An Inexpensive Gel-Filtration Chromatography Experiment A Simple Biochemical Laboratory Exercise for High School and Undergraduate
Students Who Make Their Own Column and Apply Simple Detection Techniques H. Alan Rowe
Norfolk State University, Norfolk, VA 23504
Gel-filtration chromatography (low pressure) is a topic that is rarely addressed in high school or even undergraduate biochemistry laboratory courses. This is due to the complexity and cost of constructing a gel-filtration column as well as the equipment usually needed to collect and analyze column fractions. The experiment presented here can be conducted with a minimum of supplies and equipment at the high school or undergraduate level.
Construction and Application Gel-filtration or gel-permeation chromatography is used to separate molecules, usually biological macromolecules, such as proteins and carbohydrates. It was introduced as a biochemical separation technique around 1960, and it rapidly gained popularity as a method of biochemical purification (1). It has been adapted to high-pressure liquid chromatographic (HPLC) systems with remarkable results. Gel-filtration chromatography is used to separate molecules on the basis of molecular size. A bed of gel with defined pore sizes is used. Large molecules enter the pores slightly (or not at all, as defined by the void volume) and elute off the column first. Small molecules have access to all the spaces of the gel and thus spend more time within the gel (or remain completely within the gel, as defined as the total volume). A simple model of gel-filtration theory was introduced by Flodin (2). Gels can be produced from a variety of substances, including, but not limited to, cross-linked polyacrylamide, dextrans, agarose, and combinations of these materials. Experimental Equipment and Materials
This experiment involves the preparation of a gel-filtration column, calibration using blue dextran and riboflavin (or other small colored molecules), and the analysis of a protein sample. The blue dextran and riboflavin can be detected by the presence of blue and yellow, respectively. The protein, depending on the sample, can be detected directly by color or chemical means, as described below. Making the Column
Students are to obtain a column (about 1.5 x 10 cm), a clamp for support, and a means to stop the column flow, (A small cork or piece of parafilm works well.) These columns are commercially available for approximately $1.00 each from Bio-Rad Laboratories (Poly-Prep columns), or students can make them from a disposable pipet with a small amount of glass wool in the bottom. It is important to use a pipet with a relatively large internal diameter—to facilitate fast flow rates. (A 10-mL pipet cut to size is preferred.) Gel that has been previously hydrated in buffer (e.g., 0.2 M sodium phosphate buffer, pH 7.2) by the instructor is used. A wide variety of these gels is available, and they can be re-used indefinitely if microbial growth is avoided. It is
desirable to use gel-filtration media of different pore sizes in the same class to demonstrate differences in separation. (Bio-Gel P-30 works well.) Preparing the Column
The column is poured using at least 6.0 mL of packed gel,
taking care not to let the level of the buffer drop below the gel bed.
Calibrating the Column
A flow rate is determined by collecting eluant in a 10-mL graduated cylinder while monitoring the time. This is done until there is agreement among three consecutive flow rates as expressed in mL/min.
Loading the Column A 0.50-mL sample of blue dextran (0.5%) is mixed with 0.50 mL of riboflavin (0.04%) and loaded on the column. This mixture will be green, and the blue and yellow components will separate during the column run. To load the sample, the buffer is removed from the top of the gel, and the sample is carefully layered on the exposed gel bed. Collecting Fractions Fraction collection is begun as the sample is allowed to enter the gel. Only when the sample is completely in the gel bed is buffer carefully added dropwise to the top of the column. After all of the sample is washed into the gel bed, buffer is added up to the top of the column. A delicate touch is needed for effective sample application. Buffer should be continuously added to keep the pressure head and the flow rate constant. A larger column constructed for demonstration purposes by the instructor helps the student see this separation within the column itself. Students collect 0.5-mL fractions measured by comparing the eluant in the tube to a previously measured 0.5-mL fraction. Students should add buffer to the top of the column as needed (after the 0.5 mL sample has completely entered the gel).
Analyzing the Fractions Colorimetrically After all of the color has eluted from the column, the color intensity of the fractions are rated on a 0-5 scale with 0 representing a colorless tube and 5 representing the most intense color. The peak tubes have been consistently found two fractions after the color first appears. The color of each tube is also noted in the laboratory notebook. A graph of color intensity versus fraction number (or mL or min) can be constructed. (See the figure.) Measuring the Void and Total Volumes The blue dextran, which is very large, can be used to measure the void volume (V„), and the elution position of riboflavin can be used to measure the total volume of the column (Vt). Volume 70
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Analyzing the Fractions Using Biuret Agen t The protein can be detected by adding 2.0 mL of Biuret reagent to each tube. To make the Biuret reagent, add * *
to about
3.0 g of copper(II) sulfate pentahydrate 12 g of sodium potassium tartrate (Rochelle salt) 1
L of water. Then add
600 mL of fresh 10% sodium hydroxide add water to 2 L. *
and
Fraction number
Elution profiles of blue dextran and riboflavin (open circles) and cytochrome c (closed circles) on a Bio-Gel P-30 gel chromatographic column. Cytochrome c samples were detected using the Biuret method (see text). Similar results are obtained using direct color observation of the peak tubes.
Analyzing the Results The fractions can be evaluated for color intensity to find the peak fraction (Vs) as previously described or read directly on a spectrophotometer at a wavelength of 550 nm, using the first tube collected to zero the instrument. (The peak tubes will appear brown if Nutrasweet is used as the sample.) A graph of color intensity (or A-550 if appropriate) versus fraction number (or mL) can be constructed. (See the figure.) Measuring the Kav of the Protein A Km for the protein sample can be calculated K aT
Chromatographing a Protein Sample
A sample can then be chromatographed in the using purified proteins, for example,
same
manner *
azoalbumin (10 mg/mL), noting the color of the fractions collected
*
protein samples, such as the red cytochrome c (20 mg/mL) the peptide glycytglyeine (80 mg/mL) aspartame (as in the brand Nutrasweet, 0,3 g/ml)
* *
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as
deter-
-
V0
Vt-v0
and compared to published standards to estimate the molecular weight of the sample if the column is of sufficient length. Otherwise the student can determine whether the molecular weight of the protein or peptide is large or small, depending on the calculated Kav. Molecules other than proteins or peptides can be used if they do not interact with the gel and if a method of detection is available.
Literature Cited Fisher, L. “Gel Filtration Chromatography” In The Laboratory Techniques in Biochemistry and Molecular Biology Series; Work, T, S.; Burden, R. H,, Eds.; ElsevierNorth Holland Biomedical: Amsterdam, The Netherlands, 1980. 2. Flodin, P. J. Chromatogr. 1961,5,103. 1.
The samples should be collected past the Vt mined from the first part of the experiment.
V =-
