Macro-Prep 25
Ion Exchange Resins
Instruction ManualTable of Contents

Section 1

Introduction.  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 1

Section 2

Intended Use. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 1

Section 3

Technical Specifications and Characteristics. .  .  .  .  .  .  .  .  .  . 2

Section 4

Preparation for Use.  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 4

Section 5

Column Packing .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 4

Section 6

Operation and Maintenance .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5

Section 7

Technical Assistance.  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 9

Section 8

References .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 9

Section 9

Ordering Information.  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 10

II |Macro-Prep 25 Ion Exchange Resins

Section 1 Introduction

Section 1
Introduction
Macro-Prep 25 S Strong Cation Exchange Resin and Macro-Prep 25 Q Strong
Anion Exchange Resin are designed to provide high resolution and high capacity
for both analytical and preparative separations at high flow rates. Both resins
possess the same rigid, macroporous structure of 50 µm Macro-Prep HighQ and High S Resins, but in a 25 µm bead that offers higher resolution. They
are easy to use, allow quick and reliable packing, and can be used for multiple
cycles.
Bio-Rad’s chromatography resins are manufactured in an ISO 13485 registered
manufacturing facility.

Section 2
Intended Use
Macro-Prep 25 Ion Exchange Resins are appropriate for capture, intermediate,
and polishing purification steps that remove contaminants or impurities frompartially purified targets. Due to their rigidity and unique surface chemistry,these resins are particularly suited for flow-through operations requiring highthroughput and high recovery of target molecules. The slightly hydrophobic basebead also provides selectivity for removal of trace contaminants such as residual
host cell proteins, endotoxins, and viruses.
All Macro-Prep Ion-Exchange Resins contain carboxyl groups. Depending on
the resin, pH conditions, and samples, the resins may act in mixed mode. With
proper planning, this property can be exploited to give unique selectivity, which
may increase yield and purity.

Macro-Prep 25 Ion Exchange Resins |1

Section 3 Technical Specifications and Characteristics

Section 3
Technical Specifications
and Characteristics
Macro-Prep 25 Ion Exchange Resins are rigid methacrylate, which exhibits
little shrinkage and swelling, making them suitable for medium pressure
applications with a peristaltic or higher pressure pump (see Figure 1). The
Macro-Prep 25 Ion Exchange Resins are not suitable for gravity flow. The
macroporous nature of the resin allows both small and large molecules toaccess the exchange sites located throughout the resin. This results inhigh-resolution separations with analytical or preparative sample loads.
60

7

6
40

20

4

3

40

Pressure, bar

Pressure, psi

5

2
20
1

0

0
0

60

120

180

240

300

360

420

Linear velocity, cm/hr

Fig. 1. Linear flow rate vs. operating pressure. The Macro-Prep 25 S Resin was evaluated forlinear flow rate vs. operating pressure by measuring the pressure at a series of flow rates using 1 M
NaCl on a 1.6 cm (ID) x 12.5 cm (H) column. The pressure plotted was determined by subtracting
the pressure generated by the column when empty from the pressure generated by the column
when packed.

The Macro-Prep 25 Ion Exchange Resins are available as the Macro-Prep
25 S Strong Cation Exchanger and the Macro-Prep 25 Q Strong Anion
Exchanger. Table 1 lists the properties of these resins.

2 |Macro-Prep 25 Ion Exchange Resins

Section 3 Technical Specifications and Characteristics
Table 1. Properties of Macro-Prep Ion Exchange Resins.
Macro-Prep 25 Q Resin

Macro-Prep 25 S Resin

Type of ion exchanger
Functional group
Ionic capacity
Dynamic binding capacity
Shipping counterion
Nominal particle size
Nominal pore size
Recommended linear
flow rate range

Strong anion–N+(CH3) 3
220 ± 70 µeq/ml>30 mg/ml BSA
Cl –
25 µm
725 Å
50–300 cm/hr

Strong cation–SO3–
110 ± 30 µeq/ml>40 mg/ml BSA
Na+
25 µm
725 Å
50–300 cm/hr

Maximum linear flow rate

3,000 cm/hr

3,000 cm/hr

Volume changes
pH 4–10
0.1–1.0 M NaCl

<1%
<5%

<1%
<5%

Autoclavable (121ºC, 30 min)pH stability
Storage condition

Yes*
1–10
20% ethanol

Yes
1–12
20% ethanol* Do not autoclave in the OH – form

Chemical Stability
Macro-Prep 25 Ion Exchange Resins are stable in most aqueous buffer
solutions commonly used in purification of biomolecules. These ion
exchangers also withstand treatment in solutions of acid, chaotropic agents,
detergents, and pH <10 while retaining full functional performance. We do
not recommend routine cleaning or operation of Macro-Prep 25 Q above
pH 10. Macro-Prep 25 Ion Exchange Resins should not be sanitized orstored in NaOH.

Thermal Stability
The Macro-Prep 25 Resins can be autoclaved at 121°C for up to 30 min
in deionized water as a slurry or a moist cake. Do not autoclave
Macro-Prep 25 Q in OH – form.

Macro-Prep 25 Ion Exchange Resins |3

Section 4 Preparation for Use

Section 4
Preparation for Use
Macro-Prep 25 Resins are supplied fully hydrated in 20% (v/v) ethanol. Wash
the ethanol storage solution from the resin with deionized water before packing
the column. Small volumes of the Macro-Prep 25 Resins are easily washedin a Büchner funnel with 4–5 column volumes (CV) of deionized water. For
large volumes (for example, >1 liter), it is more convenient to pour the desired
amount of resin into a suitable container, allow the support to settle, anddecant the ethanol solution. Add one CV of deionized water, resuspend theresin, allow it to settle, and decant the supernatant. Repeat this procedure
with deionized water approximately 4–5 times (or until the ethanol is removed),
and then pack the column (see Section 5 Column Packing). The resin may
be prepared by decanting the ethanol solution and resuspending it in theapplication buffer prior to column packing.

Section 5
Column Packing
The Macro-Prep 25 S and Macro-Prep 25 Q Ion Exchange Resins have anominal particle size of 25 µm, thus affording high-resolution separations
at medium pressures. In order to obtain the most resolution from theseresins, a well packed column is essential. Empty columns designed foruse at medium pressures (up to 100 psi/7 bar) such as the Bio-Scale MT
Columns (see Section 9 Ordering Information) from Bio-Rad Laboratories arerecommended. These columns must be used with bed resins with pore sizes
10 µm or smaller to prevent high backpressure due to bed support plugging.
Note: NaCl solution or buffers containing 0.5–1.0 M NaCl is recommended
for optimum column packing.
1.	 Slurry, approximately 1:1 (v/v), the prepared matrix in 0.5–1.0 M NaCland degas.
2.	 Close the outlet of column. Fill 10% of the column with degassed
0.5–1.0 M NaCl. Remove any air bubbles trapped in the bed support
or the column end piece.
3.	 Add an appropriate amount of the resin in a homogeneous slurry to
the column.
4.	 Fill the remainder of the column with 0.5–1.0 M NaCl.

4 |Macro-Prep 25 Ion Exchange Resins

Section 6 Operation and Maintenance

5.	 Connect the flow adapter to the pump, fill it with 0.5–1.0 M NaCl, and makesure it is free from air bubbles. Attach the flow adapter to the column.
Inserting it at a slight angle makes it easier to avoid trapping air bubbles.
6.	 Open the column outlet and pump 4–5 CV of 0.5–1.0 M NaCl throughthe column at the highest flow rate your pump/column system permits.
A dense, well-packed bed gives better chromatography.
7.	 Switch off the pump and close the column outlet. Disconnect the flow
adapter tubing from the pump and position the flow adapter in contact
with the resin. During this step, the packing solution will backflow throughthe adapter.
8.	 Reconnect the flow adapter tubing to the pump, open the column outlet,
and run 4–5 more CV of 0.5–1.0 M NaCl at the highest flow rate yourpump/column system will permit, then repeat steps 7 and 8 for a final
adjustment of the flow adaptor.
9.	 After equilibration in the start buffer, the column is ready forsample application.

Section 6
Operation and Maintenance
Macro-Prep 25 Ion Exchange Columns may be operated in two different
modes. In bind-elute mode, the protein of interest (target molecule) binds to
the resin during loading. It is subsequently eluted by a change of conductivity
and/or pH. This allows removal of contaminants that do not bind to the resinand contaminants that may bind more weakly or more strongly than theprotein of interest. In flow-through mode, the protein of interest does not bindduring loading while contaminants that may bind to the resin are removed.
This approach can simplify chromatography conditions, conserve buffer,and increase recovery of the target molecule.
For flow-through mode, a linear flow rate of 300 cm/hr in a 20 cm bed ispractical. If operating in a bind-elute mode, flow rates of 100–200 cm/hr are
recommended. Do not exceed 70–75% of the maximum pressure attained
during the initial column packing. If you do not have a pressure indicator, do notlet flow rates exceed 70–75% of the maximum flow rate used during packing.
All buffers used for anion or cation exchange chromatography can be used withthe appropriate ion exchange resins (see Table 2). It is best to use buffering ionsthat have the same charge as the functional group on the ion exchanger, forexample, phosphate (–) with the Macro-Prep 25 S Cation Exchanger and Tris(+) with the Macro-Prep 25 Q Anion Exchanger. Buffers should be chosen so
operating pH is within 0.5 pH unit from pK a of buffer substance.
Macro-Prep 25 Ion Exchange Resins |5

Section 6 Operation and Maintenance

The purification may be optimized by changing the pH, ionic strength ofelution buffer, gradient profile, or buffer. Typical chromatographic conditions
are use of buffer concentrations in the range of 20 to 50 mM, at least one pH
unit below the pI of the protein for cation exchange, and one pH unit above
the pI of the protein for anion exchange. Gradients are then run from 0 to 1 M
NaCl over 10–20 CV.
Proper adjustment of the sample pH and ionic strength is critical for
consistent and reproducible chromatography. For best results, the sample
should be exchanged into the start buffer or diluted to the concentration of
the start buffer.
Buffer exchange can be accomplished using Bio-Spin 6 or Bio-Spin 30
Columns, Econo-Pac 10 DG Desalting Columns, Bio-Gel P-6 DG Size
Exclusion Gel, or the Econo-Pac P6 Cartridge. The choice of productdepends on the sample volume.
Table 2. Common buffers for ion exchange chromatography.*Type of Ion Exchanger

BufferBuffering Range

Macro-Prep 25 S

Acetic acid
Citric acid
HEPES
Lactic acid
MES
MOPS
Phosphate
PIPES
Pivalic acid
TES
Tricine

4.8–5.2
4.2–5.2
7.6–8.2
3.6–4.3
5.5–6.7
6.5–7.9
6.7–7.6
6.1–7.5
4.7–5.4
7.2–7.8
7.8–8.9

Macro-Prep 25 Q

BicineBis-Tris
Diethanolamine
Diethylamine
L-histidine
Imidazole
Pyridine
TricineTriethanolamineTris

7.6–9.0
5.8–7.2
8.4–8.8
9.5–11.5
5.5–6.0
6.6–7.1
4.9–5.6
7.4–8.8
7.3–8.3
7.5–8.0* See Harris and Angal 1989, Scopes 1987, and Snyder and Kirkland 1979.

Buffer Preparation
When preparing buffers for ion exchange chromatography, it is importantthat excess conductivity is not produced during buffer pH adjustment. For
example, back-titration of Tris-HCl with NaOH elevates conductivity, which will
lower binding capacity. To prepare Tris buffer, begin with Tris base and titratewith Tris-HCl to the target pH. Apply the same principle with other buffers.
6 |Macro-Prep 25 Ion Exchange Resins

Section 6 Operation and MaintenanceColumn Equilibration
To equilibrate the column, wash with 1–2 CV of buffer containing 0.5–1.0 M of
the buffer salt used in the start buffer, then follow with 3 CV of the start buffer
or until pH and conductivity are stable.

Sample Preparation
Adjust salt concentration and pH as necessary to achieve desired selectivity
for binding of target or contaminants. If sample conductivity exceeds
conductivity of the column equilibration buffer, sample capacity may
be reduced or the target molecule may not bind. Adjustment of pH andconductivity of sample can be done by dilution or buffer exchange.

Sample Load and Adsorption
The sample load is determined empirically by loading and evaluating
breakthrough of the molecule of interest. Sample volume is not a criticalfactor. Large volumes of dilute feed such as cell culture supernatant andclarified lysates may be loaded onto the supports without prior concentration.

Wash-ThroughWhen operating in a bind-elute mode, after loading the sample onto
the column, follow with 2–5 CV of the equilibration buffer. This will washout unbound contaminants. In flow-through mode, after sample iscompletely through the column, regenerate the resin as described under
Regeneration below.

ElutionElute target molecules with a step or a linear gradient. The salt concentration
at which the desired product elutes is predetermined at small scale. With thisknowledge, the pH and salt concentration used in wash-through are adjusted
to eliminate the maximum amount of contamination before starting elution of
the target.

Regeneration
After each run, the packed bed should be washed with 2–4 CV of a high salt
buffer (0.5–2.0 M) to remove remaining bound material. Samples may thenbe loaded onto the column after reequilibration in starting buffer. If column
no longer yields reproducible results, the resins may require more thoroughcleaning and sanitization.

Macro-Prep 25 Ion Exchange Resins |7

Section 6 Operation and Maintenance

Cleaning-in-Place (CIP)
Acceptable CIP agents include 0.4 M NaCl in 1% acetic acid/1% phosphoricacid, up to 30% acetic acid, 1% Triton X-100, up to 70% ethanol or up to 30%
isopropyl alcohol, 8 M urea, and 6 M guanidine-HCl. Any of these agents canbe combined in an appropriate cleaning protocol, which is often developed
empirically. As a general guide, we recommend the following:
1.	 Use high salt buffer for regeneration, as above.
2.	 Remove additional contaminants with 0.4 M NaCl in 1% acetic acid/
1% phosphoric acid (3–5 CV) at 100 cm/hr.
3.	 For aggregated or precipitated proteins, or when dirty feedstock (forexample, crude lysate) has been used, wash with 3–5 CV of 6 M
guanidine-HCl or 8 M urea at 100 cm/hr.
4.	 For lipids or hydrophobically bound contaminants, wash with 0.1% Triton
X-100, or 20–70% ethanol or 20–30% isopropyl alcohol, or 10–30%
acetic acid. Use 3–5 CV at 100 cm/hr.
5.	 Finally, wash with 2 CV of deionized water and 4–5 CV of starting buffer.
Check the conductivity and pH of the effluent to verify that the column isequilibrated in the starting buffer before loading the sample.

Sanitization and Storage
To sanitize and store between campaigns, wash the column with 3–5 CV of
1% acetic acid/1% phosphoric acid, pH 1.5. Store the column at 4–40°C.
When not in use, store the Macro-Prep 25 Ion Exchange Resins in either
1% acetic acid/1% phosphoric acid (pH 1.5) or in 20% (v/v) ethanol solution orin 2% benzyl alcohol. The Macro-Prep 25 Ion Exchange Resins may also be
autoclaved at 121°C, 2 bar, in a neutral pH slurry, for up to 30 min and stored
in one of the above solutions.

Shelf Life
The Macro-Prep Ion Exchange Resins are stable for at least one year whenstored sealed in the original container at room temperature.

8 |Macro-Prep 25 Ion Exchange Resins

Section 7 Technical Assistance

Section 7
Technical Assistance
For additional information and technical assistance, contact your local Bio-Rad
representative. In the USA, call 1-800-4BIORAD (424-6723). Visit ourchromatography website at bio-rad.com/process for online productinformation.

Section 8
References
Harris ELV and Angal S (1989). Protein Purification Methods:
A Practical Approach (Oxford: IRL Press).
Scopes RK (1987). Protein Purification: Principles and Practice(New York: Springer-Verlag).
Snyder LR and Kirkland JJ (1979). Introduction to Modern Liquid
Chromatography (New York: Wiley).

Macro-Prep 25 Ion Exchange Resins |9Section 9
Ordering Information
Catalog #Description

Macro-Prep 25 Q Resin
1530020
Macro-Prep 25 Q Resin, 10 ml
1530021
Macro-Prep 25 Q Resin, 50 ml
1530022
Macro-Prep 25 Q Resin, 200 ml
153-0024
Macro-Prep 25 Q Resin, 5 L
Macro-Prep 25 S Resin
1530030
Macro-Prep 25 S Resin, 10 ml
1530031
Macro-Prep 25 S Resin, 50 ml
1530032
Macro-Prep 25 S Resin, 200 ml
Bio-Scale MT Column
7510081
Bio-Scale MT2 Column, 7 x 52 mm, 1.9–2.3 ml
7510083
Bio-Scale MT5 Column, 10 x 64 mm, 4.6–5.7 ml
7510085
Bio-Scale MT10 Column, 12 x 88 mm, 9.5–11.3 ml
7510087
Bio-Scale MT20 Column, 15 x 113 mm, 19.4–21.9 ml
Bio-Rad, Bio-Gel, Bio-Spin, Econo-Pac, and Macro-Prep are trademarks of Bio-Rad Laboratories, Inc.
in certain jurisdictions.
All trademarks used herein are the property of their respective owner.

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