910
Anal. Chern. 1980, 52, 910-912
Chromatography with Sephadex Gels Jos6 M. Sosa Chemistry Department, New Mexico State University, Las Cruces, New Mexico 88003
Rapid, gel-filtration chromatography using Sephadex G-25 and 6-50 is described. One-meter (‘/,-inch 0.d.) polyethylene columns packed with Sephadex gels using a stirred-slurry packer or a pressure-packing reservoir are used at linear velocities up to 470 cm/h. Column performance is evaluated by injecting mixtures of blue dextran, acetone, and d,/-tryptophan. HETP values as low as 3.0 mm are obtained routinely. Retention times (volumes) are reproducible and columns can be used up to ten days without any signs of deterioration.
T h e chromatography of many classes of compounds using Sephadex gels is well-documented. Since their introduction in 1959, these gels have practically revolutionized the science of separating biological molecules. Probably one of the few problems with these materials has been the inherent softness of the gels. Due to the ease with which these gels are compressed, their use in rapid filtration chromatography has been rather limited. Catsimpoolas and co-workers (1-3) have shown t h a t Sephadex gels can be used in rapid analytical chromatography in microbore columns. They have applied this method t o obtain molecular weights and molecular weight distributions of proteins. Linear flow velocities of 5 cm/h for columns 2.8 m m in diameter are reported. Two major advantages of rapid gel filtration are speed of the analysis and sensitivity of detection, while the major disadvantages are tailing and the development of high pressures. Edwards and Helft ( 4 ) have also shown that compressed Sephadex gels give improved resolution; however, to our knowledge, few studies have dealt with the use of these gels at pressures higher than a few Torr (5-7). Joustra (8) has investigated the dependence of flow on hydrostatic pressures up to 50 cm of H20. In general, Darcy’s law is utilized to calculate the linear velocity of the mobile phase at a given hydrostatic pressure (9). For tightly cross-linked Sephadex gels such as the G-10, G-15, G-25, and G-50, the Pharmacia Co. (Uppsala, Sweden) suggests ( 5 )that Darcy’s law is obeyed. Darcy’s law states that the linear velocity of the mobile phase ( U in cm/h) is directly proportional to the hydrostatic pressure (APin cm of H,O) and inversely proportional to the gel-bed height (1 in cm). Thus, U = K A P / l , where K is a constant that depends on the properties of the bed and the mobile phase. Hydrostatic pressures as high as 200 cm of H 2 0 for the tightly cross-linked gels have been reported (5). Although several new gel materials have been developed recently (IO),for many separations reported in the literature, Sephadex is still the preferred gel for size-exclusion chromatography. In our studies dealing with the adsorption of molecules on polymeric networks, we have found that by using polyethylene tubing (4-mm i d . ) linear velocities up t o 470 cm/h can be obtained with Sephadex G-25 and G-50. This paper describes the performance of several columns a t linear velocities up to 30 times what is normally used in gel filtration with Sephadex gels.
EXPERIMENTAL A Waters Liquid Chromatograph Model ALC 200 was used in this study. Deionized water at room temperature (22 “C) was used as the mobile phase. Approximately 80 psi are necessary to obtain a water flow of 1 mL/min when a union is used to 0003-2700/80/0352-0910$01 .OO/O
connect the injector outlet to the detector inlet in our instrument. The columns were made from I/,-inch polyethylene tubing (4-mm id.) purchased at a local hardware store. Ten micrometer porous frits were utilized to cap the ends of the column. Columns were packed by using either a Micromeritics slurry packer or a Pierce, Speed-fill, pressure-packing reservoir. Sephadex G-25 or G-50 was allowed to swell in water overnight and a slurry was poured into the reservoir of the slurry packer. A 50-cm column fitted with a porous frit at one end was connected to the cover of the reservoir. Water was then pumped using the pump of the ALC 200 at a flow rate such that the pressure gauge of the instrument was never over 80 psi. A strong light was used to see the flow of the gel beads into the column. Columns 50 cm or shorter can be packed easily with the slurry packer. Longer columns are more difficult to pack with this method owing to the fact that higher pressures are involved. The Speed-Fill, pressure packing reservoir can be used to fill columns up to 2 meters long. In this method, a column fitted with a porous frit is connected to the reservoir, which is then filled with deionized water. A slurry of the gel is added to the reservoir and allowed to flow into the column under gravity flow. The cover of the reservoir is then replaced and the system is connected to a nitrogen tank. The methacrylic reservoir is pressurized to 50-70 psi and in 5 to 10 min the column is completely packed. Flow rates of 3 to 5 mL/min are obtained at 60 psi using Sephadex G-25 (medium). The column is ready for use after removal from the reservoir and capping. A packed column can be prepared in 15 to 20 min using this technique.
RESULTS AND D I S C U S S I O N Figure 1shows the separation of a mixture of blue dextran, acetone, and d,l-tryptophan using a 1-m polyethylene column prepared with Sephadex G-25 (medium) using the Pierce Speed-Fill reservoir a t a nitrogen pressure of 65 psi. At a flow rate of 0.5 mL/min, using water as the eluent, H E T P values of 3.0,3.3,and 6.0 mm were obtained for blue dextran, acetone, and tryptophan, respectively. A flow rate of 0.5 mL/min corresponds to a linear flow velocity of 239 cm/h for this column. In order to achieve a flow of 0.5 mL/min through the tubing and fittings (without the column), approximately 40 psi are required. When the column is installed, a pressure increase was not noticeable because of the insensitivity of the pressure gauge of the ALC 200. If one assumes that Darcy’s law holds under these conditions, a AP of 304 cm of H 2 0 can be calculated for a K value of 94 (from Ref. 5) and a bed height of 100 cm. A partition coefficient, K,, (av = available), according to the theory of Laurent and Killander ( 1 1 ) can be calculated for acetone from the equation K,, = V, - Vo/V, - Vo, where V, is the elution volume, V, is the void volume and V,is the total bed volume. For this column, V , = 12.5 cm3, V,,= 4.82 cm3, V, for acetone is 10.5 cm3, and K,, = 0.74. The behavior of molecules that penetrate the gel can thus be studied in less than 0.5 h with this column. Tryptophan on the other hand is known to be strongly retarded by Sephadex gels and this fact is observed in this study as shown in Figure 1. Although deionized water is being used as an eluent, all the peaks show almost no tailing. The performance of a 50-cm polyethylene column packed with Sephadex G-25 (medium) using the slurry packer is shown in Figure 2 a t different flow rates. Figure 3 shows the variation of HETP with flow rate for blue dextran, acetone, and tryptophan. These two figures show that very fast analyses can be performed with efficiencies that could be 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL 52, NO. 6, M4Y 1980
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-___
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911
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Table I. Performance of a One-RleLer, Sephadex G-50 (Fine) Column Packed with a Pierce, Speed-Fill Reservoir Blue Dextran V,, mm
time, days initial run
12 14
___
____tryptophhii ___
HETP, cm
V,, mrn
HETP, cm
V,,rnm
HETP, cm
solvent
0.30
57.0 53.2 55.0 55 .O 54 0 54.0
0.33 0.32 LJ. 30 0.32 0.37 0.39
105.5 99.5 102.3 102 2 3 03.0 99.5
0.61
1.5 M NH,HCO, H,O
27.5 24 5 26.5 26.0 24.0 24.5
0.2 1.4 10.0
acetone
0.33 0.27
0.28 0.53 0.51
0.65 0.65 0.66
H. 0 H,O
0.72 0.73
H,O H, 0
E E
5.0]
L
L---;ij-
io 30 TIUE (min)
io
50 0.4
Separation of a three-component mixture using a polyethylene column packed with Sephadex G-25 (medium) Figure 1.
A
B
I
0
0.6
0.8
j.0
FLOW RATE I m l / m i n )
1-m
Figure 3. Plot of HETP vs.
flow rate for the data of Figure
2
far each
component
rJ
C
3
10
20
30
0
10
23
0
10
0
1C
T I M E (rntnl
Flgure 2.
Separation of blue dextran ( l ) , acetone (2), and tryptophan
(3) using a 50-cm Sephadex G-25 column at the following flow rates: A, 0.4 mL/min; 8,0.6 mL/min; C, 0.8 mL/min; D, 1.0 mL/min
considered fair. Flodin observed that in general higher flow rates produce higher H E T P values! except for very small molecules, for example HC1 (12). H E T P values of benzene and dibromobenzene have also been shown to increase with higher flow rates on Styragel columns (13). It is of interest to point out that although hundreds of separations have been performed with Sephadex gels, very few researchers give parameters that allow others reproducing experiments to compare the efficiencies of their columns. T h e use of a test mixture such as the one utilized in this study not only allows the investigator to monitor the performance of a column from day t o day, but also allows others reproducing experiments to compare the efficiencies of their systems. T h e columns prepared using the slurry packer gave higher efficiencies than those packed with the pressure-packing reservoir. Sephadex G-25 gels classified as superfine, fine, and medium were utilized. The superfine gels gave H E T P values as low as 0.5 mm for acetone. Shear degradation easily occurred with all gels when the instrument recorded pressures of 100 psi or higher. Table I shows that a t the linear velocities that were used, the columns were quite stable over a period of time. Thus, R 1-m column, packed using the pressurepacking reservoir was evaluated daily by calculating H E T P values for each component of the test mixture. -4fter the twelveth day, a significant increase in the H E T P values for the three components was observed. It is unclear why retention volumes are not changing significantly, while H E T P values increased 73% for blue dextran, 2570 for acetone, and 10% for tryptophan. It is possible that chemical degradation
oi -o Figure 4. Chromatogram sclbfilis L-4
3r
P
LO
TIME (rnin)
of a brown pigment isolated from Bacillus
occurred since no antimicrobial agent was used. Shear degradation is also possible. Since the columns are very inexpensive and readily packed, for most applications a column can be prepared, evaluated, and used for a week or so. Antimicrobial agents and/or cold storage should prolong the lifes of the columns although this has not been checked. The following applications will show that rapid gel filtration can be performed with Sephadex G-25 and G-50 with good results. Figure 4 shows the chromatogram of a brown pigment isolated from Baciilus s~lbtilz.9 168 L-4. After repeated purifications on a column having a bed volume of 3 I,, an attempt was made to determine the physicochemical properties of the material. A quick check using both G-25 and G-50 columns showed that the material contained !ow molecular weight impurities. The purification procedure involved using dimethylsulfoxide which was later found to be one of the components that was difficult t o remove from the pigment by drying (1 1). The separation was performed on Sephadex G-25, R hich excluded the pigment, but retained the smaller molecules. Figure 5 shows a plot of V , vs. log M for several Carbowax samples using Sephadex G-2; (medium). Blue dextran was used to determine the void volume. The H E T P value of the
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A N A L Y T I C A L CHEMISTRY, VOL. 52, NO. 6, M A Y 1980
\
I
0..
I
I A 1
I0
.
0
THY R O G L O B U L i N
'\
'.
11
6
8
10 V, ( m l )
12
14
Figure 5. Plot of the log of t h e molecular weight vs. elution volume ( V,) for polyethylene glycols
Table 11. Elution Volumes ( V , ) of Aromatic Compounds on a 4 mm X 500 mm Sephadex Superfine Column Packed with a Micromeritics Slurry Packer compound
V,, mL
HETP, mm
Blue Dextran mandelic acid acetone tyrosine phenylalanine tryptophan
2.76 5.0 6.4 6.3 9.0 12.2
0.80 0.9 0.9 1.0 1.is
column for acetone was 2.0 mm. The column was prepared using t h e slurry packer a t a flow rate of 1.2 mL/min. A solution of ammonium carbonate (0.015 M) was used as the eluent a t a flow rate of 0.5 mL/min. Although four samples are insufficient to establish a calibration curve, the typical behavior of a Sephadex G-25 is exemplified. A molecular weight of 4000 is totally excluded as shown by the sharp rise in the curve. A straight line can be drawn through the other three points. Gelotte and Porath (15) have shown that the elution volumes of polyethylene glycols fractionated with Sephadex G-10 are directly proportional to the log of the molecular weights. This is apparently also the case in our system. Table I1 shows the retention volumes of several amino acids and mandelic acid. A 0.5-m column packed with Sephadex G-25 (superfine) gave an H E T P of 0.9 mm for acetone a t a flow rate of 0.8 mL/min. Deionized water was utilized as the eluent. Use is made of the adsorptive properties of the dextran structure to separate the amino acids. Both tryptophan and phenylalanine are retained more strongly than acetone, tyrosine, or mandelic acid. Mandelic acid is the only compound that did not elute as a symmetrical peak. Peak leading was clearly evident, presumably due to ionization. Another example oi the usefulness of rapid chromatography with Sephadex gels can be seen in Figure 6 by the separation of thyroglobulin (mol wt 660000), cytochrome c (mol wt 11700), and insulin (mol wt 5700). These separations were performed with a 1-m plastic column which was prepared using Sephadex G-50 (fine) swollen in a 0.01 M ammonium carbonate solution and packed with the pressure-packing reservoir a t 65 psi. Using a solution of 0.01 M ammonium carbonate solution as the mobile phase, acetone gave an H E T P value of 3.0 mm at a flow rate of 0.5 mL/min (239 cm/h). The resolution factor ( R )for the separation of thyroglobulin and cytochrome c is 1.09 and for the separation of thyroglobulin and insulin, it is 1.79. Catsimpoolas and Kenney ( I ) reported the separation of thyroglobulin and cytochrome c using Sephadex G-50 in microbore columns. From their data, a resolution factor of 0.92 can be calculated. Thus, a better separation is obtained using the technique described in this paper. Another very significant improvement of the technique presented here over that presented by Catsimpoolas is seen
10
20
30
10
20
30
TIME ( M I N ) Figure 6. Chromatograms showing the separation of (A) thyroglobulin, cytochrome c , and acetone and (B) thyroglobulin, insulin, and acetone using Sephadex G-50
in the shape of the peaks. An asymmetry factor, an important parameter in measuring column performance, for cytochrome c of 1.2 can be calculated from our data and an asymmetry factor of 2.0 can be calculated from the data given in Reference 1. Columns for high performance liquid chromatography are considered acceptable if they have asymmetry values less than 1.8. Thus, increased resolution and improved peak shapes can be obtained by the methods reported here. These examples clearly demonstrate that Sephadex gels can indeed be used in rapid gel filtration with good results. Recently, a spherical silica, featuring a bonded carbohydrate monolayer was introduced as a support for rapid, aqueous GPC (Synchropak, Regis Chemical Co.). A 25 cm x 4.1 mm (i.d.1 column packed with 10-wm particles gave an H E T P of about 0.5 mm for glycyltyrosine. An analysis can be performed in approximately 7 min using these columns. Our results with a 0.5-m Sephadex (superfine) column show that we can also obtain H E T P values of 0.5 for acetone. Although further work remains to be done to determine the ultimate pressures and flow rates at which these gels can be used, it is apparent that the low cost and the simple preparative method makes this technique appear promising. Since Sephadex gels are widely used in preparative biochemistry, it may be of interest to test in small columns, in a rapid manner, the elution patterns of molecules before large columns are prepared.
LITERATURE CITED (1) (2) (3) (4) (5)
Catsimpoolas and J. Kenney, J . Chromatogr., 6 4 , 77 (1972). N. Catsimpoolas and J. Kenny, J . Chromatogr., 71, 573 (1972). N. Catsimpoolas, Anal. Biochem., 61, 101 (1974). V. H. Edwards and J. M. Helft, J . Chromafogr., 47, 490 (1970). "Gel Filtration: Theory and Practice", Pharmacia, Fine Chemicals, N.
Uppsaia, Sweden, April 1979. (6) H. Determann, "Gel Chromatography", Springer-Verlag. New York, 1969, Chapter 2. (7) L. Hagel, J . Chromatogr., 160, 59 (1978). (8) M. K. Joustra, Protides Biol. Fluids:,14, 533 (1967). (9) 2. Deyl, K. Macek, and J. Janak, Liquid Column Chromatography", Elsevier, New York, 1975, p 125. (10) W . W. Yau, J. Kirkiand, and D. Bly, "Modern Size-Exclusion Chromatography: Practice of Gel Permeation a n d Gel Filtration Chromatography", Wiley-Interscience, New York, 1979, Chapter 13. (11) T. C. Laurent and J. Killander, J . Chromatogr., 14, 317 (1964). (12) P. Flodin, J . Chromatogr. 5 , 103 (1961). (13) W. B. Smith and A. Kollmansberger, J . Phys. Chem. 69, 4157 (1965). (14) T . Barnett, P h D Thesis, New Mexico State University, 1978. (15) B. Gelotte and J. Porath. "Chromatography", E. Heftmann, Ed.. ReinhoM, New York, 1967, p 353.
RECEIVED for review December 10, 1979. Accepted February 21, 1980. The support of the Research Center of the College of Arts and Sciences (NMSU) through its Minigrant Program and of the National Institute of Health through the Minority Biomedical Program is gratefully acknowledged.
