TROUBLESHOOTING
BIA Separations, GmbH
Problem
Possible source
Action
• Increased back
pressure
Blocked disk, retaining fitting,
mixing tee, capillaries,
or detector cell
• Exchange the disk, capillary
or tee, wash the retaining
fitting with deionized water under
sonication.
• Clear out the detector cell
• Leakage from
the housing
Screw caps not tightened
enough
• Carefully tighten screw caps
O-rings damaged: Use of
strong organic solvents
• Exchange the O-rings
• Leakage at the
inlet to or at the
outlet from
the housing
Capillary fitting not tightened
enough
• Carefully tighten the fitting
Damaged threads on the
retaining fitting
• Change the retaining fitting
• O-ring falls out
O-ring swelling from using
strong organic solvents
• Try to reinsert the O-rings
into the retaining fittings. If the
O-ring will not fit, then replace
the O-rings
• Disk Changes
Color
Precipitate from the Sample
• Follow CIP Procedure in the
Product Specific Information Sheet
• Poor Resolution
(i. e. Wider
Peaks)
Various
• Perform Regeneration
and/or CIP Procedure in the
Product Specific Information Sheet
• No Apparent
Separation
Various
• Check the pH and the
composition of the buffer.
• Verify that the disk chemistry is
appropriate for your application
• Poor or no
Baseline
Separation
Various
• Check the pH and the
composition of the buffer.
• Check the HPLC System (too much
dead volume in the mixing
chamber or capillaries)
• Make the Gradient Flatter (e.-g.
0–100% buffer within 5-minutes)
• Increase the Flow Rate
CIM® DISK MONOLITHIC COLUMN
0.34 mL
INSTRUCTION MANUAL
ABOUT CIM® DISK MONOLITHIC COLUMN
Thank you for purchasing the CIM® Disk Monolithic Column. CIM® Disk
Monolithic Column consists of a rigid, highly cross-linked monolithic polymer
disk and an appropriate housing. The housing is designed according to the
Sound Engineering guidelines. The monolithic column is an innovative
chromatographic and bioconversion support that offers new ways of
performing chromatography. It can be used for the separation removal of
large molecules such as proteins, DNA, viruses, endotoxines and also for
smaller molecules such as peptides and oligonucleotides. CIM® Disk
Monolithic Column operates at high flow rates, low back pressures, and is
extremely easy to use. The advantageous features of CIM® Disk Monolithic
Column include:
• Flow-unaffected resolution
• High dynamic binding capacity unaffected by flow rate
• Extremely fast separations
• Low back pressure
• User-exchangeable monolithic support
• User-adjustable column length
• Easy to use
For any additional information please contact us:
If this has not solved your problem, please contact us at
tech-support@monoliths.com.
BEFORE YOU BEGIN
EUROPE
USA
SALES:
BIA Separations d. o. o.
Mirce 21
5270 Ajdovščina
Slovenia, EU
Tel.: +386-5-9699-504
Fax.: +386-5-9699-599
sales@biaseparations.com
www.biaseparations.com
BIA Separations, Inc.
1000 N. West St. Suite 1200
Wilmington, DE 19801 USA
Phone: 1-888-520-4CIM (4246)
Fax: 1-302-861-0362
sales@biaseparations.com
orders@monoliths.com
www.biaseparations.com
RETURN OF THE COLUMN
CHINA
SALES:
The returned Column should be accompanied with completed
Return Form (www.biaseparations.com).
sales@biaseparations.com
www.biaseparations.com
ORDERING INFORMATION
For easy ordering, please visit our website at
www.monoliths.com/catalog or simply send us an email to
sales@biaseparations.com.
Information and specifications contained herein are, to the best of our knowledge, accurate and
represented in good faith. They are intended to help you start working with the new separation technology
and are subject to change without notice. BIA Separations shall not be liable for errors contained herein or
for incidental or consequential damages in connection with the performance or use of CIM®.
For more information on our products, visit our home page at: http://www.biaseparations.com and
http://www.monoliths.com or contact your local distributor. We reserve the right to alter specification,
details, etc. without prior notice or liability.
© 2015 by BIA Separations
5
Publication #: IM-D-1506
6
IMPORTANT: Use the product according to guidelines in this Instruction
Manual. Improper use may result in malfunction, personal injury, or damage
of the product or material. Follow safety instructions, wear gloves, safety
glasses, and a lab coat during operation. For more detailed information refer
to the Material Safety Data Sheet.
Unpacking
Carefully inspect the purchased products for any damage that may have
occurred during shipping. Immediately report any such damage to your
vendor and the transport company.
CIM® Disks are shipped in a suitable storage solution at room temperature.
Upon arrival they need to be stored at +4 (39 °F) to +8 °C to (46 °F) until use.
WARNING: Do not store the CIM® Disks below 0 °C (32 °F).
ATTENTION: Occassionally, some color variation may be observed on CIM®
housings. The variation in color does not affect neither the quality of the
housing material nor the column performance.
Optimization of HPLC System for Efficient Separations
The set-up of the HPLC system is one of the crucial factors in achieving optimal
performance from CIM® Disk Monolithic Columns. Since CIM® Disk
Monolithic Columns are commonly used for gradient separations, the HPLC
system should include at least two HPLC pumps connected via capillaries and
a mixing tee to the injection valve or autosampler, a filling loop for the sample
injection, a column, a detector, and a recorder or data acquisition system.
1
Each of these components has a direct influence on the separation quality
when fast analyses are required.
Capillaries: The inner diameter of the capillaries strongly affects the peak
shape. Using smaller diameters results in sharper peaks, increases the quality
of the separation, and raises the back pressure of the LC system. Small
diameter capillaries also have smaller volume, thus reducing the total
system dead volume. Capillaries with inner diameter of 0.5 mm or less are
recommended.
Back pressure: Check the back pressure of the system at a flow rate that is up
to 2 ml/min higher than the flow rate that will be used during the actual work.
It is advisable that the back pressure of the system alone is below 20 bar
(2 MPa).
Mixing tee: A low dead volume mixing tee, (e. g. 10 µl), should be used for
fast gradient separations. This will improve the peak quality and resolution.
Detector: To obtain an optimal performance from CIM® Disk Monolithic
Column, the detector response time should be set to the lowest possible
value – which is 0.1 s for most UV detectors.
Acquisition rate: The acquisition rate depends on the analysis time. Typical
analysis times in the case of CIM® Disk Monolithic Column is just few
minutes. To obtain optimal resolution for separations of less than 1 minute,
the acquisition rate should be 5 to 10 Hz.
The HPLC system is now ready for fast analyses!
PREPARATION OF CIM® DISK MONOLITHIC COLUMN
FOR USE
CIM® Disk Monolithic Column consists of a CIM® Disk (Figure 1), a housing
(Figure 2) and a disk extractor (Figure 3). The chemistry of the disk is
determined by the ring color.
Nonporous Fitting Ring (b)
Monolithic Matrix (a)
Figure 3: Disk extractor
Assembly of CIM® Disk Monolithic Column
(Refer to Figure 2)
1. Unscrew one of the screw caps (e) from the housing.
2. Remove one of the retaining fittings (d).
3. Remove CIM® Disks from the container with a pincette and place it gently into
the housing cylinder (c). Note: Up to 4 CIM® Disks of the same or different
chemistries (e. g. ion-exchange and affinity) can be inserted into one housing
cylinder (c).
4. Reinsert the retaining fitting (d) to fix the disk approximately in the middle of
the housing cylinder (c).
5. Tighten both of the screw caps (e) by hand.
6. Connect the CIM® Disk Monolithic Column to the HPLC system preferably using
PEEK fingertight fittings (1/16” OD UNF 10-32). The flow direction is not
important due to the symmetrical design of the column.
Equilibrating CIM® Disk Monolithic Column
Before you begin working with the CIM® Disk Monolithic Column the product
should be equilibrated and the flow characteristics and backpressure checked in
accordance with the following procedure:
1. Wash the CIM® Disk Monolithic Column with at least 10 column volumes of
the binding mobile phase (e. g. for ion-exchange chromatography a low
ionic strength buffer) at one-half of the working flow rate. Note: Allow 2
column volumes to flow into a waste container. This will remove any small
particles or air bubbles that may effect the detector cell.
2. Wash the CIM® Disk Monolithic Column with at least 10 column volumes of
the eluting mobile phase (e.g. for ion-exchange chromatography a buffer
containing 1–2 M NaCl) at one-half of the working flow rate in order to elute
any bound components and impurities.
3. Finally, wash the CIM® Disk Monolithic Column with at least 10 column
volumes of the binding mobile phase at a working flow rate.
4. The CIM® Disk Monolithic Column is now ready for use.
Exchanging the Column Chemistry
Figure 1: CIM® disk
Disk Housing Cylinder (c)
Screw Caps (e)
Blind Fittings (f)
One of the unique features of CIM® Disk Monolithic Column is the ability to
quickly and easily exchange the monolith stationary phase. The exchange is
performed as follows:
1. Stop the pumps and disconnect the column from the HPLC/LC system.
2. Remove both of the screw caps (e) and retaining fittings (d).
3. Remove the disk from the housing cylinder (c) using the disk extractor
(Figure 3).
4. Place CIM® Disk in the labeled container with a suitable storage solution.
5. Insert the new CIM® Disk according to the assembly procedure (See above).
Operating Conditions
Figure 2: Housing (POM or PEEK) for the CIM® Disk Monolithic Column
1. Maximum Number of Disks per housing: 4.
2. Operating Flow Rates: up to 6 ml/min. Note: Typical flow rate is 2–4 mL/min.
3. Operating Back Pressure: up to 50 bar (5 MPa) WARNING: Exceeding 5 MPa will
2
3
Retaining Fittings (d)
seriously damage the column!
4. Mobile Phases and Organic Solvents:
• Columns using CIM® Disk Housing (POM) (Cat. #: 222.0830)
1. Water-based phases within the pH range of 2–12 and at various
ionic strengths (e. g. 6 M urea).
2. Organic solvents, such as acetonitrile should be avoided since
they will seriously damage the column housing.
• Columns using CIM® Disk Housing (PEEK) (Cat. #: 222.0850)
1. Water-based phases within the pH range of 1–13 and at various
ionic strengths (e. g. 6 M urea).
2. Concentrated organic solvents (e. g. in reversed phase chromatography), such as acetonitrile, or concentrated acid solutions can
be used without any limitations.
5. Operating Temperatures: up to 40 °C (104 °F); up to 25 °C (77 °F) for
protein disks.
NOTE: CIM® Disk Housing (PEEK) (Cat. #. 222.0850) can be thermally
sterilized in the autoclave.
WARNING: Exceeding 40 °C (104 °F) will seriously damage your
column!
CARING FOR CIM® DISK MONOLITHIC COLUMN
To extend the life of the CIM® Disk Monolithic Column, please observe the
following guidelines:
• Always use freshly prepared mobile phases (buffers).
• Always filter your mobile phases (buffers) and samples through a 0.22 µm
filter.
Cleaning and Regeneration Procedure for CIM® Disk
Monolithic Column
Please refer to the Product Specific Information Sheet which accompanied
these instructions.
Storage of CIM® Disk Monolithic Column
Up to 2 days of planned storage after column use:
• Equilibrate the CIM® Disk Monolithic Column according to the procedure
described in Equilibrating the CIM® Disk Monolithic Column (Page 3).
• Carefully seal the ends of the column with blind fittings to prevent
drying.
• Store the column at +4 °C (39 °F) to +8 °C (46 °F).
3 or more days of planned storage after column use:
• Perform CIP procedure as described in Product Specific Information
Sheet.
• Wash the column with pH neutral low ionic strength buffer (e.g. 20 mM
Tris, pH 7.4) for at least 30 min at 1 mL/min.
• Flush the column with at least 10 column volumes of deionized water at
a working flow rate.
• Wash the column with 10 column volumes of appropriate storage
solution (e.g. 20 % ethanol for DEAE, QA and SO3 chemistry or 10 mM
NaOH for C4 chemistry) at a working flow rate.
• Remove the disk from the housing and store it at +4 °C (39 °F) to +8 °C (46
°F).
WARNING: Never let the CIM® Disk Monolithic Column dry out!
WARNING: Do not store the CIM® Disks below 0 °C (32 °F).
4