Bio-Scale™ Mini
UNOsphere™ Q, S and Rapid S
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4100, 732-4102, 732-4400,
732-4104, 732-4110, 732-4401,
732-4112, 732-4114, 732-4402
For technical support, contact your local Bio-Rad office, or in the
U.S., call 1-800-4BIORAD (1-800-424-6723).
Table of Contents
Section 1...Introduction. .......................................1
Section 2...Connection to Bio-Rad’s Low-
..............Pressure Chromatography
..............Instruments ........................................5
Section 3 ..Connection to Other Liquid
..............Chromatography Systems................10
..............3.1 BioLogic DuoFlow™
Systems.................................11
3.2 HPLC Systems ......................11
3.3 FPLC Systems .......................12
Section 4 ..Preparing a Cartridge For Use..........13
..................4.1 Sample Preparation ...............14
..................4.2 General Purification Protocol..15
..................4.3 Scaling Up the Separation .....16
Section 5 ..Care of the Cartridge........................19
..................5.1 Autoclaving ............................20
..................5.2 Storage..................................20
Section 6 ..Technical Assistance ........................21
Section 7 ..Ordering Information.........................22
Section 8 ..References .......................................24
Section 1
Introduction
Bio-Scale™ Mini cartridges have a patent-pending,
double-wall design that provides extra durability and
allows easy, reliable runs with aqueous buffers most
commonly used for protein purification. The
polypropylene Luer fittings and internal sealing
surfaces assure leak-free operation at pressures up
to 45 psi. The cartridges are convenient, disposable,
and supplied ready for use. Cartridges are available
for a variety of chromatographic techniques
including desalting, ion exchange, and affinity
chromatography. See Ordering Information Section 7
for a listing of the complete Bio-Scale Mini
cartridge product line. The design of the Bio-Scale
Mini cartridges offers:
1
• Ready-to-go convenience; simply equilibrate the
cartridge in the buffer of choice
• Luer fittings for convenient connection to any
chromatography system or directly to a Luer-Lok
syringe
The Bio-Scale Mini UNOsphere™ Q, S, and Rapid S
cartridges are packed with UNOsphere ion
exchange supports. These supports are based on
hydrophilic spherical, polymeric beads designed for
the purification of proteins, nucleic acids, viruses,
plasmids, and other macromolecules. UNOsphere
beads are designed to provide high capacity, low
back-pressure, and high productivity. Detailed
product information is given in Tables 1 and 2.
2
Table 1 Bio-Scale Mini UNOsphere Q, S, &
Rapid S Cartridge Specifications.
Sizes 1 and 5 ml bed volumes
Dimensions 1 ml: 40 x 5.6 mm inner diameter
5 ml: 40 x 12.6 mm inner inner diameter
Maximum pressure tolerance 45 psi
Recommended flow rates 1 ml: 1–2 ml/min (240–480 cm/hr)
5 ml: 5–10 ml/min (140–480 cm/hr)
Maximum flow rate 1 ml: 4 ml/min (970 cm/hr)
5 ml: 20 ml/min (963 cm/hr)
Fittings Female Luer fitting inlet and
Column material Polypropylene
Frit material Polyethylene (HDPE)
Shipping condition 20% ethanol
Storage recommendation 20% ethanol
Autoclavability Not autoclavable
male Luer fitting outlet
3
Table 2 UNOsphere Q, S, and rapid S
Specifications
UNOsphere Q UNOsphere S UNOsphere Rapid S
Type of ion exchanger Strong anion Strong cation Strong cation
Functional group -N+(CH
Total ionic capacity 120 µeq/ml Ni2+260 µeq/ml 140 µeq/ml
Dynamic binding capacity*
At 150 cm/hr 180 mg BSA/ml 60 mg IgG/ml 60 mg/ml
At 600 cm/hr 125 mg BSA/ml 30 mg IgG/ml 30 mg/ml
Shipping counterion Cl
Median particle size 120 µm 80 µm 100 µm
Recommended 50–1,200 cm/hr 50–1,200 cm/hr 50–800 cm/hr
linear flow rate
Chemical stability
1.0 M NaOH (20ºC) Up to 10,000 hr Up to 10,000 hr Up to 800 hr
1.0 M HCl (20ºC) Up to 200 hr Up to 200 hr Up to 200 hr
pH stability 1–14 1–14 1–14
Antimicrobial agent 20% ethanol 20% ethanol 20% ethanol
Regeneration 1–2 M NaCl 1–2 M NaCl 1–2 M NaCl
Storage conditions 20% ethanol or 20% ethanol or 20% ethanol or
* 10% breakthrough capacity determined with 4.5 mg/ml human IgG (UNOsphere
Rapid S) and 2.0 mg/ml BSA (UNOsphere Q) in a 1.1 x 20 cm column.
3)3
-
0.1 M NaOH 0.1 M NaOH 0.1 M NaOH
-SO
Na
3
+
4
-SO
Na
3
+
Section 2
Connecting to Bio-Rad's
Low-Pressure
Chromatography
Instruments
The Bio-Scale™ Mini cartridges are ideal for use with
Bio-Rad's BioLogic™ LP system, Econo™ Gradient
Pump, and Model EP-1 Econo pump, and all lowpressure chromatography instruments. Bio-Scale
Mini cartridges can be conveniently connected
directly to the system using the Luer lock fittings on
the cartridge.
1. Install 1.6 mm ID tubing in the pumphead.
Adjust platen pressure screw (on pumphead).
Using a screwdriver or coin, turn the screw
counterclockwise as far as it will go, then turn
clockwise three full turns. Assemble with fittings
and lock rings as shown in Figure 1.
5
(Use orange lock rings and medium size barb
fittings with 1.6 mm tubing.)
Platen pressure
screw
See detail
2 3
1
9
456
7 8
0
C .
Alarm
Fig. 1. Biologic LP setup
6
Lock ring
Tubing
Luer fitting
2. To maximize gradient accuracy and apply
samples efficiently, install 1.6 mm ID tubing from
the pump to the MV-6 sample inject valve (if
available). If using the MV-6 sample inject valve,
turn the knob counterclockwise as far as it will
go so it will now correspond to the printed
diagram on the valve. (See Figure 2.)
SAMPLE LOOP
INJECT
PORT
MV-6
BIOLOGIC LP
SYSTEM OR ECONO
GRADIENT PUMP
TO
INJECT
VALVE
FILL
TO
COLUMN
WASTE
BIOLOGIC LP
SYSTEM OR ECONO
GRADIENT PUMP
Fig. 2. Connecting to a MV-6 valve.
7
SAMPLE LOOP
INJECT
PORT
FILL
MV-6
INJECT
TO
VALVE
"INJECT" POSITION"FILL" POSITION
TO
COLUMN
WASTE
3. Connect the inlet of the cartridge to the male
Luer fitting on the MV-6 sample inject valve. (See
Figure 2.) If not using the MV-6 sample inject
valve, connect a barb to male Luer fitting on the
1.6 mm ID tubing, then connect to the top of the
female Luer on the Bio-Scale mini cartridge. For
optimum performance, a cartridge should be
mounted vertically with the arrow on the
cartridge pointing downward.
4. Connect the cartridge outlet to the 1.6 mm ID
tubing leading to the BioLogic LP optics module
or Econo UV monitor. It is recommended to use
the shortest length (approximately 10 cm) of
1.6 mm ID tubing. Connect a barb to female
Luer to the 1.6 mm ID tubing, then connect to
the bottom of the male Luer on the Bio-Scale
mini cartridge.
8
Inlet fitting
Fig. 3. Column and fittings.
Outlet fitting
9
Section 3
Connecting to Other Liquid
Chromatography Systems
The Bio-Scale™ Mini cartridges can be connected
to any liquid chromatography system, provided that
the maximum pressure limit (3 bar, 45 psi, or
300 KPa) of the cartridges is not exceeded. It is
recommended that the system pressure limit be set
according to the cartridge pressure limit. Pressures
in excess of 3.4 bar are usually caused by
restrictions in tubing or detector cells downstream
from the cartridge. Bio-Rad offers two fittings kits for
easy connection of a Bio-Scale Mini cartridge to a
BioLogic DuoFlow™, HPLC-, or FPLC-type system.
10
3.1 BioLogic DuoFlow Systems
The Bio-Scale Mini cartridge to BioLogic system
fittings kit (catalog #732-0113), includes ¼–28
female to male Luer and ¼–28 female to female
Luer to connect one Bio-scale Mini cartridge to the
BioLogic DuoFlow system.
3.2 HPLC Systems
The Luer to 10–32 adaptor fittings kit (catalog
#732-0112), provides fittings necessary to connect
the cartridge to nut and ferrule type fittings found
on most HPLC systems. Alternatively, the cartridge
can be connected to HPLC systems via a low-dead
volume
1/16 in union with a new piece of stainless
steel tubing attached to the union. Simply slip a
short length of the 0.8 mm ID tubing over
1/16 in OD
stainless steel tubing to a distance of 1 cm.
11
3.3 FPLC Systems
The Luer to M6 adaptor fittings kit (catalog
#732-0111) provides fittings necessary to connect
the cartridge to the M6 fittings found on FPLC or
related systems.
Alternatively, connection can be made by using two
Upchurch P-621, ¼–28 to metric adaptors, one
Upchurch P-619, ¼–28 to male Luer, and one
Upchurch P-628, ¼–28 to female Luer. Assemble
the Luer fitting to the ¼–28 metric adaptors. Attach
the adaptor with the male Luer to the column inlet
line of the FPLC system and the adaptor with the
female luer to the FPLC column outlet. To prevent
tubing or cartridge failure, do not exceed the
maximum recommended flow rate.
12
Section 4
Preparing a Cartridge For
Use
Bio-Scale™ Mini UNOsphere™ cartridges contain
20% ethanol v/v as the storage solution. The fully
hydrated support is ready to use after equilibrating
the cartridge in the buffer of choice. To perform a
buffer exchange, connect the cartridge to a liquid
chromatography system or peristaltic pump and
condition it as instructed below:
1. Set pump flow rate to 3.0 ml/min (731 cm/hr) for
the 1 ml cartridge or 6.0 ml/min (288 cm/hr) for
the 5 ml cartridge.
2. Wash the cartridge with degassed low-salt buffer
for 2 min.
3. Wash the cartridge with degassed high-salt
buffer for 5 min.
13
4. Equilibrate the cartridge with low-salt buffer for
5 min.
5. Reduce the flow rate to the rate that will be used
in the purification protocol.
4.1 Sample Preparation
Proper pH and ionic strength is necessary for
consistent and reproducible results. Sample can be
exchanged into the starting buffer or diluted to the
starting buffer concentration. This can be achieved
by diluting the sample to the ionic strength of the
starting buffer, dialyzing against the starting buffer,
or exchanging it into the starting buffer. Buffer
exchange can be accomplished using the Bio-Scale
Mini P6 cartridge, Bio-Spin
columns, Econo-Pac
®
Bio-Gel
P-6DG gel filtration gel. The choice of
®
6 or Bio-Spin 30
®
10DG desalting columns, or
product will depend on sample volume. All samples
should be filtered through a 0.45 µm filter prior to
cartridge application.
14
Table 1 Products for Buffer Exchange.
Sample Recommended
Volume Product Use Catalog #
50–100 µl Bio-Spin 6 column Desalting proteins 732-6000
50–100 µl Bio-Spin 30 column Desalting proteins 732-6004
100 µl–3 ml Bio-Scale Mini P6 Desalting proteins 732-4502
cartridge 6 kD
Up to 3 ml Econo-Pac 10DG Desalting proteins 732-2010
desalting columns 6 kD
Unlimited Bio-Gel P-6DG gel Desalting proteins 150-0738
6 kD
30 kD
6 kD
4.2 General Purification Protocol
Ion exchange chromatography is usually performed
using increasing salt gradients or pH gradients to
elute the sample components. For best results, and
increased cartridge life, samples and buffers should
be degassed and filtered through a 0.45 µm filter.
15
Common buffers for cation exchange chromatography
are listed in Table 3.
An appropriate starting point for purifying samples is
a linear gradient from 0–0.4 M NaCl spanning
1–20 column volumes at 120 cm/hr, 0.5 ml/min for
the 1 ml cartridge, and 2.5 ml/min for the 5 ml
cartridge. The separation can be optimized by
changing the gradient profile. At the end of each run
the cartridge can be regenerated with 1.0 M NaCl
followed by starting buffer. Return to the desired flow
rate and proceed with the next separation.
4.3 Scaling Up the Separation
For quick scale up, two or three cartridges of the
same type can be connected in series. Backpressure
will increase with cartridges in series, so care
should be taken to maintain pressures £45 psi.
Bio-Scale Mini cartridges are available in 1 and 5 ml
cartridge format. The UNOsphere Q, S, and Rapid S
ion exchange media are also available in larger
16
amounts, from 25 ml bottles to bulk quantities for
scaling-up methods developed using the
cartridges. UNOsphere Q, S, and Rapid S media
are fully supported with regulatory support files. In
addition, Bio-Rad carries an extensive line of empty
chromatography columns from laboratory to
process scale.
17
Table 3 Common Buffers for Ion Exchange
Chromatography.
Type of Buffering
Cation Ion Exchanger Buffer Range
Acetic acid 4.8–5.2
Citric acid 4.2–5.2
HEPES 7.6–8.2
Lactic acid 3.6–4.3
MES 5.5–6.7
MOPS 6.5–7.9
Phosphate 6.7–7.6
PIPES 6.1–7.5
Pivalic acid 4.7–5.4
TES 7.2–7.8
Tricine 7.8–8.9
Anion
Bicine 7.6–9.0
Bis-Tris 5.8–7.2
Diethanolamine 8.4–8.8
Diethylamine 9.5–11.5
L-Histidine 5.5–6.0
Imidazole 6.6–7.1
Pyridine 4.9–5.6
Tricine 7.8–8.9
Triethanolamine 7.3–8.0
Tris 7.5–8.0
1,2,3
18
Section 5
Cleaning-in-Place (CIP) and
Sanitation
If a column no longer yields reproducible results, the
media may require thorough CIP and sanitation to
remove strongly bound contaminants. Acceptable
CIP agents include 25% acetic acid, 8 M urea, 1%
Triton X-100, 6 M potassium thiocyanate, 70%
ethanol, 30% isopropyl alcohol, 1 N HCl, 1 N
NaOH, and 6 M guanidine hydrochloride.
1. Sanitize the support in the column with 2–4 bed
volumes of 1.0 M NaOH at 50–100 cm/hr while
maintaining a minimum contact time of 40 min.
2. To reequilibrate the column, wash the column
with 2–4 bed volumes of 0.5–2 M NaCl solution
(containing 50–100 mM buffer).
19
3. If lipid removal is required, the column may be
washed with a 20–50% ethanol solution at
50 cm/hr.
5.1 Autoclaving
Bio-Scale Mini
®
cartridges are not autoclavable.
5.2 Storage
After washing the cartridges with deionized water,
Bio-Scale Mini ion exchange cartridges should be
purged and stored with PBS, containing 0.05%
NaN
, or in 20% v/v ethanol solution, and capped
3
for extended storage.
20
Section 6
Technical Assistance
For additional information and technical assistance,
contact your local Bio-Rad representative as listed
on the back cover of our catalog, or, in the USA,
call technical support at 1-800-4BIORAD.
21
Section 7
Ordering Information
Bio-Scale™ Mini Cartridges*
Description 5 x 1 ml 1 x 5 ml 5 x 5 ml
UNOsphere
UNOsphere S Support 732-4110 731-4112 731-4114
UNOsphere Rapid S Support 732-4400 732-4401 732-4402
Macro-Prep
Macro-Prep High S Support 732-4130 732-4132 732-4134
Macro-Prep DEAE Support 732-4140 732-4142 732-4144
Bio-Gel
Affi-Prep
Profinity
Affi-Gel
Affi-Gel Blue Support — 732-4642 732-4644
* For the most up to date list of cartridge offerings, please visit
• Larger package sizes of media are available for process scale
™
Q Support 732-4100 731-4102 731-4104
®
High Q Support 732-4120 732-4122 732-4124
®
P-6 Support — 732-4502 732-4504
®
Protein A Support 732-4600 732-4602 —
™
IMAC Support 732-4610 732-4612 732-4614
®
DEAE Blue Support — 732-4632 732-4634
us online at www.bio-rad.com/cartridges/.
chromatography. Inquire with your local Bio-Rad representative.
22
Fittings Kits
Catalog # Description
732-0111 Luer to M6 Adaptor Fittings Kit, includes Luer to
732-0112 Luer to 10–32 Adaptor Fittings Kit, includes Luer
732-0113 Luer to BioLogic™ System Fittings Kit, includes
M6 fitting to connect to an FPLC system
to polypropylene/PTFE 10–32 fittings to connect
1 cartridge to an HPLC system
¼–28 female to male luer and ¼–28 female to
female Luer to connect one cartridge to the
BioLogic DuoFlow™ system
23
Section 8
References
1. Harris ELV and Angal S, Protein Purification
Methods: A Practical Approach, IRL Press,
Oxford (1989)
2. Scopes RK, Protein Purification: Principles and
Practice (Second Edition), Springer-Verlag, New
York (1987)
3. Snyder LR and Kirkland JJ, Introduction to
Modern Liquid Chromatography (Second
Edition), Wiley, New York (1979)
4. Gagnon P, Avoiding Instrument-associated
Aberrations in Purification Scale-up and
Scale-down, BioPharm 10, 42–45 (1997)
24
FPLC is a trademark of GE Healthcare Group Companies. Luer-Lok
is trademark of Becton, Dickinson and Co.
Triton is a trademark of Union
Carbide Corp. Upchurch is a trademark of Upchurch Scientific.
25
Life Science
Group
Bulletin 0000 Rev A US/EG
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 4BIORAD
Australia 61 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11
Brazil 55 21 3237 9400 Canada 905 364 3435 China 86 21 6426 0808
Czech Republic 420 241 430 532 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84 0
Greece 30 210 777 4396 Hong Kong 852 2789 3300
36 1 455 8800 India 91 124 4029300 Israel 03 963 6050
Hungary
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99
Portugal 351 21 472 7700 Russia 7 495 721 14 04
Singapore 65 6415 3188 South Africa 27 861 246 723
Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55
Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000
00-0000 0000 Sig 0308
10005454 Rev E
Loading...