Separations Utilizing Gel Filtration and lon-Exchange Chromatography Salvatore F. Russo and Angie Radcliffe Western Washington University, Bellingham, WA 98225 Biochemical separations must be performed under mild conditions to avoid damage to the desired components. Gel filtration (also known as gel exclusion chromatography or gel nermeation chromatoeraohv), relies on differences in size . L o n g molecules. On h e other hand, ion-exchange chromatography is dependent on charge properties. They are both commonly used in the purification of biological molecules because moderate conditions of temperature, ionic strength, and pH can he used. Experiments that apply gel filtration to biochemical systems have appeared in the Jourml.'J In addition, rhe-separation of~glycohemoglobinsby ion-exchange chromatography has been described.' An experiment has been published' that uses carboxymethyl (CMJ-Sephadex at pH 6.0 with a mixture of blue dextran, cytochrome C , and 2.4-dinitro~henvl(DW-elvcineas the samole. However, only cytoc&om;~'is retalrnkd by the matr;x, so it was important to devise a sample where a t least two components could be sequentially eluted since this would better illustrate the ion-exchange technique. In the experimentthat will be described in this paper the sample contains blue dextran, DNP-glycine, chymotrypsinogen, and cytochrome C. The blue dextran and DNP-glycine separate by a gel filtration mechanism, whereas chymotrypsinogen and cytochrome C are resolved by ion-exchange chromatography. Thus, this laboratory experiment has the advantage of illustrating both separation techniques.
168
Journal of Chemical Education
Theory
Ion-exchange chromatography is a type of partition process that makes use of the different affinity of ions in solution (movingphase) for insoluble charged polymers (stationary phase). In cation exchange, a cation from the solution replaces another positively charged component ionically bonded to the neeativelv chareed immobile ~ h a s e . In this experiment ~ ~ - ~ e ~ iisaused d easxthe stationary phase. Anionic and nonionic solutes are not attracted to the negatively charged resin. Cations, on the other hand, bind ionically to the CM-Sephadex with the extent of binding dependent on their charge density. The bound cations can be seauentiallv removed bv . varying . -the ionic strendh . of the buffe; used aieluant. Gel filtration utilizes porous beads made of an insoluble but highly hydrated carbohydrate or polyacrylamide. Small molecules can enter the beads, whereas large ones cannot. Thus, large molecules only pass through the volume between
' Hurlbut, J. A.; Schonbeck, N. D. J. Chem. Educ, 1981.61, 1021-
11199
Smith, R. A. J. Chern. Educ. 1988.65.902-903. Jackman, L. E. J. Chem. Educ. 1981,58.77-78. Clark, Jr. J. M.; Switzer, R. L. Experimental Riochemlstv, 2nd ed.: Freeman: San Francisco. 1977:p 26.
Sample Composltlon
Name
Colw
elm D s m n Chymohypsinogen Cytochrome C DNP-glycine DNP-glyclnehasma structure
BIW
CDlorless Red Yellow
MoI. Wt.
Ionic Character
2,000,000 23.240 12.400 241
Nonlonlc p l = 9.1 p i = 10.7 p K = 3.0
the beads (void volume), but small ones can penetrate the beads as well as occupy the void volume. The net result is greater column retention of smaller molecules. These considerations a ~ d to v the anionic and noniouic solutes used in this experiment. T h e sample consists of blue dextran, chymotrypsinogen, cytochrome C, and DNP-glycine. The color, molecular weight, and ionic character of each component are listed in the table. A.
-
Experimental
Equipment A glass column that is 20 cm long with 1.3-cm internal diameter with attached Tygon tubing is needed. A screw clamp can be used to adjust flow rate through the tubing, and the column is attached to a ring stand via a buret holder. In addition, glass wool is needed to prevent escape of the resin from the column. A 500-pL microsyringe is useful for sample application, and three graduated centrifuge tuhes (15 mL) far colledion of colored fractions are needed. A UV spectrophotometer with l-em sample cell is required. Also, 20 test tuhes each witha capacity of 3 mL are usedfor collectionof fractions containing chymotrypsinogen. Reagents CM-Sephadex (C-50) may be purchased from Pharmacia, Inc. Allow the ion exchanger to swell in excess 0.01 M potassium phosphate huffer at pH 5 for 2 days. Remove the supernatant, and replace with fresh huffer several times during the swelling period. Then degas the slurry immediately before use. A bed volume of approximately 30 mL per grsm of exchanger is expected. Prepare 0.01 M potassium phosphate buffer at pH 5, 0.01 M potassium phasphate buffer at pH 5 with 0.25 M KCI, and 0.01 M potassium phosphate huffer at pH 5 with 1.0 M KCI. Blue dextran is available from Pharmacia, Inc., and chymotrypsinogen, cytoehrome C, and DNP-glycine may be purchased from Sigma Chemical Co. Each component in the sample is dissolved in the same portion of 0.01 M potassium phosphate buffer at pH 5 to give a final concentration of 5 mg1mL for each component. Expermental procedure ColumnPreporation. Using a g l m rod, push asmall piece of glass wool that has been premoistened in 0.01 M potassium phosphate huffer inta the bottom of the column. Add approximately 5 mL of the same huffer with no effluent flowing. Then add a well-mixed slurry of CM-Sephader in 0.01 M potassium phosphate huffer to the solvent in the column. Allow to drip slowly while adding additional increments of the slurried CM-Sephadex until the settled resin bed reaches a height of 15 cm. Do not allow the column town dry, i.e., do not allow the top fluid surface topenetrate inta the settled resin bed. Instead add small sliquots of buffer to keep the fluid surfaceabove the resin bed.
Sample Application. Once a 15-emdefined resin bed has accumulated, allow the column to drip until the fluid surfacejust reaches the resin bed. Immediatelv add 0.5 mL of the colored samde to the inside glass wall so that itforms a thin hand on top of theresin. Be careful not to disturb the top of the resin bed. Allow the solution to penetrate until the fluid surface is again momentarily dry, then gently add a few drops of the 0.01 M potassium phosphate huffer at pH 5. Allow this buffer to penetrate, and then slowly fill up the rest of the column with huffer. Replenish the upper fluid volume as necessary while observing the course of the elution. Collection ofFractions. Collect the blue and yellow colored drops in separate 15-mL graduated centrifuge tuhes. Then begin adding 0.01 M potassium phosphate buffer containing025 M KCI. Collect 20 fractions, each with 3-mL volume. Test these fractions for the presence of colorless chymotrypsinogen by UV absorption, and pool those with absorbance 20.1 at 282 nm. Calculate the milligrams of chymotrypsinogen using an absorptivity of 2.0 mL/mg cm-' at 282 nm5. After these fractions have been collected, change to 0.01 M potassium phosphate buffer containing 1.0 M KCI, and collect the red material in a graduated centrifuge tube. The resin should be saved after elution of all components since it can be reequilibrated in low ionic strength buffer and reused for future experiments.
Discussion T h e predominant charge on a solute component can be obtained from the data in the table. DNP-glycine carries a negative charge since p H 5 of the buffer is greater than pK = 3.0 for the carboxyl group. Blue dextran is nonionic. Neutral solute molecules like hlue dextran and negatively charged solute molecules like DNP-elvcine will show no affinitv for -~~~ the stationary phase, and tLey would he expected to move with the eluting buffer. However, a gel filtration mechanism must also be considered toexplain why hlue dextranemerges before DNP-elvcine. T h e CM-Se~hadex(C-50) is derived from ~ e p h a d &type G-50. heref fore, the hlue dextran of molecular weight 2,000,000 is excluded from the gel heads, but DNP-glycine can penetrate and will be retarded in elution. Chymotrypsinogen with p I = 9.1 and molecular weight 23,240 will he positively charged a t pH = 5. When the ionic strength is increased by addition of 0.25 MKCI to the huffer, the K+ will replace the chymotrypsinogen. Cytochrome C with DI = 10.7 and molecular weieht 12.400 has hieher ~ o s i t&e~Ehargedensity than does c ~ y m o t W p s i u o g ae ~ n d will onlvemereefrom the column when 1.0 M KClis added to the buffer. ~ < student e will observe shrinkingof the column bed with increasing ionic strength since the repulsion between carboxylate side chains on the resin is reduced a t the higher ionic strendh. One of t i e challenges of teaching is to provide lab experiences that illustrate ~ r i n c i ~ l but e s that are not excessively s can be performed in 3 h time-consuming. ~ h f experiment by students working in pairs. It provides practical experience with column techniques and requires analysis of both gel filtration and iou-exchange mechanisms. ~~
~
Wllcox. P. E.; Cohen. E.; Tan. W. J. Biol. Chem. 1957, 228. 9991019.
Volume 68
Number 2
February 1991
169
