In the Laboratory
A Problem-Solving Approach to Chromatography in the Biochemistry Lab
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Frank R. Gorga Department of Chemical Sciences, Bridgewater State College, Bridgewater, MA 02325;
[email protected] Many biochemistry lab manuals include experiments involving the purification of enzymes; wheat germ acid phosphatase (1, 2) and lactic dehydrogenase (3, 4) are common “targets”. Most of these experiments rely mainly on precipitation techniques to effect purification and include, at most, a single chromatographic step. However, the purification of proteins in the “real world” usually involves a number of chromatographic steps and a number of chromatographic media (in addition to many other techniques). Thus, the typical experiment does not expose students to the idea of multiple-linked chromatographic steps in purification. These experiments also tend to be “cookbook” exercises in which the student follows a detailed protocol and does not have to directly address the “big picture”. The purification of enzymes is also a poor choice for introducing students to the correlation between the physical properties of molecules and their chromatographic behavior. Biological samples are complex mixtures of proteins, most of which are not visibly colored. Thus, students are unable to watch separations in real time and it is difficult to correlate the chromatographic behavior of a particular protein with its properties. At least one attempt to deal with these issues in the undergraduate biochemistry lab has been reported (5). However, this experiment requires the preparation of labeled proteins, a time-consuming step that many instructors might prefer to avoid. Herein, I describe an experiment in which students must devise and carry out a separation protocol for a five-component mixture using multiple, sequential chromatographic steps. The components to be separated are well defined, commercially available, visibly colored compounds. Thus, students are able to watch the separation of the compounds and to correlate their movement on various chromatographic media. Students are given mixtures of colored molecules and three chromatographic matrices (a cation exchange medium, an anion exchange medium, and a size exclusion medium). After an extensive prelab lecture dealing with the mechanics of chromatography, students are challenged to devise (and carry
Table 1. Properties of Materials to Be Separated Component of Color Mixture
Compound Blue dextran
1, 2
Blue
Yellow dextran
2
Yellow
Cytochrome c
1
Vitamin B12
2 1
Yellow
DNP-glycine aDNP
264
a
MW/Da >500,000
Ionic Character at pH 6 Very strong anion
~40,000
Strong anion
Red-orange
12,400
Strong cation
Cherry red
1,357
Weak cation
241
Weak anion
stands for dinitrophenyl.
out) a procedure for the separation of the five colored molecules whose properties they are given (see Table 1). Students are told to proceed in two stages over two 3-hour lab periods. For the first stage, each student is given 1-mL samples of two test mixtures, each of which contains three components (see Table 1). They are instructed to run a portion of each sample on each of the chromatographic matrices provided, to collect fractions, and to make careful observations of the separations. Students use the data they collected during the first week and the interval before the second lab period to devise a multicolumn separation of a mixture containing all five components shown in Table 1. The instructor is available for consultation during this phase and students are actively encouraged to avail themselves of this help. When students arrive for the second lab period they are given 0.5 mL of the five-component mixture and are told to present the instructor with five test tubes, each containing a single component, by the end of the period. This approach gives students experience in making careful observations, using those observations to draw conclusions, and testing those conclusions by performing a multi-column separation of all five components in the mixture. Materials and Methods This experiment requires no special equipment. The materials, including the chromatographic media, are inexpensive and are used in small quantities. In addition to the sample components, eluting solutions consisting of potassium acetate buffers of various concentrations and HCl solutions to 6.0 M are required. The chromatographic media used are carboxymethyl (CM) Sephadex G25, diethylaminoethyl (DEAE) cellulose, and Sephadex G25. Small reusable glass and plastic columns are used in my lab. However, glass Pasteur pipets with small glass wool plugs are a viable alternative to specialized columns. Results There is no single correct solution to this exercise. Resolution of the five components can be accomplished in a number of ways. Typically, the macromolecules (the dextrans and cytochrome c) may be separated from the small molecules (vitamin B12 and DNP-gly) but not from each other by gel filtration. Resolution of the macromolecules is readily accomplished by anion exchange chromatography on DEAE-cellulose. The cytochrome c is not retained. The yellow dextran can be eluted with 1.0 M potassium acetate (KAc), pH 6. The blue dextran is eluted with either 2.0 M or 6.0 M HCl (depending on the patience of the student).
Journal of Chemical Education • Vol. 77 No. 2 February 2000 • JChemEd.chem.wisc.edu
In the Laboratory
Resolution of the small molecules can be accomplished by either cation exchange or anion exchange. Vitamin B12 does not bind to DEAE cellulose in 0.1 M KAc, pH 6, while DNP-gly can be eluted from this column with 1.0 M KAc, pH 6. Both of these compounds can also be separated on CM Sephadex by eluting with 0.1 M KAc, pH 6. Vitamin B12 is weakly bound under these conditions and thus elutes significantly later than the unretained DNP-gly. I have used this experiment with two groups of students (totaling 25 individuals). Initially, students expressed some discomfort at the idea of an open-ended “noncookbook” experiment. However, all students were able to complete the exercise satisfactorily. Of course, some needed more coaching or advice from the instructor than others. Comments obtained at the end of the exercise (and before grades were issued) were uniformly positive. Students commented on learning the “thought processes” in addition to the mechanics.
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Supplemental Material
The following supplemental material for this article is available in this issue of JCE Online: instructor’s notes including instructions for preparation of the required solutions, the material given to students, and the short PowerPoint presentation used in the prelab lecture. Literature Cited 1. Minich, M. J. Experiments in Biochemistry: Projects and Procedures; Prentice Hall: Englewood Cliffs, NJ, 1989; pp 169–176. 2. Smith, R. A. J. Chem. Educ. 1988, 65, 902–903. 3. Anderson, A. J. J. Chem. Educ. 1988, 65, 901–902. 4. Dryer, R. L; Lata, G. F. Experimental Biochemistry; Oxford University Press: New York, 1989; pp 386–392. 5. Chakravarthy, M.; Snyder, L.; Vanyo, T.; Holbrook, J.; Jakubowski, H. V. J. Chem. Educ. 1996, 73, 268–272.
JChemEd.chem.wisc.edu • Vol. 77 No. 2 February 2000 • Journal of Chemical Education
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