Showa Denko Shodex PROTEIN LS-G 4J User manual

Operation Manual Shodex PROTEIN LS-G 4J Version 190422E
Operation Manual
Shodex™ PROTEIN™ LS-G 4J
Europe, Middle East, Africa, Russia
www.shodex.de
Sales Office:
Showa Denko Europe GmbH
Shodex Business
Konrad-Zuse-Platz 3
81829 Munich
Germany
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 PROTEIN LS-G 4J Version 190422E
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Operation Manual
Shodex™ PROTEIN™ LS-G 4J
(Please read this manual carefully to obtain 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.
Caution! 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 that there is no damage on the package and outside of the column.
(2) Please check if the product name and serial number written on the column package, column name
tag, and enclosed Certificate of Analysis (CoA) are correct.
1. Introduction
Thank you for purchasing the ShodexTM product.
Shodex PROTEIN LS-G 4J is a guard column for high performance Shodex PROTEIN LW-403 4D column
which is designed to be used in gel filtration chromatography for precision separation of proteins
and/or enzymes. This guard column can not only protect the analytical column, but also suppress the
injection noise* that often occurs when using a light scattering detector.
*Injection noise: A ghost peak that appears near the holdup volume due to pressure fluctuation associated with
sample injection
2. Column Components
3. 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.
Attention! A drastic change in the eluent compositions may remove substances adsorbed on the
system and they may enter and deteriorate the column.

Operation Manual Shodex PROTEIN LS-G 4J Version 190422E
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4. Column Installation
(1) Connect the column to LC system by following the "flow direction arrow" (->) indicated on the
column name tag. 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 allow the sample to spread out and result in wide peaks.
(3) Set the initial flow rate at less than 0.17 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.
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.
Attention! 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.
Attention! 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 results in creating an empty space in the column and may deteriorate the column.
5. Column Specifications
Product code
Product name
Size (mm)
Particle size
(μm)
Pore size
(Å)
Plate Number
(per column)
ID
Length
F6700134
PROTEIN LS-G 4J
4.6
20
1.9
-
(Guard column)
Base material: Spherical porous particles of silica (bound with hydrophilic group polymer)
Column material: Stainless steel (SUS 316)
Screw type: Unified Thread Standard No. 10-32 UNF
Shipping solvent: Pure distilled water
6. Usable Conditions
Product name
Flow rate
Max.
pressure
pH range
Temperature
range
Standard
Max.
PROTEIN LS-G 4J
0.35 mL/min
0.5 mL/min
20 MPa
3.0 - 7.5
4 - 45°C
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.

Operation Manual Shodex PROTEIN LS-G 4J Version 190422E
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7. Eluents
(1) The following solvents are usable:
Various buffer solutions such as phosphate buffer solution, tris-HCI buffer solution, acetate buffer
solution with salts are generally used. Salts to be added include sodium chloride, sodium sulfate and
ammonium sulfate. The suitable salt concentration is 0.1 to 0.3 M.
(2) Acetonitrile and methanol can be used up to 100%.
(3) Protein denaturation reagents such as urea or guanidine hydrochloride, and surfactants such as
SDS or Brij can also be used. Since the addition of urea or guanidine hydrochloride in the eluent
increases the viscosity, so set the flow rate at less than 0.17 mL/min.
(4) Degas the eluent fully to prevent the formation of air bubbles.
(5) 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 0.45 μm disposable filter to
prevent the problems.
(6) Start the system at less than 0.17 mL/min when replacing the solvent.
(7) 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 "6. Usable Conditions".
Attention! When using a mixture of organic solvent and buffer solution, make sure that there is no
precipitation of salt in the eluent before use.
Attention! Frequent solvent replacement can deteriorate the column, and thus not recommended.
Attention! When using highly corrosive salts such as sodium chloride, wash out the salts after the
analysis. The metal parts of the devices and/or the columns may rust.
Attention! Since protein denaturants may remain on the column, it is recommended to use the same
eluent conditions without replacing other eluent conditions.
Note! Use of on-line degasser is recommended.
Note! It is recommended to set the pump limiter to avoid use exceeding the maximum pressure.
8. 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, but fully
dissolves or dilutes the sample.
(2) Filter the sample solution using disposable 0.45 µm filter to prevent the column from clogging or
deteriorating.
9. 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.
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. 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 0.17 mL/min. A suggested cleaning solvent
volume is 5 - 10 times the column volume. Make sure to clean only the guard without the analytical
columns.

Operation Manual Shodex PROTEIN LS-G 4J Version 190422E
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(1) Removal of ionic substances:
Flow about 0.5 M salt solution or phosphate buffer solution of about pH 3.0 through the column as
cleaning solvent.
(2) Removal of hydrophobic substances:
Flow the eluent to which about 10 - 20 % of water-soluble organic solvents such as acetonitrile or
methanol is added through the column as cleaning solvent.
Attention! When cleaning with added organic solvents in the eluent, make sure that there is no
precipitation of salt in the eluent before use.
Attention! If some proteins remain in the column, it may be precipitated by cleaning.
10. 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 "7. Eluents" when replacing the eluent.
Attention! Never allow the inside of the column to dry. It can damage the column.
11. Column Inspection
Column inspection method is described in the Certificate of Analysis (CoA).
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.

Operation Manual Shodex PROTEIN LS-G 4J Version 190422E
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13. Warranty (Version 4)
1) Showa Denko K.K. warrants that the ShodexTM 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.

Operation Manual Shodex PROTEIN LS-G 4J Version 190422E
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Product Information: Shodex SEC Columns (aqueous GPC)
Shodex SEC column series cover the analysis of water-soluble molecules like proteins, enzymes,
biomolecules, polysaccharides, surfactants and polymers. They have different target molecular weight
ranges and exclusion limits depending on the pore size. Guard columns are available for each series.
Please refer to the individual operation manuals for detailed specifications or ask for technical support
for your application.
PROTEIN KW-800 series
Silica-based
Suitable for the analysis of proteins and enzymes
Range of 5,000 to 4,000,000 molecular weight
KW400 series
Reduced particle size enhances column performance
Three to four-fold higher sensitivity than KW-800 series
KW405-4F is applicable analyzing samples with molecular weight up to 20,000,000
PROTEIN LW series
High performance analysis of antibody drugs
For analyzing proteins with a molecular weight of several hundred of thousand
High lot-to-lot reproducibility
OHpak SB-800 series
Polymer-based
Supports a wide range of molecular weight sample analysis
Suitable for modified proteins, PEGs, polysaccharides, surfactants
The eluent can be replaced with DMF, enabling the analysis of polar polymers
OHpak LB-800 series
For light scattering detectors (MALS)
Asahipak GS series
Multi-mode SEC columns
With the choice of eluent additional separation by reversed phase, HILIC, and ion exchange modes
Suitable for the separation of peptides or nucleic acids with similar molecular weights
Suitable for desalting samples or substituting buffer in protein analysis
Asahipak GF series
Multi-solvent SEC columns
Polymer-based SEC columns with high solvent durability
Works well with both aqueous and organic solvents
Calibration Standard: Pullulan
STANDARD P-82 kit with 8 standards for aqueous SEC
Unbranched polysaccharide standard
High solubility in water eliminates the possibility of recrystallization