6.1 Separation of Metal Ions................................... 18
6.2 Glucose Binding on AG 1-X8 Resin ................ 21
Section 1
Introduction
AG 1, AG MP-1 and AG 2 resins are strongly basic
anion exchangers. They are capable of exchanging
anions of acidic, basic, and neutral salts, and ampholytes
on the basic side of their pI. Strong anion exchange
resins are used for sample preparation, enzyme assays,
metal separations, and peptide, protein, and nucleic acid
separations.
Section 2
Technical Description
Strongly basic anion exchange resins are available
as Analytical Grade AG 1 and AG 2 resins, AG MP-1
macroporous resin, and Biotechnology Grade AG 1
resin. The Analytical Grade AG 1, AG MP-1 and AG 2
resins have been exhaustively sized, purified, and converted to make them suitable for accurate, reproducible
analytical techniques. Biotechnology Grade AG 1 resin
is analytical grade resin which is certified to contain less
than 100 microorganisms per gram of resin.
1
AG 1 and AG 2 resins are strongly basic anion
exchangers with quaternary ammonium functional
groups attached to the styrene divinylbenzene copolymer lattice. The amount of resin crosslinkage determines
the bead pore size. A resin with a lower percentage of
crosslinkage has a more open structure permeable to
higher molecular weight substances than a highly
crosslinked resin. It also has a lower physical resistance
to shrinking and swelling, so that it absorbs more water
and swells to a larger wet diameter than a highly
crosslinked resin of equivalent dry diameter. For example, the lower crosslinked resins, particularly AG 1-X2
2% crosslinked resin, are useful for the sorption and
fractionation of relatively high molecular weight substances such as peptides, ribo- and deoxyribonucleotides, and uranium. The higher crosslinked resins,
particularly AG 1-X8 8% crosslinked resin, are used for
sorption, exchange, and separation of low molecular
weight inorganic anions, and in applications such as
cyclic nucleotide assays and fractionation of organic
acids. Table 1 shows the approximate molecular weight
2
exclusion limits in water for resins of various crosslinkages.
Table 1. Approximate Molecular Weight Exclusion
Limits for Ion Exchange Resins in Water
PercentApproximate MW Exclusion Limit
Crosslinkingfor Globular Molecules
2%2,700
4%1,400
8%1,000
10%800
12%400
AG 2 resin is similar to AG 1 resin, but is slightly
less basic and slightly less resistant to oxidation due to
differences in the structure of the quaternary functional
group. It offers advantages in certain applications. For
example, it is capable of separating sugars, sugar alcohols, and glycosides using a step gradient and borate
buffers without isomerizing some sugars, as AG 1 resin
tends to do.
3
Each AG 1 resin is supplied in the chloride form.
Selected resins are available in the acetate, formate, and
hydroxide form. These ionic forms may be considered
more activated forms than the chloride form, as may be
deduced from the order of selectivity information given
in Tables 2 and 3. AG 1 resins purchased in the more
active forms may be converted to any other form. The
chloride ion, because of its higher selectivity for the
resin, is relatively difficult to replace with formate,
acetate, hydroxide, or fluoride. Thus, if various ionic
forms are to be used, the formate or acetate forms provide flexibility and convenience (see Table 3). Formate
and acetate forms may be used to separate most low
molecular weight biological compounds, such as
nucleotides, hormones, peptides, and carboxylic acids.
AG MP-1 resin is the macroporous equivalent of AG 1
resin. Its effective surface area approximates 23 square
meters per dry gram, 20% porosity.
The physical properties of the resins are listed in
Table 2. The anion exchange resins are thermally stable
and resistant to solvents (alcohols, hydrocarbons, etc.),
reducing agents, and oxidizing agents.
4
Table 2. Guide to Analytical Grade Anion
Exchange Resins
ResinActiveOrder ofThermalSolvent to Oxidizing
TypeGroupSelectivity StabilityStability Agents
AG 1
R-CH2N+>phenolateOH-form,Very good Slow
and(CH3)3>HSO4>ClO3fair to 50 °C;solution
AG MP-1>NO3>Br>Cl-and otherin hot 15%
ResinsCN>HSO3>forms, good HNO3or
CN>HSO3>to 150 °CHNO3 or
NO2>Cl>OHconc. H2O
>IO3>H2COO
>Ac>F
-
Resistance
solution
Section 3
Mechanism
In an ion exchange procedure, the counterions on
the resin are replaced by sample ions that have the same
charge. With anion exchange resins such as AG 1 and
5
2
2
AG MP-1, neutral species and cations do not interact
with the resin. In the chloride form of AG 1, AG MP-1,
and AG 2 resin, the counterion on the resin is Cl
-
. 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 3 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.
Finally, the order of selectivity can be used to estimate
the difficulty of converting the resin from one form to
another. Conversion from a highly selected to a less
highly selected form requires an excess of the new ion.
6
Table 3. Relative Selectivity of Various Counterions
The AG 1 and AG MP-1 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. The larger the mesh size number, the smaller the particle size. Table 4 shows wet mesh and equivalent micron diameters.
Large mesh material (20-50 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) may be used in
8
batch as well as column applications. Medium mesh is
an ideal, general purpose particle size for use in analytical and preparative scale column chromatography. Fine
mesh material (200-400 and minus 400 mesh) is used
for high resolution analytical separations.
9
Section 4
Resin Conversion
Table 5 outlines common techniques for converting
ion exchange resins from one ionic form to another.
Table 5. Techniques for Resin Conversion
ResinFrom→To
ConversionSol’n/Vol.Flow Rate
AG 1 andCl
AG MP-1OH
ResinsCl
Cl
AG 2 ResinCl
Cl–→ NO
(1)
Reagent Usedof Resincm/min of Bed Exchange
–
–
→ OH
–
→ formate1 N formic acid22NpH<24pH>4.8
–
→ formateUse Cl–→ OH–and20IX-NpH>4.8
–
→ acetate same as formate exceptIX-NpH<24pH>4.8
–
–
→ OH
–
3
(2)
1 N NaOH
then OH–→ formate2pH>4.8
use 1 N HAc
(2)
1 N NaOH
0.5 N NaNO
3
1. Typical conversions are listed. The same reagents can be used to con-
vert from other ionic forms. Two steps regeneration, ion exchange followed by neutralization, is included because of ease of conversion and
saving on expensive reagents.
2. Use U.S.P. or C.P. grade (low chloride).
3. N = Neutralization; IX = Ion exchange; IXN = two step process: Ion
exchange to acid or base form followed by neutralization with appropriate base or acid of salt, example (Step 1) Resin-Cl
10
Volumes ofLinearTest forRinse:Vol.Test for
–
+ NaOH →
Resin conversion is most efficiently carried out in the
column mode. However, when choosing a column,
remember that the resin may shrink, or it may swell as
much as 100%, depending on the conversion.
4. For 50-100 or finer mesh resin. For 20-50 mesh about
is recommended
5. Test for Cl
HNO
or too basic.
–
in effluent: Acidify sample with a few drops of conc.
. Add 1% Ag NO3solution. White ppt indicates Cl–, yellow Br
3
–(5)
–(5)
–(5)
4pH<9
4pH>9
4
1
⁄5 the flow rate
11
–
Conversions to ionic forms not listed in Table 5 can
be achieved using the information supplied in Table 3,
which lists relative selectivities of various counterions
for AG 1, AG MP-1, and AG 2 resin. To convert a resin
to an ionic form with a higher selectivity, wash the resin
with 2-5 bed volumes of a 1 M solution of the desired
counterion. For conversion to an ionic form with a lower
relative selectivity for the resin, the necessary volume of
counterion solution will depend on the difference in
selectivity. As a general rule, use 1 bed volume of 1 M
counterion solution for each unit difference in relative
selectivity. For example, converting AG 1-X8 resin
from the formate form (relative selectivity 4.6) to the
hydroxide form (relative selectivity 1.0) would require
4-5 bed volumes of 1 M NaOH.
In some cases, it is more economical and more efficient to go through an intermediate counterion when
converting to a counterion of much lower selectivity.
One example of this is the conversion of AG 1-X8 resin
from the chloride form (relative selectivity 22) to the
formate form (relative selectivity 4.6). The resin is first
converted to the hydroxide form (relative selectivity 1.0)
12
using 20 bed volumes of 1 N NaOH. The resin has a
very low selectivity for hydroxide, making the conversion to the formate form relatively simple (2 bed volumes of 1 N formic acid). Another conversion method is
to first convert to an ionic form of intermediate selectivity. When converting from the chloride form (relative
selectivity 22) to the hydroxide form (relative selectivity
1), the resin can first be converted to the bicarbonate
form (relative selectivity 6.0).
The easiest method to test for completeness of conversion depends on the particular conversion.
Conversion is complete when the first ion is no longer
detected in the effluent. In many cases, this can be monitored by pH or by simple qualitative tests. When conversion is complete, the resin should be rinsed with deionized water, then with starting buffer, until a stable pH is
obtained. The resin is then equilibrated to the desired
starting conditions.
13
Section 5
Instructions for Use
AG 1 and AG MP-1 resins may be used in a batch
method or a column method. The batch method consists
of adding the resin directly to the sample and stirring.
The column method requires packing a column with
resin, and passing the sample through.
5.1 Batch Method
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.
5.2 Column Method
The column method involves pouring a column with
the resin and passing the sample through to achieve the
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 sample ionic concentration is unknown, begin
with 5 grams of resin for 100 ml of sample, and then
optimize the volumes after looking at the results of
the first separation.
2. Insure that the resin is in the proper ionic form to
allow the sample ions to be exchanged onto the
resin. If conversion of the resin into another ionic
form is necessary, use the guidelines described for
resin conversion (see Table 5).
3. Prepare the initial buffer so that the pH and ionic
concentration will allow the sample ions to be
14
15
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. 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 the 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 enough buffer to just 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 por-
16
tions 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 sample.
7. The actual flow rate that is used will depend upon
the application, the resin, and the column cross-section. To obtain flow rates for any given size column,
multiply the suggested flow rates in Table 6 by the
column cross-sectional area. Table 6 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 off the resin with a solution containing a counterion of higher selectivity than the bound
anion.
17
Table 6. Suggested Flow Rates for
Ion Exchange Resin Columns
Linear
Flow Rate
Application(cm/min)
Removing trace ions5-10
Separations with very few components1-3
Separations of multi-component samples0.3-1.0
Using high resolution resins
with small particle size1-2
Section 6
Sample Protocols for
Anion Exchange Resins
2, Trail Edition, Laboratory Manual of the Chemical
Education Material Study.
Materials
AG 1-X8 resin, 50-100 mesh, 10 grams
Glass column approximately 12 mm ID, 30-40 cm
long, and resistant to 9 N HCl
HCl approximately 9 N, 5 N, and 0.5 N
Sample solution - 0.1 meq Co2+, Ni2+, and Fe
pared from 238 mg CoCl
and 271 mg FeCl
3
·6H2O; 238 mg NiCl2·6H2O;
2
·6H2O in 10 ml 9 N HCl
3+
pre-
Cobalt test solution - 10% NH4SCN in acetone
Nickel test solution - 1% KSCN or 1% NH
SCN
4
Test tubes or vials, 12
6.1 Separation of Metal Ions
This experiment was suggested by Professor Harold
Walton, University of Colorado, and Professors Charles
Koch and George Pimental, University of California at
Berkeley. It is a modification of Experiment 32, Volume
18
Procedure
1. Slurry the resin in distilled water.
2. Pour the resin into a column that is resistant to 9 N
HCl. The resin bed should be approximately 10 cm
deep.
19
3. Equilibrate the resin by passing approximately 15 ml
of 9 N HCl over the resin bed.
4. Adjust the flow rate to no faster than 1 drop/second
or 4 ml/min.
5. When the level of 9 N HCl has reached the top of
the resin bed, either shut the screw clamp, or add 2
ml of sample (0.2 meq of each ion).
6. Do not allow air into the resin bed because air may
cause channeling with uneven flow of subsequent
solutions.
7. After the sample has soaked into the resin bed, add
approximately 20 ml of 9 N HCl.
8. Begin collecting 5 ml aliquots.
9. The color of the eluant should intensify and then the
eluant should become nearly colorless in the third or
fourth aliquot.
10. Continue elution using 20 ml of 5 N HCl, and then
20 ml of 0.5 N HCl, in the manner described above.
11. In each case, 5 to 10 ml more of the eluant may be
added if the eluant is still strongly colored after 20
ml of acid has soaked into the resin bed.
20
12. Observe and record the colors in the resin bed and in
the eluant.
13. Test each aliquot for each of three ions:
+2
Co
test - 1 drop eluant plus 1 drop cobalt test solu-
tion. Strong test is a bright blue color.
+2
Ni
test - 1 drop eluant plus 1 drop nickel test solu-
tion. Neutralize with concentrated NH
. A bright red
3
ppt is a positive test for nickel.
+3
Fe
test - 1 drop eluant plus 1 drop iron test solu-
tion. A bright red color is a positive test.
6.2 Glucose Binding on AG 1-X8 Resin
This procedure demonstrates the binding and elution
of glucose on AG 1-X8 resin.
Materials
Poly-Prep®disposable chromatography column, 2 ml
1 N NaOH
AG 1-X8 resin, 200-400 mesh, formate form, 2-3 ml
Glucose sample, 100 mg/ml
21
Ames Keta-Diastix test for glucose
1 M NaCl
Procedure
1. Pack the Poly-Prep chromatography column with
2 ml AG 1-X8 resin, 200-400 mesh, formate form.
2. Convert the resin to the OH form by washing it with
10 bed volumes of 1 N NaOh, then with 5 bed volumes of distilled water.
3. Add 1 ml of the glucose sample.
4. Wash with 3 bed volumes of water.
5. Check for the presence of glucose.
6. Elute the glucose with 1 M NaCl.
7. Check for glucose.
Section 7
Applications
Strong anion exchange resins are used for sample
preparation, enzyme assays, metal separations, and peptide, protein, and nucleic acid separations. The tables
below summarize the applications.
Table 7. Anion Exchange Resins for
Sample Preparation
ApplicationResinReference
Recovery of P
glucose-phosphateresinBiochem., 24, 6, 2304 (1985).
Extraction of AG 1-X8 Dombro, R. S. and Hutson, D. G.,
5-hydroxy-indole resinClin. Chim. Acta, 100, 231 (1980).
acetic acid from
CSF and urine
Anion removal from AG 1-X8 Torben, K. and Penderson, J. S.,
porphyrin in urineresinScand. J. Clin. Lab. Invest., 38, 279
Purification of cyclicAG 1-X8Shanfield, J., Jones, J. and
nucleotidesresinDavidovitch, Z., Anal. Biochem.,
from AG 1-X4 Stroop, S. D. and Boyer, P. D.,
i
(1978).
113, 256 (1981).
22
23
ApplicationResinReference
Purification of AG 1-X8 Rajagopalan, T. G., Moore, S. and
carboxylated resinStein, W. J., J. Biol. Chem., 241,
pepsinogen4940, (1966).
Separation of cAMP AG 1-X8 Kuehl, F. A., Jr., Ham, E. A. and
from cGMPresin Zanetti, M. E, et al., Proc. Nat.
Concentration AG 1-X8 Minkler, P. E., Ingalls, S. T.,
of aminesresinKormos, L., et al., J. Chromatog.,
Removal of triiodideAG 1-X8 Basciano, L. K., Berenstein, E. H.,
Concentration of Ag 1-X8 Tyler, T. A. and Shrago, R. R.,
niacin prior to HPLC resinJ. Liq. Chromatog., 3, 269 (1980).
analysis
Removal of organic AG 1-X8 Marescau, B., De Deyn, P., Van
acids and carbo-resinGorp, L. and Lowenthal, A., J.
hydrates from Chromatog., 377, 334 (1986).
guanidino compounds
resinKmak, L. and Siraganian, R. P., J.
Acad. Sci. USA, 71, 1866 (1974);
Fallon, A. M. and Wyatt, G. R.,
Anal. Biochem., 63, 614 (1975).
336, 271 (1984).
Biol. Chem., 261, 11823 (1986).
ApplicationResinReference
Removal of thyroid AG 1-X8 Stanley, F., Tsai, J. R. and
hormone from serumresinSamuels, H. H., J. Biol. Chem., 261,
AG 2-X8 Stringer, B. M. J. and WynfordresinThomas, D., Hormone Res., 16, 392
Concentration AG 1-X8 Ellis, R. and Morris, E. R., Cereal
of phytateresinChem., 63, 58 (1986).
Removal of ATP from AG 1-X8 Woldegiorgis, G. and Shrago, E., J.
proteoliposomesresinBiol. Chem., 260, 7585 (1985).
Removal or concen-AG 1-X8 Chen, P. M., Richardson, D. G. and
tration of organic acids resinMellenthin, W. M., J. Amer. Soc.
9400 (1966).
(1982).
Hort. Sci., 107, 807 (1982).
Table 8. Metal Separation on Anion Exchangers
MetalsResinEluted IonsReference
Ni, Mn (ii), AG 1-X8 Ni - 12 M HCl:Kraus, K. A., and
Co (ii), resinMn - 6 M HCl; Moore, G. E., J. Amer.
Cu (ii), Cu - 2.5 M HCl; Chem. Soc., 75, 1460
Fe (iii),Fe - 0.5 M HCl; (1953).
Zn (ii)Zn - 0.005 M HCl
Recommended Eluant and
24
25
MetalsResinEluted IonsReference
Ni, Co, Cu, AG 1-X8 Ni - 96% MeOH, Fritz, J. S. Pietrzyk,
Znresin0.2 M HCl; Co - D. J., Talanta, 8, 143
Mn, Co, Ni AG 1-X8 Mn, Co, Ni - 8.5 Morie, G. P., and
Fe, Mo,resinx 10
(also Cr, Zn,Fe - tartaric acid Chromatog., 16, 201
Cd, Hg)in 0.1 M HCl; (1964).
Th, Hf, Zr, AG 1-X8 Th - 0.7 N Strelow, F. W. E. and
MoresinH
V, Th, FeAG 1-X8 Absorbed as Korkisch, J. and
Recommended Eluant and
55% IPA, 1.3 M (1961).
HCl; Cu - 55%
IPA, 0.1 M HCl;
Zn - 0.005 M HCl
-2
M tartrate; Sweet, T. R., J.
Mo - 3 M NaOH
; Hf - 1.25 Bothma, C. J. C.,
2SO4
N H2SO4; Zr - Anal. Chem., 39, 595
2.0 N H2SO4; (1967).
Mo - 2.0 N NH4;
NO3, 0.5 N NH
resincitrate com-Krivanec, H., Anal.
plexes; Th - 8 M Chim. Acta, 83, 111
HCl; Fe - IBMK, (1976).
acetone, 1 N HCl
(1:8:1 v/v);
V - 1 M HCl
3
MetalsResinEluted IonsReference
Bi, Pb, Cd, Ag 1-X8 Pb, Cd, Zn -Strelow, F. W. E.,
ZnresinHBr-HNO
Recommended Eluant and
Bi - EDTA(1978).
;Anal. Chem., 50, 1359
3
Table 9. Peptide and Protein Separations
on Anion Exchangers
ApplicationResinReference
Separation of smallAG 1-X2 Titani, K., Koide, A., Ericsson,
peptides from rabbit resinL. H., et al., Biochem., 17, 5680
muscle(1978).
Separation of peptides AG 1-X2Ozuls, J., Craig, G. and Nobrega,
from horse liver cyto- resinF. G., J. Biol. Chem., 251, 6767
chromes(1976).
Purification of fungal AG 1-X4 Bhella, R. S. and Altosaar, I., Anal.
glucoamylaseresinBiochem., 140, 200 (1984).
26
27
Table 10. Anion Exchange Resins
in Enzymatic Assays
EnzymeSubstrate ProductResinReference
NADaseNADNicotinamideAG 1-X2Moss, J., Manganiello, V. C. and Vaughn,
resinM., Proc. Nat. Acad. Sci. USA, 73, 4424
(1976).
Cyclic 3',5' - cAMPAdenosineAG 1-X2Brooker, G., Thomas, L. J., Jr. and
nucleotideresinAppelman, M. M., Biochem., 12, 4177
phosphodiesterase(1968); Ong, K. K. and Rennie, P. I. C., Anal.
Biochem., 76, 53 (1976); Thompson, W. J.,
Teraski, W. L., Epstein, P. M. and Strada, S.
J., Advan. Cyclic Nucleotide Res., 9, 69
(1978).
Sucrose synthetase;UDP-glucoseSucrose + UDP;AG 1-X4Salerno, G. L., Gamundi, S. S. and Pontis,
sucrose phosphateand fructose;UDP + resinH. G. Anal. Biochem., 93, 196 (1979).
synthetaseUDP-glucosesucrose -6-P
and fructose-6-P
GuanylateGTPcGMPAG 1-X8Krishnan, N. and Krishna, G., Anal.
cyclaseresin;Biochem., 70, 18 (1976).
neutral
alumina
28
29
Table 10. Anion Exchange Resins
in Enzymatic Assays (
continued
)
EnzymeSubstrate ProductResinReference
HexokinaseMannoseMannose 6-PAG 1-X8Li, E., Jabas, I. and Kornfeld, S., J. Biol.
resinChem., 253, 7762 (1978).
CholineACh + ATPPhosphoryl-AG 1-X8Kato, A. C., Collier, B. Ilson, D. and Wright,
kinasecholineresinJ. M., Can. J. Physiol. Pharmacol., 53, 1050
(1975).
HMG-CoA reductase HMG-CoAMevalonolactoneAG 1-X8Edwards, P. A., Lemongello, D. and
resinFogelman, A. M., J. Lipid. Res., 20, 40
(1979)
Glutamine synthetase GlutamateGlutamineAG 1-X8Pishak, M. R. and Phillips, A. T., Anal.