0.0547 g albumin per 1.00 g sample, fall within one standard deviation of the reported value of 3 g protein per 4 g serving (0.75 g protein per 1.00 g sample), since the protein content in egg white is primarily due to albumin. However, students should be cautioned that the reaction of bromophenol blue with albumin is primarily a screening test and should never be considered as an absolute measure of the quantity of unknown present in the absence of confirmatory testing.
Hazards Preparation of 0.2 M acetic acid will require dilution of glacial acetic acid, which is corrosive and can cause burns with skin contact. This chemical should be used under a hood if possible. Bromophenol blue can cause skin and eye irritation and should not be ingested. Standard safety precautions, including the use of safety goggles should be followed during this experiment. All solutions are dilute and sufficiently nontoxic to be disposed of by flushing down the sink with adequate quantities of water after completion of the experiment. Learning Outcomes This experiment familiarizes students with several basic laboratory techniques and facilitates development of scientific thought processes. The following scientific processes are incorporated into the experiment. The student demonstrates safe laboratory practices; the student collects data and makes measurements with precision; the student expresses and manipulates chemical quantities using mathematical procedures; the student organizes, analyzes, and evaluates data; the student communicates a valid conclusion; and the student makes responsible choices in selecting everyday products using scientific information (analysis of a common food product). The following scientific concepts are taught by this experiment. The student investigates and identifies properties of mixtures and pure substances; the student investigates and compares the physical and chemical properties of ionic and covalent compounds (binding of dye to protein); the student investigates solubility in aqueous solutions; the student evaluates the significance of water as a solvent in living organisms and in the environment (solubility of biomolecules); and the student analyzes household products using indicators to classify the products as acids or bases (use of pH 4 buffer solution for the analysis).
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Conclusion Learning goals are well met by this experiment. When the experiment was performed by high school chemistry students late in the spring semester, it served as an excellent review of the concepts of solution preparation, solubility, pH, and qualitative and quantitative analysis of a common food product. The students were observed to use safe laboratory techniques, collect and analyze data using proper scientific methodology, and obtain accurate results. The students also enjoyed performing this experiment. Student outcome was also evaluated by the instructor in the form of a weekly quiz in which the students were asked to discuss applications of colorimetric analysis to other common situations. Examples given by the students included: swimming pool and aquarium test kits, dipsticks used in medical analyses, use of color intensity to monitor concentrations of soft drink preparations, use of color intensity to measure serving sizes of food, and measurement of water depth and turbidity by visual observation of devices lowered into water. The quantitative determination of albumin in powdered egg white with bromophenol blue using a Duboscq technique is simple, rapid, and generates no hazardous wastes. All chemicals, glassware, and equipment necessary for this analysis can be purchased for less than a total cost of $150. Acknowledgments This procedure was originally developed by undergraduate students in response to the American Chemical Society’s 2003 National Chemistry Week “Chemvention” challenge to
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In the Laboratory
“develop a colorimetric procedure for the analysis of albumin in dried egg white for a total cost of less than $250” (4). The financial assistance of The Robert A. Welch Foundation, Chemistry Departmental Grant AS-0012 is gratefully acknowledged. The authors wish to thank Susan V. Alexander of Comanche High School, Comanche, TX and the students in her high school chemistry classes for testing this experiment in the spring of 2005 and for her helpful suggestions. W
Supplemental Material
Student instructions, notes for the instructor, safety and hazards, and sample data are available in this issue of JCE Online.
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Literature Cited 1. Kaplan, A.; Szabo, L. L. Clinical Chemistry: Interpretation and Techniques; Lea & Febiger: Philadelphia, 1979; pp 172–173, 421. 2. Graff, L. A Handbook of Routine Urinalysis; J. B. Lippincott: Philadelphia, 1983; pp 29–31. 3. Ewing, G. W. Instrumental Methods of Chemical Analysis; McGraw Hill: New York, 1975; p 47. 4. Charlton, A. K.; Sevcik, R. S. Chemvention: Colorimetric Determination of Albumin in Powdered Egg Whites with Bromophenol Blue. In Abstracts of 229th National Meeting of the American Chemical Society; San Diego, CA, March 13–17, 2005.
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