Bio-Beads®S-X Beads
Gel Permeation Chromatography
Instruction Manual
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............................................................ 8
Collecting Fractions........................................... 8
Regeneration....................................................... 9
Chemical Resistance .......................................... 10
Applications........................................................10
References...........................................................18
Product Information..........................................21
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.
Technical Description
Bio-Beads S-X beads are neutral, porous styrene
divinylbenzene copolymer beads. The beads in the BioBeads S-X series have exclusion limits from 400 to 14,000
daltons. This range makes them particularly suitable for the
fractionation and separation of low molecular weight organic polymers and other hydrophobic substances. The amount
of divinylbenzene crosslinkage determines the pore size,
and hence the molecular weight exclusion limit of a particular gel in this series. The beads are available with crosslinkages from 1-12%. Pore dimensions and exclusion limits are
also influenced by the eluant employed; maximal expansion
1
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. Large
compounds, greater than the molecular exclusion limit, pass
through the column unhindered, whereas small compounds,
within the molecular weight operating range, will be
retained in the column. The small compounds permeate the
pores of the Bio-Beads S-X beads, and thus they take longer
to pass through the column. This mechanism requires an
eluant which is mobile, and, therefore, Bio-Beads S-X beads
must always be used in a column mode.
2
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.
Swelling the Beads
Before use, swell the Bio-Beads S-X beads in an organic solvent, such as:
Aromatics Methylene chloride
Benzene Orthodichlorobenzene
Carbon tetrachloride Perchloroethylene
Dimethylformamide Tetrahydrofuran
Ketones Trichlorobenzene
These organic solvents allow maximal swelling of the
beads. If polar solvents, such as water or methanol, are used,
3
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. Solvents should generally be degassed prior to use and protected from
atmospheric contamination, to prevent later outgassing during the chromatographic run.
Bio-Beads S-X1 beads will swell considerably, and
should be placed in at least six times the resin weight of solvent (w/w). The higher crosslinked resins will not swell as
much, so they will not require as much elution solvent. The
swelling should always be done in the presence of excess
solvent to prevent the resin from drawing up all the solvent,
and possibly not swelling fully. The higher crosslinked
resins will require more time to become fully swollen.
Bio-Beads S-X12 and S-X8 beads may require swelling
overnight, whereas the lower crosslinked resins will be fully
swollen in a few hours. Complete swelling is necessary to
prevent swelling after packing, which could break the col-
4
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.
When the beads are used for the first time, low molecular weight polystyrene trapped inside the pores of the beads
will tend to cause slow column equilibration. Although
swelling the beads in one of the solvents listed above will
remove some of the low molecular weight polystyrene, several column volumes of the running buffer may be necessary
to reach baseline equilibration.
Packing the Column
Metal columns are often used in gel permeation
chromatography, though glass columns offer the important
advantages of visibility of packing and therefore are better
suited.
5
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.
The Bio-Beads S-X beads should be placed in an
approximately 50% slurry of beads and elution solvent. A
good practice at this point is to degas this mixture by sonicating it under vacuum. Safety precautions should be
observed. Double check that the stopcock below the column
is closed. Swirl the slurry to get a homogeneous mixture,
then slowly and consistently pour the slurry into the column.
It is best to pour a long narrow column in sections, in order
to obtain a uniform and reproducible column. A glass rod
may be used to facilitate pouring the mixture down the side
of the column. This will also help eliminate the formation of
bubbles. When it is clear that a few centimeters of packed
bed have settled, open the stopcock below the column and
allow the mobile phase to begin flowing. Begin with a slow
flow rate and gradually increase to 10-15% above the final
operating flow rate, if possible. The purpose of this elevated
flow rate is to pack the column under more pressure than
6
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. If a small column does go
dry, add extra solvent, cap both ends, invert several times until
the resin is fully slurried, allow the resin bed to settle, and begin
flow as normal. An alternative technique, for larger columns, is
to backwash the resin. This will resuspend the beads and allow
repacking. This technique should only be used if the packed bed
fills less than half the column. Simply connect a piece of tubing
to the column outlet, and, at a very slow flow rate, flow the elution solvent into the column. This will resuspend the packing
material. Care must be taken that enough space is available for
the resuspended material, as well as the additional solvent.
When the material is fully suspended, and the air pockets have
risen above the packing material, stop the flow. Allow the material to settle.
Note: Bio-Beads S-X beads will float in high density solvents,
e.g. chloroform. First swell the media in tetrahydrofuran and
pack the column as previously described. Insert an adjustable
flow adaptor into the column to constrain the beads in a packed
7
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.
Collecting Fractions
Separated compounds are often collected for further analysis. Fractions can be constantly collected, for example in 2 ml
increments, or they can be collected specifically when the compound of interest is eluting. In order to know when the compounds are eluting from the column, pass the eluant through a
detector. Ultraviolet (UV) detectors are commonly used, though
a preparative cell may be necessary so that the flow is not
restricted too much. Use the information from the UV chro-
8
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.
After long periods of storage, small loose polystyrene
which had been trapped inside the beads will leach out. This
polystryrene (2-10 chain length) has a very high extinction coefficient, so even trace amounts will have a high UV absorption.
To eliminate this problem, wash with several bed volumes of
solvent.
Chemical Resistance
Bio-Beads S-X beads are highly chemically resistant,
though explosive mixtures may form in the presence of strong
oxidizing reagents such as chromic acid, nitric acid, and hydrogen peroxide. The beads may be autoclaved.
9
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.1Remarkable separations can therefore be achieved at low flow rates.
Many different types of compounds have been separated on
Bio-Beads S-X beads. The beads have been used for analysis
and quantification of pesticides
Beads S-X beads are the basis of the official EPA procedure for
the measurement of organic priority pollutants in sludge.8The
beads are useful for separation of polycyclic aromatic com-
9-11
pounds,
assessment of tissue reaction to biomaterial,
fractionation of halogenated environmental contaminants.
Other applications include separation of tall oil components,
19, 20
lipids,
alkalines,21fatty acids,22a variety of hydrocarbons,
and polystyrenes.24These beads can also be used to determine
polymer molecular weights and molecular weight distribu-
24-28
tions.
They have been used for the separation of low molecular weight trimethylsilylated silicic acid,29and for the isolation
of low molecular weight polar organics in fatty tissues for subsequent GLC-MA analysis.30They have also been used for the
2-5
10
and rodenticides.
6,7
12, 13
Bio-
and
14-17
18
23
analysis of fish lipid extracts,31and for fractionation of food
grade poly (vinyl chloride) resin.
32, 33
A variety of lipophilic polymer substances has been suc-
cessfully purified on beds of Bio-Beads S-X beads (Figure
11
5
3
2
1
Relative solute concentration
4
7
6
1).
Relative effluent volume
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 = tri-
caprylin; 5 = tricaproin; 6 = hexadecane; 7 = undecane.
12
21
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.
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.
Resin acid
Fatty acid dimer
Fatty acid
Resin acid dimer
Recorder response
90 100 110 120 130 ml
Elution volume
0.2 0.3 0.4 0.5 0.6 0.7
Fig. 2. Gel permeation chromatogram of tall oil.
13
18
Ault and Spurgeon used Bio-Beads S-X3 beads for specific
separation of chlorinated pesticides form animal fat.
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.
14
35
34
References
Reference Application
1. Harmon, D. J., Sep. Sci., 5, 403 (1970). Bead swelling in
2. Stalling, D. L., et al., J.A.O.A.C., 55, 32 (1972). Pesticides
3. Johnson, L. D., et al., J.A.O.A.C., 59, 174 Pesticides
(1976).
4. Steinwandter, H., Fresenius, Z. Anal. Chem., Pesticides
313, 536 (1982).
5. Blaha, J. J. and Jackson, P. J., J.A.O.A.C., Pesticides
68 (6), 1095 (1985).
6. Hunter, K., J. Chromatog., 299, 405 (1984). Rodenticides
7. Hunter, K., J. Chromatog., 321, 255 (1985). Rodenticides
8. Haile, C. L. and Lopez-Avila, V., U.S. Envi- Sludge
ronmental Protection Agency, Project
Summary No. 600/S4-84-001, March, 1984.
9. Friley, B. K., Phelps, J. B. and Kincaid, J. R., Polycyclic aromatics
J. Chromatog., 258, 310 (1983).
10. Chamberlain, W. J., Snook, M. E., Baker, J. L. Polycyclic aromatics
and Chortyk, O. T., Anal. Chim. Acta, 222,
235 (1979).
11. Snook, M. E., Chamberlain, W. J., Severson, Polycyclic aromatics
R. F. and Chortyk, O. T., Anal. Chem., 47,
1155 (1975).
15
various solvents
Reference Application
12. Hood, C. I., Schoen, F. J., Coleman, S. E. and Tissue reaction
Mickley, L. D., J. Biomed. Materials Res., 18,
1031 (1984).
13. Schoen, F., et al., J. Biomed Materials Res., 20, Tissue reaction
709 (1986).
14. Stalling, D. L., Smith, L. M. and Petty, J. D., Halogenated
ASTM Special Publication No. 686, page 302 environmental
(1979). contaminants
15. Musial, C. and Uthe, J. F., J.A.O.A.C., 69 (3), Halogenated
462 (1986). environmental
16. Ault, J. A., Schofield, C. M., Johnson, L. D. Halogenated
and Waltz, R. H., J. Ag. and Food Chem., 27, environmental
825 (1979). contaminants
17. LeBel, G. and Williams, D. T., J.A.O.A.C., Halogenated
69 (3), 451 (1986). environmental
18. Chang, T-L., Anal. Chem., 40 (6), 989 (1968). Tall oil
19. Tipton, C. L. Paulis, J. W. and Pierson, M. D., Lipids
J. Chromatog., 14, 486 (1964).
20. Hirsch, J., Colloq. Int. Centre Nat. Res. Sci., Lipids
99, 11 (1960).
21. Chang, T-L., Anal. Chim. Acta, 39, 519 (1967). Alkalines
22. Chang, T-L., Anal. Chim. Acta, 42, 51 (1968). Fatty acids
16
contaminants
contaminants
Reference Application
23. Henfrickson, J. G., J. Chromatog., 32, 543 Hydrocarbons
(1968).
24. Cantow, M. J. R., et al., J. Polymer Sci., 5, 987 MW determination
(1967)
25. Coll, H., Separ. Sci., 5, 273 (1970). MW determination
26. Pickett, H. E., et al., J. Applied Polymer Sci., MW determination
10, 917 (1966).
27. Cantow, M. J. R., et al., J. Polymer Sci., MW determination
(Part C), 16, 13 (1967).
28. Cantow, M. J. R., et al., J. Polymer Sci., MW determination
(Part A-1), 5, 1391 (1967).
29. Shimono, T., Toshiyuki, I. and Tarutani, T., Trimethysilylated
J. Chromatog., 179, 323 (1979). silicic acid
30. Kuehl, D. H. and Leonard, E. N., Anal Chem., Polar organics
50, 182 (1978). in fatty tissues
31. Burns, B. G., et al., J.A.O.A.C., 64, 282 (1981). Fish lipid extracts
32. Gilbert, J., Shepherd, M. J. and Wallwork, Food grade PVC
M. A., J. Chromatog., 320, 361 (1985). fractionation
33. Waliszewski, S. M. and Szymczynski, G. A., Food grade PVC
J. Chromatog., 321, 480 (1985). fractionation
34. Ault, J. A. and Spurgeon, T. E., J.A.O.A.C., Chlorinated pesticides
67 (2), 284 (1984). from animal fat
35. Aitzetmuller, K., J. Chromatog., 71, 355 Triglycerides
(1972).
17
Product Information
Catalog Product Mesh Exclusion Operating ml/g
Number Description Size Limit Range Benzene
152-2150 Bio-Beads S-X1 200-400 14,000 600-14,000 7.5
152-2151 Bio-Beads S-X1 200-400 14,000 600-14,000 7.5
152-2750 Bio-Beads S-X3 200-400 2,000 up to 2,000 4.75
152-3350 Bio-Beads S-X8 200-400 1,000 up to 1,000 3.1
152-3650 Bio-Beads S-X12200-400 400 up to 400 2.5
Beads, 100 g
Beads, 1 kg
Beads, 100 g
Beads, 100 g
Beads, 100 g
M.W. M.W. Bed Vol.
18
Swollen
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT263 Rev C
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