Bio-Rad Bio-Beads S-X Media User Manual

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

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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.

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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,

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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-

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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.

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