Showa Denko Shodex C18U 2B User manual

Operation Manual Shodex C18U 2B, 2D Version 20191015E
Operation Manual
Shodex™ C18U 2B, 2D
Europe, Middle East, Africa, Russia
Showa Denko Europe GmbH
Shodex Business
Konrad-Zuse-Platz 3
81829 Munich
Germany
www.shodex.de
Technical Support:
[email protected]
+49 (0)89 / 93 99 62 37
Quotes and Orders:
[email protected]
+49 (0)89 / 93 99 62 34

Operation Manual Shodex C18U 2B, 2D Version 20191015E
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Operation Manual
Shodex C18U 2B, 2D
(Please read this manual carefully to achieve the best and consistent column performance for a long time)
Important Handling Instructions
Caution! • Please consult the Safety Data Sheet (SDS) of reagents and solvents used with the
column and understand their proper handling methods to prevent potential health
hazards or death from occurring.
• Please wear appropriate personal protective equipment such as lab goggles and
gloves when handling organic solvents and acid and alkaline reagents. Avoid any direct
physical contact to prevent chemical injuries.
Before Using the Column
(1) Please visually inspect the package and outside of the column for any damage.
(2) Please check if product name and serial number written on the column package, column name tag,
and enclosed Certificate of Analysis (CoA) are matching and correct.
1. Introduction
Thank you for purchasing the Shodex™product.
Shodex™C18U series is reverse phase chromatography column in which octadecyl group is bonded to
organic and inorganic hybrid gel. Compared to conventional silica-based reversed phase columns, it
has excellent separation performance and high durability, so it can be used under a variety of analytical
conditions and is suitable for the separation of a wide variety of compounds.
2. Column Components

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3. Device Settings
The C18U series is a column for ultra high performance liquid chromatography (UHPLC) packed with
1.9 μm particles. Pay attention to the pressure resistance of the pump and connecting tubing.
Generally, it is suitable for use in UHPLC compatible systems with a pressure resistance of about
60 MPa or higher. Sample diffusion in the flow path of device (diffusion outside the column) affects
the column performance greatly. Optimize the operating condition of the device as shown below.
(1) Keep the tubing from the injector to the column and from the column to the detector as short as
possible, use tubing with small inner diameters (0.15 mm I.D. or less).
(2) Use a semi-micro injector or micro injector and minimize the sample loop.
(3) Use a low-volume type flow cell for the detector, such as semi-micro or micro type. The response
of the detector should be 0.1 sec or less and the data sampling rate should be 10 points / sec or more
to detect the earliest eluting sharp peak properly.
4. HPLC System Preparation
Wash entire LC system prior to the column installation, including all flow-lines and sample loop by
switching the valve, and then replace the washing solution with the eluent to be used. When desired
new eluent has low miscibility/solubility to the eluent of previous analysis, first use the eluent that is
miscible/soluble to both eluents, and then replace it with the desired eluent.
(Example) When replacing buffer solution (or salt solution) to acetonitrile/water, first run 100 % pure
water and then replace it with the desired eluent.
Attention! • If the eluent left in the system is not compatible with the column to be used, it may
damage the column.
• A drastic change in the eluent compositions may remove substances adsorbed on
the system and they may enter and deteriorate the column.
5. Column Installation
(1) Connect the column to LC system by following the “flow direction arrow” () indicated on the
column name tag. If guard column is used, position the guard column in front (before the inlet) of the
analytical column.
(2) Make sure to insert the tubing all the way to the end fitting and secure it with the male nut. It is
important that there is no extra space between the tubing and the column side of the end fitting.
Presence of an extra space will let the sample to spread out and may result in wide peaks.
(3) Set the initial flow rate at less than 0.2 mL/min and start the system. If using the column at an
elevated temperature, keep a low flow rate until the temperature of the column reaches to the set
temperature, and then gradually increase the flow rate to the desired value.

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Caution! • Verify that there is no solvent leak. It may cause electronic leakage, rust, and/or
chemical injury.
Attention! • Make sure not to let air bubbles enter the column while installing the column. The
air bubbles may damage the column.
• When restarting the system after column installation or after holding the eluent
flow, start the system at half of the recommended flow rate. A rapid increase in
pressure can damage the column.
• If the column was used at an elevated temperature, lower the flow rate to less than
half of the recommended flow rate at the end of analysis. Then, turn off the column
oven, and let the column temperature return to room temperature before stopping
the pump. If the pump was stopped while the eluent inside the column was still hot,
as the eluent temperature decreases, its volume also decreases. This may result in
creating an empty space in the column and deteriorates the column.
6. Column Specifications
Product Code
Product Name
Column Size
ID x Length
Particle Size
Carbon Load*
Pore Size
F6654011
C18U 2B
2.0 x 50 mm
1.9 µm
20 %
120 Å
F6654012
C18U 2D
2.0 x 150 mm
1.9 µm
20 %
120 Å
* Includes 8 % carbon content of organic and silica hybrid gel
Base Material: Spherical organic and silica hybrid particles modified with
octadecyl groups (C18)
Column Housing: SUS-316 (stainless steel)
Screw type: Internally-threaded type No. 10-32 UNF (Parker Type)
Shipping solvent: Acetonitrile
The guard column for the C18U series
Product Code
Product Name
Content
F6709801
EXP® cartridges
3 Cartridges
F6709802
EXP® direct connect holder
1 Holder (with 2 titanium hybrid ferrules and 1 nut)
7. Usable Conditions
Product
Name
Recommended Flow
Rate (mL/min)
Maximum
Pressure (MPa)
pH
Range
Temperature Range (°C)
C18U 2B
0.2 to 0.8
100
1 to12
90°C (pH 1 to 7) / 50°C (pH 7 to 12)
C18U 2D
0.2 to 0.8
100
1 to 12
90°C (pH 1 to 7) / 50°C (pH 7 to 12)
For details on the guard column for the C18U series, see the instruction manual that comes with the
guard column.

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Attention! • Use the column within above stated flow rate, pressure, and temperature ranges.
Using the column outside the given range may damage the column and lower its
performance.
• Continuous use near the upper limit of the operating pressure and sudden pressure
changes can shorten column life.
8. Eluents
(1) It is possible to use water, 100 % each of organic solvents, and a mixture of water and organic
solvents in any proportion. Organic solvents that can be generally used include acetonitrile, methanol,
and tetrahydrofuran (THF).
(2) Degas the eluent fully to prevent the formation of air bubbles.
(3) Presence of small debris or insoluble substances may result in deterioration of the column and/or
they appear as noise on the chromatograms. Filter the eluent with a 0.2 μm disposable filter to prevent
the problems.
(4) Check miscibility/solubility of the desired new eluent and eluent previously used in the system. If
the new eluent has low miscibility/solubility to the eluent previously used, first use the eluent that is
miscible/soluble to both eluents, and then replace it with the desired eluent.
(Example) When replacing buffer solution (or salt solution) to acetonitrile/water, first run 100 % pure
water and then replace it with the eluent.
(5) Start the system at less than 0.2 mL/min when replacing the solvent.
(6) Use of viscous solvents will increase the column pressure. Make sure to adjust the flow rate and/or
column temperature so that thecolumn pressure remains below the maximum column pressure stated
in section “7. Usable Conditions”.
(7) As the eluent composition changes during the gradient elution, it may increase the column pressure
to above the column’s maximum pressure. Make sure to adjust the flow rate and/or column
temperature so that the column pressure remains below the maximum column pressure.
Attention! • Always use freshly prepared solvents. Solvents stored for a long time may have
changed their compositions and may influence elution patterns and/or damage the
column.
• Frequent solvent replacement can deteriorate the column, and thus not
recommended.
• Generally, the higher the column temperature, buffer solution and additive
concentration, and the lower the organic solvent concentration, there is a risk of
deterioration.
Note • Use of on-line degasser is recommended.
•It is recommended to set the pump limiter to avoid exceeding the maximum
pressure.
• In the case of long-term use in alkaline conditions, it is recommended that using low
concentration organic buffer such as 1-10 mM trimethylamine or glycine at low
temperature (30°C or less). The recommended organic solvent is methanol.

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9. Sample Preparation
(1) If possible, use the eluent for analysis to dissolve or dilute the samples. If this is difficult, use a
solvent which has a composition that is as close as possible to the eluent's composition. When gradient
elution is used, it is recommended to use the initial eluent to prepare the sample.
(2) Filter the sample solution using disposable 0.2 μm filter to prevent the column from clogging or
deteriorating.
(3) Recommended sample injection volume is less than 10 μL per column.
(4) For samples containing polymer compounds such as proteins and polysaccharides and samples with
a lot of impurities, pretreatment (clean up) by solid phase extraction or the like in advance.
Note • Use of guard column is recommended to protect the analytical column.
10. Column Cleaning
Problems of peak shapes and elution timing changes or elevated column pressure etc. are often caused
by insoluble or adsorbing components in the eluent and reagents being deposited inside the column.
These problems may be resolved by cleaning the column. If a guard column is used with the analytical
column, first remove the guard column and check the performance of the analytical column alone. If
the problem is solved, most likely the cause is from the guard column. In this case, clean the guard
column. If the problem is not resolved after the cleaning, replace the guard column with a new one. If
the problem is not solved by removing the guard column, clean both guard and analytical columns.
(1) Insoluble components that block the column inlet may be removed by reversing the direction and
introducing the eluent from the column outlet with the flow rate at less than half of the recommended
flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the
direction and waste the washing solution directly from the column outlet into the waste container
without the detector and reduce the flow rate at less than 0.2 mL/min. A suggested cleaning solvent
volume is 5 - 10 times the column volume. Make sure to clean the guard and the analytical columns
separately.
(Example 1) [After using eluent not containing buffer salts/additives]
Wash the column with solution containing a higher ratio of organic solvent that constitutes
the eluent. A cleaning solution containing THF might be effective when removing highly
hydrophobic (lipid-soluble) substances that are adsorbed onto the gel.
(Example 2) [After using mobile phase containing buffer salts/additives]
First replace with a water/organic solution containing no buffer salts or additives (A ratio of
water to organic solvent should be set at the same proportion as eluent). Then flush the
column in accordance with the method described above.
Attention! • Flushing with 100 % water after using the column around the pH limit might shorten
column life, so flush the column with water/organic solution.

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11. Column Storage
Remove the column from the system after replacing the in-column solvent with the shipping solvent.
Securely tighten the end caps and store the column in a location with stable temperature (a cool and
dark space is recommended). Please refer to section “8. Eluents” when replacing the eluent.
Attention! • Never allow inside of the column to dry. It can damage the column.
12. Additional Warnings
(1) Do not remove end fittings.
(2) Do not make a strong impact on the column. Do not drop or hit the column on a hard surface.
Refer to the Shodex website (www.shodex.de) for product details and their applications.
Contact through the Shodex website or Shodex partners for additional assistance.

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13. Warranty (Version 4)
(1) Showa Denko K.K. warrants that the Shodex™product, at the time of delivery to the user, will
conform to the specification of the attached CERTIFICATE OF ANALYSIS, if the Shodex product is used
in accordance with the attached operating manual. The foregoing warranty is exclusive and is in lieu
of all other warranties with respect to the Shodex product, whether written, oral, implied, statutory or
otherwise. No warranties by Showa Denko K.K. are implied or otherwise created, including, but not
limited to, the warranty of merchantability and fitness for particular purposes.
(2) Any claim of inconformity to the specification must be notified to Showa Denko K.K. within ten (10)
days after delivery to the user. User’s exclusive remedy and Showa Denko K.K.‘s exclusive liability for
such claim are limited to the replacement of the Shodex product in question. In no event is Showa
Denko K.K. liable for any indirect, incidental or consequential damage arising out of in connection with
the Shodex Column, whether or not such damage is allegedly based on breach of warranty, negligence
or otherwise.
(3) No warranty is made in any of the following cases:
3-1 If the Shodex product is not used in accordance with the operating manual
3-2 If the Shodex product is remodelled by anyone other than person or firm designated by Showa
Denko K.K.
3-3 If the Shodex product is disposed of
3-4 If the Shodex product is resold by the user without giving prior written notice to Showa Denko K.K.
3-5 If the performance of the Shodex product is not conform to the specification of the attached
CERTIFICATE OF ANALYSIS due to any of the reasons below:
a) Computer virus
b) Impurities contained in the sample, reagent, gas air or cooling water provided by the user
c) Breakdown or malfunction of equipment, apparatus or component used in combination with the
Shodex product
d) Force majeure such as fire, earthquake, flood, other natural disaster, crime, riot, act of terrorism,
war or radioactive contamination
(4) In no event is Showa Denko K.K. liable for
4-1 the results of analyses or preparations using the Shodex product or any portion of the same,
including, but not limited to, the reliability, accuracy, efficacy and safety of said results, and
4-2 the occupational hazard in the use of the Shodex product, whether or not such use is made in
accordance with the attached Conditions for use.
(5) The Shodex Column is for laboratory use only. It must not be used for clinical diagnosis. Showa
Denko K.K. is not liable for any use of the Shodex product except laboratory use.
Shodex is a registered trademark of SHOWA DENKO K.K.
EXP is a registered trademark of Optimize Technologies, Inc.