LIT263C 9/2/98 1:13 PM Page A Bio-Beads® S-X Beads Gel Permeation Chromatography Instruction Manual
LIT263C 9/2/98 1:13 PM Page B Table of Contents Page Introduction........................................................ 1 Technical Description ........................................ 1 Mechanism.......................................................... 2 Instructions for Use............................................ 3 Swelling the Beads.............................................. 3 Packing the Column........................................... 5 Flow Rate ..........................................
LIT263C 9/2/98 1:13 PM Page 1 Introduction Bio-Beads S-X beads are a series of porous crosslinked polystyrene polymers used for gel permeation separations of lipophilic polymers and low molecular weight, hydrophobic materials in the presence of organic solvents. These nonaqueous spherical beads are used in much the same way aqueous gels are used, except that they are swollen with organic solvents during the separation.
LIT263C 9/2/98 1:13 PM Page 2 of the matrix is achieved with relatively nonpolar, aromatic solvents. The beads are typically used with benzene, toluene, xylene, carbon tetrachloride, and mixtures of solvents. Mechanism Gel filtration, also called gel permeation, is the mode of separation which occurs with Bio-Beads S-X beads.
LIT263C 9/2/98 1:13 PM Page 3 Instructions for Use Bio-Beads S-X beads are supplied dry, and must be swollen prior to packing into a chromatographic column. The general instructions are: 1. Swell the beads. 2. Assemble the column. 3. Pour the beads into the column. 4. Add the sample and proceed with the separation. The instructions below describe swelling the beads and packing the column in details.
LIT263C 9/2/98 1:13 PM Page 4 the Bio-Beads S-X beads will not swell, and the pore size will be minimal. The chosen solvent should be the one used for the separation, and the same as the solvent in which the sample is dissolved. The solvents should be of highest quality available, and preferably redistilled if non-volatile matter is present in them. Some solvents, e.g. tetrahydrofuran, develop peroxides on standing in contact with air.
LIT263C 9/2/98 1:13 PM Page 5 umn. If the amount of swelling is unknown, it can be checked by swelling a known weight of beads and measuring the volume. After the beads are fully swollen, they are packed into a chromatographic column and washed with the solvent in which they were swollen. Normally, the sample is dissolved and the elution is performed with this same solvent, to prevent swelling or shrinking of the resin during the run. If the beads swell during the run, a glass column may break.
LIT263C 9/2/98 1:13 PM Page 6 Put together a clean column assembly. Place a small amount of elution solvent in the column to prevent bubble formation at the base of the poured column packing. Prepare a solvent reservoir, which should be placed at an elevation higher than the top of the column. The solvent reservoir may be connected to the column by tubing. Any type of connection must be air-tight and clean.
LIT263C 9/2/98 1:13 PM Page 7 will be used during the separation. (This is not necessary for Bio-Beads S-X1 beads.) Maintain at least a few centimeters of liquid above the resin bed. Never allow the packed beads to become dry, because this will cause air pockets and channeling within the bed, resulting in poor efficiency and low resolution.
LIT263C 9/2/98 1:13 PM Page 8 position. Then, displace the tetrahydrofuran with 3 bed volumes of chloroform. Flow Rate Bio-Beads S-X beads have the capacity for different flow rates, depending on the crosslinkage. The 1% and 2% crosslinked resins (Bio-Beads S-X1 beads) are very soft when fully swollen and should only be used in gravity flow procedures. Bio-Beads S-X3 beads can withstand 5 ml/min with a backpressure of 300 psi. Bio-Beads S-X8 and S-X12 beads can withstand up to 5,000 psi backpressure.
LIT263C 9/2/98 1:13 PM Page 9 matogram to establish the time periods to use when collecting a specific fraction. Regeneration Regeneration of Bio-Beads S-X beads may be necessary if compounds have become trapped within the pores of the resin, for example if a series of eluants has been used. Bio-Beads S-X beads are hydrophobic, and can also absorb compounds. To wash the resin, swell it to its maximum with a solvent such as methylene chloride, toluene, or tetrahydrofuran.
LIT263C 9/2/98 1:13 PM Page 10 Applications Harmon reviewed the many different types of gels that are available to the chromatographer, and reported that the relatively soft gels, such as the lower Bio-Beads (particularly S-X1 ) products swell appreciably in many solvents.1 Remarkable separations can therefore be achieved at low flow rates. Many different types of compounds have been separated on Bio-Beads S-X beads.
LIT263C 9/2/98 1:13 PM Page 11 analysis of fish lipid extracts, 31 and for fractionation of food grade poly (vinyl chloride) resin.
LIT263C 9/2/98 1:14 PM Page 12 Relative solute concentration 6 7 5 3 1 2 4 Relative effluent volume 1). Fig. 1. Separation of triglycerides and hydrocarbons on Bio-Beads S-X1 and S-X2 beads in benzene (two beds in series). 1 = tristearin; 2 = trimyristein; 3 = trilaurin; 4 = tricaprylin; 5 = tricaproin; 6 = hexadecane; 7 = undecane.
LIT263C 9/2/98 1:14 PM Page 13 90 0.2 Resin acid Fatty acid Resin acid dimer Recorder response Fatty acid dimer Bio-Beads S-X2 and S-X8 beads have been used in multicolumn system for separating various components of tall oil, wood resin, and gum resin by gel permeation chromatography.18 Figure 2 shows the fractionation of tall oil with Bio-Beads S-X beads. All of the injected acid sample was fully recovered, and no delay in elution time was noticed. 100 110 120 Elution volume 0.3 0.4 0.5 0.
LIT263C 9/2/98 1:14 PM Page 14 Ault and Spurgeon used Bio-Beads S-X3 beads for specific separation of chlorinated pesticides form animal fat. 34 Aitzetmuller has used Bio-Beads S-X beads in benzene to fractionate a 1 g sample of polymeric trioleins. The large pore size of the gel effectively resolved dimeric triglycerides (m.w. ca 1,800) from monomeric triglycerides.
LIT263C 9/2/98 1:14 PM Page 15 References Reference Application 1. Harmon, D. J., Sep. Sci., 5, 403 (1970). 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Bead swelling in various solvents Stalling, D. L., et al., J.A.O.A.C., 55, 32 (1972). Pesticides Johnson, L. D., et al., J.A.O.A.C., 59, 174 Pesticides (1976). Steinwandter, H., Fresenius, Z. Anal. Chem., Pesticides 313, 536 (1982). Blaha, J. J. and Jackson, P. J., J.A.O.A.C., Pesticides 68 (6), 1095 (1985). Hunter, K., J. Chromatog., 299, 405 (1984).
LIT263C 9/2/98 1:14 PM Page 16 Reference Application 12. Hood, C. I., Schoen, F. J., Coleman, S. E. and Mickley, L. D., J. Biomed. Materials Res., 18, 1031 (1984). 13. Schoen, F., et al., J. Biomed Materials Res., 20, 709 (1986). 14. Stalling, D. L., Smith, L. M. and Petty, J. D., ASTM Special Publication No. 686, page 302 (1979). 15. Musial, C. and Uthe, J. F., J.A.O.A.C., 69 (3), 462 (1986). 16. Ault, J. A., Schofield, C. M., Johnson, L. D. and Waltz, R. H., J. Ag. and Food Chem., 27, 825 (1979). 17.
LIT263C 9/2/98 1:14 PM Page 17 Reference Application 23. Henfrickson, J. G., J. Chromatog., 32, 543 (1968). 24. Cantow, M. J. R., et al., J. Polymer Sci., 5, 987 (1967) 25. Coll, H., Separ. Sci., 5, 273 (1970). 26. Pickett, H. E., et al., J. Applied Polymer Sci., 10, 917 (1966). 27. Cantow, M. J. R., et al., J. Polymer Sci., (Part C), 16, 13 (1967). 28. Cantow, M. J. R., et al., J. Polymer Sci., (Part A-1), 5, 1391 (1967). 29. Shimono, T., Toshiyuki, I. and Tarutani, T., J. Chromatog., 179, 323 (1979).
LIT263C 9/2/98 1:14 PM Page 18 Product Information Catalog Number Product Description Swollen M.W. M.W. Bed Vol. Exclusion Operating ml/g Limit Range Benzene Mesh Size 152-2150 Bio-Beads S-X1 200-400 14,000 Beads, 100 g 600-14,000 7.5 152-2151 Bio-Beads S-X1 200-400 14,000 Beads, 1 kg 600-14,000 7.5 152-2750 Bio-Beads S-X3 200-400 2,000 Beads, 100 g up to 2,000 4.75 152-3350 Bio-Beads S-X8 200-400 1,000 Beads, 100 g up to 1,000 3.
LIT263C 9/2/98 1:14 PM Page 19 Bio-Rad Laboratories, 2000 Alfred Nobel Dr.
