Showa Denko Shodex OHpak LB-800 Series User manual

Operation Manual Shodex OHpak LB-800 series Version 210215E
Operation Manual
Shodex™ OHpak LB-800 series
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 OHpak LB-800 series Version 210215E
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Operation Manual
Shodex™ OHpak LB-800 series
(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 OHpak LB-800 series is a size exclusion
chromatography used with aqueous solvents. The column series is suitable for the separations of
various water-soluble high molecular weight polymers, proteins, and/or oligomers, and molecular
weight distribution analysis of high molecular weight polymers. Successfully reduced baseline noise in
the light scattering (LS) detector by suppressing the column bleeding as much as possible made it to
provide accurate molecular weight measurements. The columns with different exclusion limits are
available. Please select columns with target molecular weight ranges (see below) that meet the
molecular weight range of your samples.
2. Column Components

Operation Manual Shodex OHpak LB-800 series Version 210215E
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3. Column Specifications
Product
code
Product Name
Column Size
(mm)
Particle
Size
(μm)
Theoretical
Plate
Number (Per
Column)
Target
Molecular
Weight Range*1
Exclusion
Limit*1
Light
Scattering
Noise*2
(V)
I.D.
Length
F6429206
OHpak LB-802.5
8.0
300
6
≥16,000
500 to 10,000
10,000
≤5.0 × 10-5
F6429201
OHpak LB-803
8.0
300
6
≥16,000
1,000 to 100,000
100,000
≤2.0 × 10-5
F6429204
OHpak LB-804
8.0
300
10
≥16,000
5,000 to 400,000
1,000,000
≤5.0 × 10-5
F6429203
OHpak LB-805
8.0
300
13
≥12,000
100,000 to
1,000,000
(4,000,000)*3
≤5.0 × 10-5
F6429205
OHpak LB-806
8.0
300
13
≥12,000
100,000 to
(20,000,000)*2
(20,000,000)*3
≤5.0 × 10-5
F6429202
OHpak LB-806M
8.0
300
13
≥12,000
500 to
(20,000,000)*2
(20,000,000)*3
≤2.0 × 10-5
F6709434
OHpak LB-G 6B
6.0
50
13
(Guard
Column)
-
-
-
LB-806M 1s a mixed-gel column, suitable for the analysis ofw1de molecular weight ranges.
*1Reference value only / Measured with pullulan
*2Taken by using 0.1 M aqueous sodium nitrate solution as the eluent
*3Estimated value
Base Material: Spherical porous particles of polyhydroxymethacrylate
Column Housing: SUS-316 (stainless steel)
Screw Type: Internally-threaded type No.10-32 UNF
Shipping Solvent: Water
4. Usable Conditions
Product Name
Flow Rate (mL/min)
Maximum Pressure
(MPa) (Per Column)
pH Range
Temperature
Range (°C)
Recommended
Maximum
OHpak LB-802.5
0.5 - 1.0
1.2
5.5
3 - 10
4 - 80
OHpak LB-803
OHpak LB-804
3.5
OHpak LB-805
OHpak LB-806
OHpak LB-806M
OHpak LB-G 6B
-
Usable solvents are listed below
(1) Buffers and aqueous solutions of different salts can be used separately or together. They include
phosphate, acetate, citrate, and Tris buffers and aqueous solutions of sodium chloride, sodium sulfate,
potassium sulfate, and ammonium sulfate. Please keep total concentration of salts under 0.5 M. The
recommended concentration ranges are 0.05 - 0.3 M.
(2) Up to 100 % (v/v) acetonitrile, methanol, and dimethylformamide (DMF) is usable.
(3) Salts such as lithium bromide can be added to DMF. Their recommended concentration ranges
are 10 - 50 mM.
(4) Aqueous solutions of protein denaturants such as urea and guanidine hydrochloride can also be
used. However, since highly concentrated solutions are usually used, there is a risk of column
deterioration from the solvent replacement. It is recommended to dedicate the column for this specific
use.
(5) Surfactants such as SDS and Brij-35 can also be added to the eluent. However, since surfactants
tend to remain on the column, solvent replacement after their use takes a longer time than
replacement from general solvents. The replacement time can be shorten by using 30 to 50 % (v/v)
methanol.

Operation Manual Shodex OHpak LB-800 series Version 210215E
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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.
• When using a mixture of salt solution and organic solvent, make sure there is no
precipitation of salt.
• When adding chloride ions, make the eluent pH 6 or higher.
• Do not use borate buffer as it forms a complex with dial group of the packing
material.
• 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.
• Shear degradation is more likely to occur in larger molecular weight compounds.
When shear degradation occurs, the measured molecular weight may be lower than
the actual value or it may influence the repeatability of the analysis. If shear
degradation is suspected, use a lower flow rate.
Note • Baseline noise in LS detector is often improved by using buffer or aqueous salt
solutions rather than using water alone as an eluent. The recommended eluent for
noise reduction is 0.1 M aqueous sodium nitrate solution.
5. 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.
(2) Filter the sample solution using disposable 0.45 μm filter to prevent the column from clogging or
deteriorating.
(3) To prepare a sample with molecular weight larger than 1,000,000, first allow the sample to stand
in the eluentof analysis for 1 day until it becomes fullyswollen. Next, slowly agitate the sample solution
to completely dissolve the sample. Be careful as aggressive agitation can cut the polymer chains of the
analyte.
(4) Recommended injection volume is 10 to 100 μL per column.
(5) Viscosity of high molecular weight compound is largely influenced by its molecular weight and
concentration. Samples with high viscosity cause peak broadening and elution delay, and this makes it
difficult to obtain their accurate molecular weight distributions. In general, the larger the molecular
weight of the compound, the higher its viscosity becomes. To suppress the influence from high
viscosity, it is recommended to lower the sample concentration. Please use the below table as a
reference when preparing samples for molecular weight distribution analyses.
Molecular Weight Range
Optimal Concentration (w/v)
≤5,000
≤ 1.0 %
5,000 -
25,000
≤ 0.5 %
25,000 -
200,000
≤ 0.25 %
200,000 -
2,000,000
≤ 0.1 %
≥2,000,000 -
≤ 0.05 %
Note •Use of guard column is recommended to protect the analytical column.

Operation Manual Shodex OHpak LB-800 series Version 210215E
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6. Column Usage Procedure
6.1. 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 chloroform to water, first run methanol and then replace it with water.
e.g. When replacing highly concentrated buffer solution or aqueous salt solution to water/acetonitrile,
first run water and then replace it with water/acetonitrile.
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.
6.2. Column Installation
(1) Connect the column to LC system by following the “flow direction arrow” () indicated on the
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 0.3 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.
(4) Multiple number of columns can be connected in series. When connecting multiple columns with
each having different exclusion limits, set the column with higher exclusion limit at the upper stream
position. However, please note that LB-806M is prepared by using gels with different pore sizes to
provide linear calibration curves. Therefore, combining columns with different exclusion limits may
alter their linearity. When improved separation is required, use multiple number of LB-806M in series.
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 0.3 mL/min. 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
0.3 mL/min 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.

Operation Manual Shodex OHpak LB-800 series Version 210215E
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6.3. Eluents
(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 0.45 μm disposable filter to
prevent the problems.
(3) When replacing the eluent with miscible solvents, first use 1:1 mixture of the current solvent and
the new solvent, and then replace it with 100 % of the new eluent.
e.g. When replacing highly concentrated buffer solution (or aqueous salt solution) to methanol, first
run 1:1 mixture of buffer (or aqueous salt solution) and methanol, then replace it with methanol.
(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.
e.g. When replacing highly concentrated buffer solution (or aqueous salt solution) to
water/acetonitrile, first run water, and then replace it with water/acetonitrile.
(5) When replacing the solvent, start the system at less than 0.3 mL/min.
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.
Solvents left in an opened bottle for a long time should not be used. The content may
have been changed 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.
• Frequent solvent replacement deteriorates the column, and thus not recommended.
• 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.
Note • Use of on-line degasser is recommended.
•It is recommended to set the pump limiter to avoid exceeding the maximum
pressure.
6.4. 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.
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 solved by removing the guard column, clean both guard and analytical columns.
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 at less than 0.5 mL/min. In general, the solvent volume introduced
should be 5 to 10 times the column volume.
Method 1 Adsorption of hydrophobic compounds (when using aqueous eluent)
Introduce the eluent with a high polar organic solvent (acetonitrile or methanol) added.
Method 2: Adsorption of ionic compounds
Introduce the eluent with a higher salt concentration.

Operation Manual Shodex OHpak LB-800 series Version 210215E
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7. Column Storage
Remove the column from the system keeping the solvent used for the analysis. 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 6.3 Eluents for how to replace the eluent.
Attention! • Never allow inside of the column to dry. It can damage the column.
8. Column Inspection
Inspection method is described in the Certificate of Analysis (CoA). Theoretical Plate Number (N) and
Asymmetry Factor (Fas) were calculated using the below equations.
(1) Theoretical Plate Number (N)
(2) Asymmetry Factor (Fas)
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 (www.shodex.de) 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.
10. 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.

Operation Manual Shodex OHpak LB-800 series Version 210215E
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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.
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
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