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AG®3, AG 4, and Bio-Rex®5
Anion Exchange Resin
Instruction Manual
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT207 Rev B
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AG 3, AG 4, and Bio-Rex 5 Resin
Weakly Basic and Intermediate Basic
Anion Exchange Resin Instructions
Introduction
AG 3-X4 and AG 4-X4 resins are weakly basic
anion exchangers. These resins can exchange anions,
such as organic acids, with weak interactions, allowing
easy elution and regeneration. They will also adsorb
mineral acids, such as hydrochloric acid and perchloric
acid, to yield a neutralized solution. Bio-Rex 5 resin is an
intermediate basic anion exchanger, which also
exchanges anions, such as iodide, bromide, and chloride,
with weak interactions, allowing easy elution and
regeneration.
Technical Description
AG 3-X4, AG 4-X4, and Bio-Rex 5 resins are all
analytical grade purity. They have been exhaustively
sized, purified, and converted to make them suitable for
accurate, reproducible analytical techniques. AG 4-X4
resin is also available in Biotechnology Grade,
Analytical Garde resin which has been further processed
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and is certified to contain less than 100 microorganisms
per gram of resin.
AG 3-X4 resin and AG 4-X4 resin are both 4%
crosslinked and have a tertiary amino functional group.
The resins differ in their backbone resin matrix material.
AG 3-X4 resin has a styrene-divinylbenzene copolymer
lattice matrix, and AG 4-X4 resin has an acrylic matrix.
These two matrices differ in their physical and
mechanical strength, and in their inherent hydrophilic
nature. The styrene-divinylbenzene matrix (AG 3-X4
resin) is very strong mechanically, but is hydrophobic,
which can cause adsorption of large organic molecules or
proteins. The acrylic matrix (AG 4-X4 resin) is softer,
and therefore sensitive to crushing from excess flow rates
or very large column sizes. This material is hydrophilic
and is suitable for use with high molecular weight
organic compounds, proteins, and carbohydrates.
Bio-Rex 5 intermediate base resin contains primarily
tertiary, but also some quaternary, amines on a styrenedivinylbenzene lattice. Bio-Rex 5 resin can be used to
separate organic acids from neutral sugars. When the
resin is regenerated with base, the quaternary ammonium
2
groups will be in the hydroxide form, while the tertiary
amine groups will be in the free base form.
The physical properties of the resins are listed in
Table 1. These resins are thermally stable and resistant to
solvents (alcohols, hydrocarbons, etc).
Table 1. Summary of Properties
Resin Type Active Group Selectivity Stability Stability
Bio-Rex 5 R-N(CH
intermediate HSO
base NO
anion HSO
exchanger >HCO
AG 4-X4 R-CH
AG 3-X4 CrO
weakly basic tartaric>oxalic
anion >H
exchangers H
N+(CH3)2HSO3>HCit> Good to Good
2
Order of Thermal Solvent
I>phenolate> Good to Good
3)2
>CIO3> 60 °C
4
>Br>CN>
3
>NO2>CI
3
>IO3>
3
COO>HC>
H
2
OH>F
3>H2SO4
>
3PO4
AsO4>HNO
4
>HI>HBr>HCl
>HF>HCOO>
HAc>H2CO
3
> 60 °C
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Mechanism
Both AG 3-X4 and AG 4-X4 resin are available in
the free base form. Here the functional group is neutral,
and is not charged. When a mineral acid is passed over
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the resin, the nitrogen in the functional group becomes
protonated, allowing the adsorption of the mineral acid.
When either of these two resins is washed with base, the
resin goes back into the free base form. Figure 1 shows
the adsorption of hydrochloric acid onto the resin.
CH
3
R – CH2– N + H+CI
CH
3
Resin in free-base form Mineral Acid Adsorption of acid
Fig. 1. Adsorption of hydrochloric acid.
-
←
R – N – HCI
→
CH
CH
3
3
Weakly basic anion exchangers, such as AG 3-X4
and AG 4-X4 resins, will act as anion exchangers when
equilibrated and used at or below pH 7. In an ion
exchange procedure, the counterions on the resin are
replaced by sample ions that have the same charge. The
functional group on these resins is a tertiary amine,
which will be positively charged at or below pH 7. The
corresponding anion is the counterion which will be
exchanged with the anion in the sample. Neutral
compounds and cations will not interact with the resin,
and should pass through in the void volume. A resin can
be converted from one ionic form to another. Usually the
resin is used in an ionic form with a lower selectivity for
the functional group than the sample ions to be
exchanged. The sample ions are then exchanged onto the
resin when introduced, and can be eluted by introducing
an ion with higher affinity for the resin or a high
concentration of an ion with equivalent or lower affinity.
Table 1 shows the relative selectivity of various
counterions. In general, the lower the selectivity of the
counterion, the more readily it exchanges for another ion
of like charge. The order of selectivity can also be used
to estimate the effectiveness for different ions as eluants,
with the most highly selective being the most efficient.
To convert from a highly selected to a less highly
selected form requires an excess of the new ion. Table 2
outlines common techniques for converting ion
exchange resins from one ionic form to another. Resin
conversion is most efficiently carried out in the column
mode.
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Table 2. Techniques for Resin Conversion
Resin: AG 4-X4 resin
Conversion from
Reagent used: 0.5 NaOH
Volumes of solution per volume of resin: 2
(3)
Flow rate
Test for completeness of conversion: Cl
Rinse: volume DI water per volume resin: 4
Test for completion of rinsing: pH <9
ml/min/cm2of bed: 1
(1)
: Cl
AG 3-X4 resin
Bio-Rex 5 resin
-
→
free base+OH
(2)
-
-(4)
(1) Typical conversions are listed. The same reagents
can be used to convert from other ionic forms. Two
step regeneration, ion exchange followed by
neutralization, is included because of ease of
conversion and savings on expensive reagents.
(2) Use U.S.P. or C.P. grade (low chloride).
(3) For 50-100 or finer mesh resin. For 20-50 mesh,
about 1/5 the flow rate is recommended.
(4) Test for Cl
drops of conc. HNO
White ppt indicates Cl
-
in effluent: Acidify sample with a few
. Add 1% AgNO3solution.
3
-
, yellow Br-or too basic.
All three resins are available in several particle size
ranges. The flow rate in a chromatographic column
increases with increasing particle size. However, the
attainable resolution increases with decreasing particle
size and narrower size distribution ranges. Particle size is
given either in mesh size or micron size. Mesh refers to
the number of openings per inch on the screens used to
size ion exchange resins. Therefore, the larger the mesh
size number, the smaller the particle size. Table 3 shows
wet mesh and equivalent micron diameters.
Table 3. Wet Mesh and Equivalent Micron Diameters
Wet mesh 16 20 40 50 80 100 140 200 270 325 400
(U.S. Standard)
Micron diameter 1,190 840 420 297 177 149 106 74 53 44 37
Large mesh material (20-50 mesh and 50-100 mesh)
is used primarily for preparative applications and batch
operations where the resin and sample are slurried
together. Medium mesh resin (100-200) is also used in
batch operations, although the primary use of medium
mesh resin is for column chromatography in analytical
and laboratory scale preparative applications. Fine mesh
material (200-400 mesh) is used for high resolution
analytical separations.
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Instructions for Use
AG 3-X4, AG 4-X4, and Bio-Rex 5 resin may be
used in the batch method or the column method. The
batch method consists of adding the resin directly to the
sample and stirring. The column method requires
preparing a column filled with resin, and passing the
sample through.
Batch Method Instructions
The batch method is performed by adding the resin
directly into the sample and stirring. The resin should be
in the correct ionic form prior to beginning.
1. Weigh out about 5 grams of resin for every 100 ml
of sample. For larger scale applications or when an
exact amount of resin is needed, calculate the resin
volume based on the resin capacity.
2. Add resin to the sample and stir or shake gently for 1
hour.
3. Filter or decant the sample from the resin.
Column Method Instructions
The column method involves pouring a column with
the resin and passing the sample through to achieve the
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separation. Particle size will determine the flow rate,
which will affect the separation. The resin should be in
the correct ionic form and equilibrated prior to adding
the sample.
1. Calculate the amount of resin required based on the
expected resin capacity and sample concentration. If
the ionic concentration of the sample is unknown,
begin with 5 grams of resin for 100 ml of sample,
and optimize the volumes after obtaining the results.
2. Insure that the resin is in the proper ionic form,
which will allow the sample ions to be exchanged
onto the resin. If conversion of the resin to another
ionic form is necessary, use the guidelines in Table 2
for resin conversion.
3. Prepare the initial buffer, so that the pH and ionic
concentration will allow the sample ions to be
exchanged onto the column. For unknown solutions,
use deionized water.
4. Slurry and pour the resin into the column.
Equilibrate the resin in the initial buffer using 3 bed
volumes of buffer. Poorly equilibrated resin will not
give reproducible results.
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Alternatively, equilibration can be done by the batch
technique, prior to pouring the column. First, convert the
resin to the appropriate form, then suspend it in the
starting buffer. Check the pH with a pH meter while
stirring continuously. Adjust the pH by adding acid or
base dropwise to the buffer until the desired pH is
obtained. Then transfer the resin to the column and pass
1 bed volume of starting buffer through the column.
5. Slurry the resin in the initial buffer and pour the
column. Allow excess buffer to pass through the
column, leaving just enough buffer to cover the top
of the resin bed.
6. Apply the sample dropwise to the top of the column
without disturbing the resin bed. Drain the sample
into the top of the bed and apply several small
portions of starting eluant, being very careful to rinse
down the sides of the column and to avoid stirring up
the bed. Drain each portion to the level of the resin
bed before the next portion is added. Never allow the
liquid level to drain below the top of the resin bed.
7. The actual flow rate that is used will depend upon
the application, the resin, and the column cross
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section. Table 4 gives typical flow rates of analytical
grade resins.
8. If an anion free solution is the goal, collect the
effluent. If the concentrated anions are of interest,
allow all of the sample to pass through the column.
Then, elute the anions from the resin with a solution
containing a counterion of higher selectivity than the
bound anion.
Table 4. Suggested Flow Rates for Ion Exchange Resin
Columns
Application Flow Rate
Removing trace ions 5-10
Separations with very few components 1-3
Separations of multi-component samples 0.3-1.0
Using high resolution resins with small particle size 0.1-0.2
ml/ min/ cm
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Sample Protocol
Separating Neutral Sugars and Acids from Wine
and Grape Must Samples
This procedure describes a method for separating
organic acids and neutral sugars using Bio-Rex 5 resin,
according to the protocol described by McCord, et al.
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Since some of these compounds co-elute during HPLC,
this simple fractionation technique was developed.
Protocol
1. In a test tube, carefully pipet:
1.00 ml wine or grape must
0.100 ml 5% formic acid
0.100 ml 5% propylene glycol
0.2 ml 5 N ammonium hydroxide
Mix well. Insure that the pH is between 8 and 9;
adjust if necessary. Keep closed.
2. Prepare the column as follows (do not let the column
dry out):
a. Slurry 1 g Bio-Rex 5 100-200 mesh resin (Cl
form) in 3 ml water.
b. Pour slurry into a polypropylene column.
c. Using a syringe, expel excess liquid, then wash
bed with 5 ml water.
3. Neutral fraction.
a. Collect in a clean vial (scintillation vials work
well) and use a syringe to hasten flow through
the column.
b. Add the sample to column, drain through.
c. Wash with water (1 ml, 4 ml, 4 ml).
d. Cap and mix well. Store at 4 °C.
4. Acid fraction.
a. Change to a fresh vial.
b. Carefully add 1 ml of 10% sulfuric acid.
c. Wash with water (1 ml, 4 ml, 4 ml).
d. Cap and mix well.
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Technical Information
If you have any questions or need additional
information, contact your local Bio-Rad representative.
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Reference
1. McCord, J. D., Trousdale, E. and Ryu, D. D. Y., Am. J.
Enol. Vitic., 35, 28 (1984).
Product Information
Minimum
Catalog Mesh Ionic Pkg Capacity Diameter Density
Number Description Size Form Size meq/ml (microns) g/ml
140-4341 AG 4-X4 Resin, 100-200 Free Base 500 g 0.8 75-150 0.70
Analytical Grade
140-5341 AG3-X4 Resin, 100-200 Free Base 500 g 1.0 106-250 0.70
Analytical Grade
140-7841 Bio-Rex 5 Resin, 100-200 Chloride 500 g 1.1 75-150 0.70
Analytical Grade
140-7851 Bio-Rex 5 Resin, 200-400 Chloride 500 g 1.1 45-75 0.70
Analytical Grade
143-3341 AG 4-X4 Resin, 100-200 Free Base 100 g 0.8 75-150 0.70
Biotechnology
Grade
Wet Nominal
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