Showa Denko Shodex GPC HK-400 Series User manual

Operation Manual Shodex GPC HK-400 series Version 201210E
Operation Manual
Shodex™ GPC HK-400 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 GPC HK-400 series Version 201210E
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Operation Manual
Shodex™ GPC HK-400 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 GPC HK-400 series is an ultra-rapid analysis
type size exclusion chromatography used with organic solvents. The column series is suitable for the
separations of organic compounds and/or oligomers, and molecular weight distribution analysis of
high molecular weight polymers. 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. The columns fulfill USP L21 requirements.
2. Column Components
3. Column Specifications
Product Code
Product Name
Column Size (I.D.
x Length) (mm)
Particle Size
(µm)
Pore
Size (Å)
Theoretical Plate
Number (Per Column)
F6025010
GPC HK-401
4.6 x 150
3
50
≥ 9,000
F6025020
GPC HK-402
4.6 x 150
3
300
≥ 12,000
F6025030
GPC HK-403
4.6 x 150
3.5
550
≥ 9,000
F6026040
GPC HK-404L
4.6 x 150
3.5
2,000
≥ 9,000
F6025050
GPC HK-405
4.6 x 150
3
5,000
≥ 7,000
F6025060
GPC HK-406
4.6 x 150
6.5
10,000
≥ 5,000
HK-404L is a mixed-gel column, prepared using gels with different pore sizes.

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Base Material: Spherical porous particles of styrene divinylbenzene copolymer
Column Housing: SUS-316
Screw Type: Internally-threaded type No.10-32 UNF
Shipping Solvent: Tetrahydrofuran (THF)
Product Code
Product Name
Description
Contens
F6700200
GPC HK-G
Guard filter (cartridge type)
One holder and one filter
F6700100
GPC HK-G filter
Replacement filters
Three filters
Holder Material: SUS-316
Filter Material: SUS-316
Filter Pore Size: 0.5 µm (sintered filter)
Holder End-screw Type: Internally-threaded type No.10-32 UNF
Product Code
Column Name
Target Molecular Weight Range
Exclusion Limit
F6025010
GPC HK-401
100 - 1,500
2,000
F6025020
GPC HK-402
200 - 10,000
20,000
F6025030
GPC HK-403
2,000 - 700,000
100,000
F6026040
GPC HK-404L
100 - 1,000,000
1,000,000
F6025050
GPC HK-405
10,000 - 2,500,000
4,000,000
F6025060
GPC HK-406
30,000 –8,000,000
10,000,000
Reference value only / Measured with polystyrene
4. Usable Conditions
4.1 System Settings
Product Name
Flow Rate (mL/min)
Maximum Pressure
(MPa) (Per Column)
Temperature (°C)
Recommended
Maximum
Recommended
Maximum
GPC HK-401
0.3 - 1.0
2.0
25
40
60
GPC HK-402
1.5
20
GPC HK-403
2.0
25
GPC HK-404L
GPC HK-405
GPC HK-406
4.2 List of Applicable Solvents
Solvent
Column
HK-401, HK-403, HK-404L, HK-405, HK-406
HK-402
Tetrahydrofuran (THF)
O
O
Chloroform
O
O
Dimethylformamide (DMF)
O
O
Toluene
O
O
30% HFIP/Chloroform
O
O
Hexafluoroisopropanol (HFIP)
O
X
O: Can be used X: Cannot be used
The standard solvent is THF. Please use THF with stabilizer (BHT) whenever possible.
Caution! • THF gradually produces peroxides when it reacts with oxygen in the air. There is a
danger of explosion when peroxide is concentrated. An extra caution should be taken
when concentrating the collected sample after separation.

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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.
• THF without BHT easily forms peroxides and this causes unstable baseline. If
necessary to use THF without BHT, use the fresh solvent from an unopen bottle.
• Do not use the THF with BHT, when its bottle is left open for a while as it may contain
peroxides.
• THF absorbs moisture easily. Be careful not to let the solvent absorb the moisture
from air during the analysis.
• 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.
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 1 to 25 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 %
(6) When sample contains water, methanol, or hexane, remove them prior to the injection.
Note •Use of guard filter is recommended to protect the analytical column.
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

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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 water to chloroform, first run methanol and then introduce chloroform.
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
different exclusion limits, set the column with higher exclusion limit at the upper stream position.
When connecting HK-404L, use multiple number of the same columns in series. This is because HK-
404L is prepared by using several different gels with different pore sizes, designed to provide linear
calibration curves over a set molecular weight range. If a HK-404L was used together with columns
with different pore sizes, this changes the mixing ratio of the gels and consequently it may deform the
linear calibration curve.
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.
Guard filter (HK-G): Installation
(1) At the first time use, connect the flow line to the guard filter inlet alone (do not connect to the
analytical column), set the flow rate at 0.3 mL/min and start the system. Leave it for about 1 minute.
(2) Stop the system and connect the outlet of the guard filter to the inlet of the analytical column,
tighten by hand. Place wrenches on the parts indicated by ★and tighten additional 30 to 60°. After

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connecting the guard filter and the analytical column, they can be kept connected together during the
storage time.
Attention! • Tightening too much with wrenches can damages the thread.
• Do not place a wrench on the part indicated by ◎to tighten or remove the guard
filter. This may loosen the end fitting of the analytical column and results in solvent
leak.
Guard filter (HK-G): Filter replacement
(1) Place wrenches on the parts indicated by ★and untighten the connection. Detach the guard filter
from the analytical column. Cap the inlet of the analytical column.
(2) Use wrenches to untighten the guard filter. Replace the old filter with a new filter.
(3) Tighten the holder by hands and then use wrenches to tighten additional 180°.
(4) Refer to the steps described in “Guard filter (HK-G): Installation” to connect the guard filter to the
analytical column.
Attention! • Make sure to use an official replacement filter (product code: F6700100). Product
performance cannot be guaranteed if unofficial filter was used.
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 chloroform to DMF, first run 1:1 mixture of chloroform and DMF and then
introduce 100% DMF.
(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 THF to DMF with LiBr, first follow step (3) above, and then replace 100% DMF to
DMF with LiBr.
(5) When replacing the solvent, set the column temperature at 40 to 60°C and start the system at less
than 0.3 mL/min.
Attention! • 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.
• Frequent solvent replacement deteriorates the column, and thus not recommended.
Note • Use of on-line degasser is recommended.
•It is recommended to set the pump limiter to avoid exceeding the maximum
pressure.

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6.4. Column Cleaning
Problems in peak shapes and elution timing 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 filter (HK-G) is used with the analytical column, first remove the guard filter and check the
performance of the analytical column alone. If the problem is solved, most likely the cause is from the
guard filter. In this case, replace the filter in the guard filter. Refer to section 6.2 Column Installation
“Guard filter (HK-G): Filter replacement” for how to replace the filter. If the problem is not solved by
removing the guard filter, replace the filter in the guard filter and clean analytical columns. Make sure
to clean the analytical columns after removing the guard filter. 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>
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.
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).
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)

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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 (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.
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.