Shodex C18U 2B User manual

ver.210601E
SHOWA DENKO K.K. (https://www.shodex.com/)
1
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
3. Column Specifications
Product
Code Product Name Column Size (mm) Particle Size (μm) *Carbon (%)
I.D. Length
F6654011 C18U 2B 2.0 50 1.9 20
F6654012 C18U 2D 2.0 150 1.9 20
*Includes 8% carbon content of organic and silica hybrid gel
Base Material : Spherical organic and silica hybrid particles modified with octadecyl group
Column Housing : SUS-316
Screw Type : Internally-threaded type No.10-32 UNF (Parker Type)
Shipping Solvent : Acetonitrile

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4. Usable Conditions
Usable solvents are listed below.
(1) A mixture of water and organic solvents of any ratio can be used. Up to 100 % water, or organic solvents
can be used.
(2) Organic solvents that can be generally used include acetonitrile, methanol, and tetrahydrofuran (THF).
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.
· When using a mixture of buffer (or aqueous solution of salt) and organic solvent, make
sure there is no precipitation of salt.
· When using highly corrosive salts such as sodium chloride, wash out the salts at the end
of analysis. The metal parts of the devices and/or the columns may rust.
·Column pressure is influenced by the eluent composition, flow rate, and column
temperature. When changing the eluent compositions, adjust the flow rate and column
temperature so that the column pressure remains below the usable maximum pressure.
·Continuous use near the upper limit of the operating pressure and sudden pressure
changes can shorten column life.
· 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 · 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.
5. Eluent Preparation
(1) Degas the eluent fully to prevent the formation of air bubbles.
(2) 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.
Attention! · Whenever water is required, use ultra-pure water freshly generated by a water purification
system or water from a newly opened HPLC grade distilled water bottle. Use HPLC grade
organic solvent whenever possible. Solvents left in an opened bottle for a long time should
not be used. The content may have been changed, absorbed moisture, or has been
contaminated.
· 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.
Note · Use of on-line degasser is recommended.
6. Sample Preparation
(1) If possible, use the eluent for analysis to dissolve or dilute samples. If this is difficult, use a solvent which
has a composition that is as close as possible to the eluent's composition, but which fully dissolves or dilutes
the sample. 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 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.
Product
Name
Recommended
Flow Rate (mL/min)
Maximum
Pressure (MPa)
(Per Column)
pH Range Temperature (°C)
Recommended Maximum
C18U 2B 0.2 - 0.8 100 1 - 12 20 - 40 90 (pH1 - 7)
50 (pH7 - 12)
C18U 2D 0.2 - 0.8 100

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7. Column Usage Procedure
7.1 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.
7.2 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. If 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.
e.g. When replacing highly concentrated buffer solution or salt solution to water/acetonitrile, first run water and
then replace it with the 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.
7.3 Column Installation
(1) Connect the column to LC system by following the “flow direction arrow” (➡) indicated on the column name
tag.
(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.
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 less than half of the recommended flow rate. A rapid increase in pressure
can damage the column.

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· 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.
Note · It is recommended to set the pump limiter to avoid exceeding the maximum pressure.
7.4 Solvent Exchange
(1) When replacing the solvent, start the system at less than 0.2 mL/min. Recommended solvent volume to
introduce at each step is 3 to 5 times of the column volume.
(2) Check miscibility/solubility of the desired new solvent and the solvent currently filled in the column.
(3) When replacing the current solvent with a solvent with low miscibility/solubility to the current solvent, first
use a solvent that is miscible/soluble to both eluents, and then replace it with the new solvent.
e.g. When replacing highly concentrated buffer solution or salt solution to water/acetonitrile, first run water
and then replace it with the eluent.
(4) When using a gradient method, changes in the eluent compositions may increase the column backpressure.
Adjust the flow rate and column temperature so that the column backpressure remains below the usable
maximum pressure.
7.5 Column Cleaning
Problems in peak shapes and elution time changes or elevated column pressure etc. are often caused by
insoluble or adsorbing components present in the eluent and reagents being deposited inside the column. These
problems may be resolved by cleaning the column.
Make sure to clean the guard and the analytical columns separately. When washing the column, let the washing
solution flow from the column outlet go directly into the waste container and not let the solution go through the
detector.
<Cleaning Method>
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e.,
introducing the eluent from the column outlet, with 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 flow direction
and use the flow rate less than 0.2 mL/min. Introduce 5 to 10 times column volume of the washing solvent.
Method 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.
Method 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.
8. 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 at a location with stable temperature (a cool and dark space is
recommended). Refer to section 7.4 Solvent Exchange for how to replace the eluent.
Attention! · Never allow inside of the column to dry. It can damage the column.

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9. 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 (https://www.shodex.com/) for product details and their applications.
For additional assistance, contact the distributor from whom you purchased the column or contact your regional
Shodexsupport office (https://www.shodex.com/en/support_office/list/).

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10. Warranty (Ver. 4COLUMN)
(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 product, 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 remodeled 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 analysis 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 product 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.
Following names are trademarks or registered trademarks of SHOWA DENKO K.K.
Shodex, AFpak, Asahipak, AXpak, CLNpak, CXpak, HILICpak, MSpak, ODP, OHpak,
ORpak, RSpak, SUGAR, USPpak.
Products described in the operation manual are not intended for medical use or medical applications.