Page 1
Bio-Rex®Weakly Acidic
Cation Exchange Resin
Instruction Manual
Catalog Numbers
142-5822, 142-5832,
142-5842, 142-5852,
143-5832, 143-5852,
Page 2
Table of Contents
Section 1 Introduction ..........................................1
Section 2 Technical Description...........................1
Section 3 Resin Equilibration ..............................6
Section 4 Resin Conversion..................................7
Section 5 Instructions for Use..............................7
5.1 Batch Method..................................................8
5.2 Column Method..............................................8
Section 6 Resin Regeneration.............................11
Section 7 Applications.........................................11
7.1 Protein, Peptide,
and Amino Acid Separations........................12
Section 8 Product Information...........................15
Page 3
Section 1
Introduction
Bio-Rex 70 resin is a weakly acidic cation
exchanger. This resin is used for the purification and
fractionation of peptides, proteins, antibiotics, and other
cationic molecules.
Section 2
Technical Description
Bio-Rex 70 resin is available as both Analytical
Grade and Biotechnology Grade resin. The Analytical
Grade Bio-Rex 70 resin has been exhaustively sized,
purified, and converted to make it suitable for accurate,
reproducible analytical techniques. Biotechnology Grade
Bio-Rex 70 resin is an Analytical Grade resin which is
certified to contain less than 100 microorganisms per
gram of resin.
The resin contains carboxylic acid exchange groups
on a macroreticular acrylic polymer lattice. The high
porosity of the resin allows large protein molecules to
penetrate the pores and have access to the exchange sites
located throughout the matrix. In an ion exchange
procedure the counterions on the resin are replaced by
1
Page 4
sample ions that have the same charge. On a cation
exchange resin such as Bio-Rex 70 resin, neutral
molecules and anions do not interact with the resin.
Normally Bio-Rex 70 resin is supplied in the sodium
form, but the 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 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
monovalent and divalent counterions, as well as some
nominal properties of the Bio-Rex 70 resin.
Table 1. Properties of Bio-Rex 70 Resin
Wet density 0.6 ml/g (Na+and H+forms)
+
% moisture 65-74% (Na
form); 55-65%
(H+ form)
Swelling
1 ml (H+form); 1.7 ml (Na+ form)
Capacity 10 meq/dry g
Na+ form 0.5 meq/ml
H+ form 2.4 meq/ml
Pore volume 0.10 cc/cc
Pore size range 700-4,000 Å
Thermal stability Up to 100 °C
Normal operating
pH range 5 to 14
Order of selectivity
for monovalent ions H>>Ag>K>Na>Li
Order of selectivity
for divalent ions H>>Fe>Ba>Sr>Ca>Mg
2
3
Page 5
Bio-Rex 70 resin has a high capacity for
macromolecules, yet does not bind proteins so tightly
that they are difficult to elute. The acrylic polymer
matrix is hydrophilic, and generally free of nonspecific
binding or denaturing effects on proteins. Figure 1 shows
the titration curve. Note that the optimal operating pH
range is above pH 5.
pH
Bio-Rex 70
meq. NaOH added per dry gram.
Fig. 1. Bio-Rex 70 titration curve.
Bio-Rex 70 resin is 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, the smaller the particle size. Table 2 shows wet
mesh and equivalent micron diameters.
Table 2. Wet Mesh and Equivalent Micron
Diameters
Wet Mesh
(U.S. Standard) 16 20 40 50 80 100 140 200 270 325 400
Micron Diameter
(1 µ=0.001 mm) 1190 840 420 297 177 149 105 74 53 44 37
Large mesh material (20-50 mesh and 50-100 mesh)
is used primarily for large preparative applications, and
batch operations where the resin and sample are slurried
together. Medium mesh resin (100-200 mesh) is used
primarily in column chromatography, for analytical and
laboratory scale preparative applications. Fine mesh
material (200-400 mesh and minus 400 mesh) is used for
high resolution analytical separations.
4
5
Page 6
Section 3
Resin Equilibration
Especially when Bio-Rex 70 resin is used to separate
multi-component samples, it is very important to
equilibrate the resin to the initial buffer conditions before
the sample is applied. In general, unequilibrated resin
will not give reproducible results. Due to its weakly
acidic functional groups. Bio-Rex 70 resin is slow to
equilibrate. The following procedure is recommended:
1. Place the Bio-Rex 70 resin in a beaker with the
buffer in which it is to be equilibrated. The volume
of buffer should be four to five times the volume of
the resin.
2. Allow the resin to equilibrate for at least 30 minutes.
Adjust the pH with acid or base. Re-equilibrate.
Repeat until pH is stable.
3. When pH is stable, decant the buffer off and repeat
steps 1 and 2 using fresh buffer. Repeat until no pH
change is noted when fresh buffer is added. This may
take several buffer changes.
4. Decant excess buffer off and pour the resin into the
column. Pass 2-3 bed volumes of buffer through the
column, monitoring both pH and conductivity. When
the pH and conductivity of the effluent are the same
as that of the influent, the resin is fully equilibrated
and ready for sample application.
Section 4
Resin Conversion
Bio-Rex 70 resin can be converted to other ionic
forms by washing with a 0.5-1.0 M solution of the desired
counterion. Conversion to a counterion of relatively high
selectivity (see Table 1) will be rapid (2-3 bed volumes),
while conversion to a counterion of relatively low
selectivity will be slower (3-5 bed volumes). Conversion
is complete when the starting counterion is no longer
detected in the effluent. In most cases, this can be
monitored by pH or simple qualitative tests.
Section 5
Instructions for Use
Bio-Rex 70 resin may be used with either 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.
6
7
Page 7
5.1 Batch Method
The batch method is performed by adding the resin
directly to the sample and stirring. The resin should be in
correct ionic form and equilibrated 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 of
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 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 described
in Section 4 for resin conversion.
3. Prepare the initial buffer, so that the pH and ionic
concentration will allow the sample ions to be
exchanged on to the column. For unknown solutions,
use deionized water.
4. Slurry and pour the resin into the column. Resin
should be equilibrated according to the instructions
outlined above. Poorly equilibrated resin will not
give reproducible results.
5. 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 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
8
9
Page 8
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 3 by the
column cross-sectional area. Table 3 gives typical
flow rates of analytical grade resins.
8.
If a cation-free solution is the goal, collect the
effluent. If the concentrated cations are of interest,
allow all of the sample to pass through the column,
then elute the sample with a solution containing a
counterion of higher selectivity than the bound cation.
Table 3. Suggested Flow Rates for Ion
Exchange Resin Columns
Flow Rates
Application ml/min/cm
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
Section 6
Resin Regeneration
Bio-Rex 70 resin is most efficiently regenerated in a
column. Regenerate to the sodium form by washing with
3 bed volumes of 0.5 N NaOH. The flow rate should not
exceed 1 ml/min per cm2 cross-sectional area of the
column. Conversion is complete when the pH becomes
greater than 9. Rinse with 4 bed volumes of deionized
water and equilibrate according to the procedure above.
Remember that the resin volume will approximately
double when converting from the hydrogen form to the
sodium form.
Section 7
Applications
2
Bio-Rex 70 resin has been used for the purification
and fractionation of enzymes, histones, endonucleases,
and toxins. Table 4 lists a variety of applications using
Bio-Rex 70 resin.
10
11
Page 9
7.1 Protein, Peptide, and Amino Acid
Separations
Proteins may be separated from one another using
the macroporous Bio-Rex 70 cation exchange resin,
because this resin has pores of sufficient size to admit the
proteins. The effective exclusion limit of this resin has
not been established, however, from the many
separations reported, the limit exceeds 75,000 daltons.
Table 4. Applications of Bio-Rex 70 Resin
Purification of the rep pro- Scott, J. F. and Kornberg, A., J. Biol. Chem.,
tein of E. coli 253, 3292 (1978).
Purification of human saliva Vasstrand, E. N. and Jensen, H. B., Scand. J.
lysozyme Dent. Res., 88, 219 (1980).
Purification of neurotoxins Karlsson, E., Eaker, D., Fryklund, L. and
from sea snake venom Kadin, S., Biochemistry, 11, 4628 (1972).
Purification of initiation Stringer, E. A., Chaudhuri, A. and Maitra,
factor 2 from calf liver U., J. Biol. Chem., 254, 6845 (1979).
Purification of histones
Purification of restriction Green, P. J., et al., Nucleic Acids Research,
endonucleases 5, 2373 (1978).
Purification of human Savage, C. R. Jr. and Harper, R., Anal.
epidermal growth factor Biochem., 111, 195 (1981).
Purification of human C1q Tenner, A. J., Lasavre, P H. and Cooper, N.
D’Anna, J. A., Strniste, G. F. and Gurley, L.
R., Biochemistry, 18, 943 (1979); Thompson,
J. A., Stein, J. L., Kleinsmith, L. J. and Stein,
G. S., Science, 194, 428 (1976).
R., J. Immunol., 127, 648 (1981).
Table 4. (Continued)
Purification of inhibitory pro- Fearon, D. T., Proc. Nat. Acad. Sci. USA, 76,
tein of human erythrocytes 5867 (1979).
Measurement of glyco-
sylated hemoglobin
Purification of protein aller- King, T. P., Sobotka, A. K., Alagon, A.,
gens of white-faced yellow Kochoumiam, L. and Lichtenstein, L. M.,
hornet, and yellow jacket Biochemistry, 17, 5165 (1978).
venoms
Purification of oximdoleala- Shrake, A. and Rupley, J. A., Biochemistry,
nine -62 lysozyme 19, 4044 (1980).
Isolation of gentamicin Habbal, Z. M., Clin. Chim. Acta, 95, 301
from serum (1979).
Isolation of brain histamine Lewis, S. J., Fennessy, M. R., Laska, F. J.
Determination of thiamine Ellefson, W. C., Richter, E., Adams, M. and
in food Baillies, N. T., J.A.O.A.C., 64, 1336 (1981).
Purification of toxins
Isolation of siderophores Frederick, C. B., Szaniszlo, P. J., Vickrey,
from fungus P. E., Bentley, M. D. and Shive, W.,
Purification of E. coli photo- Koka, P., Biochemistry, 20, 2914 (1984).
reactivity enzyme
Purification of polypeptides Hamilton, S. L., Yatani, A. and Hawkes,
from croatalus atrox venom M. J., et al., Science, 229, 182 (1985).
Purification of a yeast Johnson, A. W. and Demple, B., J. Biol.
DNA repair enzyme Chem., 263, 18009 (1988).
Trivelli, L. A., Ranney, H. M. and Lai, H. T.,
N. E. J. Med., 284, 353 (1971); Schifreen, R.
S., Hickingbotham, J. M. and Bowers, G. N.
Jr., Clin. Chem., 26, 466 (1980).
and Taylor, D. A., Agents and Actions, 10,
197 (1980).
Levinson, S. R., Curatolo, C. J., Reed, J. and
Raftery, M. A., Anal. Biochem., 99, 72 (1979).
Biochemistry, 20, 2432 (1981).
12
13
Page 10
Table 4. (Continued)
DNA A protein of E. coli Sedimizu, K., et al., J. Biol. Chem., 263,
Hemoglobin from blood Ersser, R. S., et al., Biomedical Chrom., 1,
Peptides derived from Wilson, S. P., J. neurosci. Methods, 15, 155
proenkephalin A (1985).
Hemocyanin Moore, M. D., et al., J. Biol. Chem., 261,
Urogastrone Savage, C. R. and Harper, R., Anal.
Lysozyme Matei, L., Rev. Roium. Biochem., 23, 45
Nonhistone chromosomal Wen, L. and Reeck, G. R., J. Chromatog.,
proteins 314, 436 (1984).
Catabolite gene activator Blazy, B. and Ullmann, A., J. Biol. Chem.,
proteins of E. coli 261, 11645 (1986).
FLP recombinase protein Bruckner, R. C. and Cox, M. M., J. Biol.
from S. cerevisiae Chem., 261, 11798 (1986).
Amine and amino acid
determination
Alpha and beta subunits of Ong, L. J. and Glazer, A. N., J Biol. Chem.,
R-phycocyanin II 262, 6323 (1987).
Extraction of GIIIA Cruz, L. J., et al., Biochem., 28, 3437
7136 (1988)
183 (1986).
10511 (1986).
Biochem., 111, 195 (1981).
(1986).
Carlucci, F. V. and Karmas, E., J. A. O. A. C.,
71, 564 (1988).
(1989).
Section 8
Product Information
Minimum
Catolog Dry Mesh Ionic Pkg. Capacity Diameter (Nominal)
Number Designation Form Size (meq/ml) (microns) gm/ml
Analytical Grade Bio-Rex 70 Resin
142-5822 20-50 Sodium 500 g 2.4 300-1,180 0.70
142-5832 50-100 Sodium 500 g 2.4 150-300 0.70
142-5842 100-200 Sodium 500 g 2.4 75-150 0.70
142-5852 200-400 Sodium 500 g 2.4 45-75 0.70
Biotechnology Grade Bio-Rex 70 Resin
142-5832 50-100 Sodium 500 g 2.4 150-300 0.70
142-5852 200-400 Sodium 500 g 2.4 45-75 0.70
Wet Density
14
15
Page 11
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT-206 Rev B
Loading...