Bio-Rad Bio-Rex Reactor Grade Resins User Manual

Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules CA 94547

LIT205 Rev B

AG®501-X8 and

Bio-Rex®MSZ 501(D)

Mixed Bed Resin

Instruction Manual

Introduction

Mixed bed resins are used for deionizing water or
other non-ionic substances such as urea, acrylamide, for­mamide, or glyoxal. Deionization is the complete removal
of all ionic species from a solution.

Technical Description

AG 501-X8 mixed bed resin for deionization consists
of equivalent amounts of AG 50W-X8 strong cation
exchange resin H
exchange resin OH-form. Bio-Rex MSZ 501(D) resin is
similar to AG 501-X8 resin, but the uniform bead size
minimizes pressure drops, increases resin capacity, and
allows shorter regeneration times. Molecular Biology
Grade AG 501-X8 resin is certified to be endo- and
exonuclease-free and to contain no ligase inhibitors.
Biotechnology Grade AG 501-X8 resin is certified to con­tain less than 100 micro-organisms per gram. AG 501­X8(D) resin and Bio-Rex MSZ 501(D) resin have a blue
dye irreversibly bound to the anion exchange resin, which
turns from blue to gold when the exchange capacity is
exhausted. The resin specifications are given in Table 1.

+

form and AG 1-X8 strong anion

1

Table 1. Mixed Bed Resin Specifications

AG 501-X8 AG 501 -X8 (D) MSZ 501(D)

Resin Resin Resin

+

Chemical H
form

Resin type Mixed bed Mixed bed Mixed bed
Physical form Light and dark Blue and gold Blue and brown

Minimum total 1.5 meq/ml 1.5 meq/ml 1.5 meq/ml
capacity

Particle 300-1,180 µm 300-1,180 µm 600±100 µm
diameter

Mesh size 20-50 20-50 25-35
Matrix Styrene divinyl- Styrene divinyl- Styrene divinyl-

-

&OH

gold beads beads beads

benzene benzene benzene

H+&OH

Bio-Rex

-

H+&OH

-

All of the mixed bed resins are used for deionization.
Deionization can be performed by exchanging the solute
cations for hydrogen on the resin and the solute anions for
hydroxyl on the resin. The resulting neutralization yields
water.

Resin-H + Resin-OH + NaCl → Resin-Na + Resin-Cl + H2O

Instructions for Use

The batch method or the column method may be used

to deionize a sample with mixed bed resin.

Batch Method

The batch method is the addition of resin directly into

the sample followed by stirring to achieve deionization.

1. Weigh out about 5 g of fresh resin for every 100 ml of
sample.

2. Add resin to sample and stir or shake for 1 hour.

3. Filter or decant sample from resin.

Note: If samples contain sugars or polyhydric alcohols, it
is important to use the minimum quantity of resin required
for deionization, and to remove the resin from the sample
after the deionization process to minimize adsorption of
the sample. If the sample contains extremely hydrophilic
proteins, the resin should be placed in dialysis tubing to
avoid direct contact of the resin with the sample. If the
anion resin beads change from blue to gold prior to deion­ization, the quantity of resin should be increased.

32

Column Method

Use mixed bed resin in a column to deionize larger
volumes (liter volumes) of mobile phase such as water.
The following procedure gives guidelines for column
deionization for a range of volumes; use Table 2 to select
the column size and quantity of resin.

1. Select a column with a length to diameter ratio of at

least 5 to 1. Refer to Table 2.

2. Weigh out approximately 0.6 grams of resin for every

1 ml of column volume.

3. Using the resin as it comes from the bottle, pour a

small portion of resin into the column. Pour the col-

umn in sections, a scoop at a time, to prevent separa-

tion of the anion and cation resins. Add water,

keeping about 1/4 inch of water above the resin.

4. Repeat step 3, alternating resin and water, until the

total amount of resin is added. The resin will have a

tiger's tail appearance; more bands will mean a higher

efficiency. Remove any trapped air bubbles by tap-

ping the column during packing.

5. When the column is packed, wash the resin with 3

bed volumes of deionized water. Discard wash.

4

6. Slowly pour the solution to be deionized into the
reservoir above the column, then elute slowly through
the column taking care not to disturb the resin bed.
Discard the first 1-2 bed volumes of the solution.

7. The quality of the deionized solution may be verified
by measuring its conductivity against the conductivi­ty of the starting solution.

Table 2. Guidelines for Column Deionization

Column Size Weight of of Deionized Water*

(diameter x length) Resin (g) (liters)

0.5 x 20 2.6 1.5

1.0 x 50 27.0 6.7

1.5 x 100 118.0 29.0

2.5 x 100 325.0 80.0

5.0 x 100 1,300.0 325.0

15.0 x 120 13.6 3,500.0

* Based on using 100 ppm NaCl. This volume will vary depending on ini-

tial water quality.

Approximate Volume

The dye (D) form of resin may be used to deionize

solutions until the dye begins to fade from blue to gold,
indicating capacity is exhausted. In a large column, the
dye will change color starting at the top of the column,
and deionization will continue until the color change

5

occurs at the bottom of the column. If the column is not
used for more than 1 week, it should be washed with 3
bed volumes of water prior to use.

Regeneration

Mixed bed resin used in laboratory-scale applications
is not normally regenerated because the of the difficulty in
separating the mixed anion and cation resins, the large
volumes of regenerants required for the anion resin, and
difficulty in accurately remixing chemically equivalent
resins. The following procedure may be used and is cost­effective for large-scale applications.

To regenerate a mixed bed resin, separate the anion
exchange resin from the cation exchange resin by back­washing the resin in a column. First use a low flow rate to
expand the bed. This separates the bed into two regions:
the anion exchanger on top and the denser cation exchang­er on the bottom. Then slowly increase the flow rate to
carry the anion exchanger out the top of the column and
into a separate container. An alternative separation method
is to shake the resin in twice its volume of water, let it set­tle, and decant the top layer containing the anion exchang­er. This procedure is repeated until separation is complete.

Regenerate the cation exchanger using 3 bed volumes
of 3 N HCl and rinse with 4 bed volumes of deionized
water or until the effluent is >pH 5. Regenerate the anion
exchanger with at least 10 bed volumes of 3 N NaOH and
rinse to <pH 9 with deionized water. Regeneration flow
rates should be about 2 ml/min/cm

2

.

Mix the resins thoroughly in the original column by
gently backwashing with deionized water while agitating
with a stirring rod or air, then stopping the upflow and
continuing to stir as the resin settles.

Shelf Life

Mixed bed ion exchange resins are stable for 2 years
when stored at 21 °C and protected from exposure to
ultraviolet light. The shelf life may be extended by storing
the resin at 4 °C.

Applications

AG 501-X8 and Bio-Rex MSZ 501(D) mixed bed
resins may be used to prepare non-ionic reagents for criti­cal analytical applications. Either the batch or column
method may be used to obtain purified urea, acrylamide,

6

7

formamide, glyoxal, or PEG, although the batch tech­nique is much more common.

Batch Deionization of Formamide, Acrylamide,
and Glyoxal

This procedure was originally described by Maniatis,
et al. [Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Lab (1982)] for deionization of form­amide, and can be used for any nonionic reagent such as
acrylamide, glyoxal, urea, or water.

1. Weigh 5 grams of resin for every 100 ml of for-

mamide or acrylamide solution to be deionized. For

glyoxal, use 1 gram of resin per ml of glyoxal. This

quantity of resin will be sufficient for any concentra-

tion of these solutions.

2. Wash resin briefly with the solution to be deionized,

using about 1 ml solution per ml of resin. Discard the

solution.

3. Add resin to sample and stir for 1 hour. Check pH

with pH paper to insure deionization is complete.

Repeat Step 3 using new resin if necessary.

4. Filter or decant sample from resin.

Note: Formamide interferes with the color change of
the dye, but will not affect the deionization capacity
of the resin.

Batch vs. Column Deionization of Urea

Table 3 compares deionizing urea both by the batch
and the column technique. The decrease in conductivity
using the batch method is plotted in Figure 1.

70

Conditions
Resin:

Sample: 100 ml 6 M urea

Conductivity µmhos/cm

10

15 10

Fig. 1. Decrease in conductivity using batch method.

AG 501-X8 resin, 5 g

Hours

8

9

Table 3. Deionization of Urea by Batch and
Column Methods

Batch Column

Sample 100 ml 6 M urea 100 ml 6 M urea
Starting conductivity 70 µmho/cm 70 µmho/cm
Amount of mixed bed resin 5 grams 5 grams (~8 ml)
Final conductivity 5.0 µmho/cm 0.2 µmho/cm
Time ~5 hours ~10 minutes

Ampholyte Removal

Carrier ampholytes may be quantitatively removed
from protein fractions derived from isoelectric focusing
using mixed bed resin. Mixed bed ion exchange chro­matography represents a method for the quantitative
removal of carrier ampholytes. Figure 2 illustrates the
separation of proteins from ampholytes and sucrose. A 3
ml sample was applied to a 0.9 x 25 cm column of AG
501-X8 resin. AG 501-X8 resin has also been shown to be
useful for separating peptides of greater than 4,000 dal­tons from ampholytes.

AG 501-X8 resin has also been shown to be useful
for separating peptides of greater than 4,000 daltons from
ampholytes.

Fig. 2. Separation of hemoglobin from ampholytes
and sucrose on a mixed bed ion exchange column
(AG 501-X8 resin). A hemoglobin blank in the sucrose

determination gives an absorbance of about 0.1. Neither
sucrose nor ampholyte emerged from the column in the
total volume tested.

3

10

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References

1. Kapp, O. H. and Vinogradov, S. N., Anal. Biochem., 91, 230 (1978).

2. Bakker, J. A., Vanden Brande, J. L. and Haggerbrugge, C. M., J. Chromatog., 209, 273

(1971).

3. Brown, W. D. and Green, S., Anal. Biochem., 34, 593 (1970).

Product Information

Catalog Mesh Diameter Density Pkg.
Number Description Size (µm) (g/ml) Size

142-6424 AG 501-X8 Resin 20-50 300-1,180 0.75 500 g
142-6425 AG 501-X8 (D) Resin 20-50 300-1,180 0.75 500 g
142-7425 Bio-Rex MSZ 501 (D) 25-35 500-700 0.75 500 g

143-7424 Biotechnology Grade 20-50 300-1,180 0.75 100 g

143-7425 Biotechnology Grade 20-50 300-1,180 0.75 100 g

143-6424 Molecular Biology 20-50 300-1,180 0.75 100 g

143-6425 Molecular Biology 20-50 300-1,180 0.75 100 g

Resin

AG 501-X8 Resin

AG 501-X8 (D) Resin

Grade AG 501-X8
Resin

Grade AG 501-X8 (D)
Resin

12

Nominal