Showa Denko Shodex Asahipak GF-HQ Series User manual

Operation Manual Shodex Asahipak GF-HQ series Version 2106029E
Operation Manual
Shodex™ Asahipak GF-HQ 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 Asahipak GF-HQ series Version 2106029E
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Operation Manual
Shodex™ Asahipak GF-HQ 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 Asahipak GF-HQ series is a polymer-based size
exclusion chromatography column. They work well with both aqueous and organic solvents (multi-
solvent column).
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)
F7600000
Asahipak GF-210 HQ
7.5 x 300
5
180
≥ 19,000
F7600001
Asahipak GF-310 HQ
7.5 x 300
5
400
≥ 19,000
F7600002
Asahipak GF-510 HQ
7.5 x 300
5
2,000
≥ 19,000
F7600003
Asahipak GF-710 HQ *
7.5 x 300
9
10,000
≥ 11,000
F7600004
Asahipak GF-7M HQ
7.5 x 300
9
10,000
≥ 13,000
F6710018
Asahipak GF-1G 7B
7.5 x 50
9
-
(guard column)
Asahipak GF-7M HQ is a mixed-gel column, suitable for the analysis of wide molecular weight ranges.
* Column size marked with asterisk is available until end of 2021
Base Material: Spherical porous particles of polyvinyl alcohol
Column Housing: SUS-316
Screw Type: Internally-threaded type No.10-32 UNF
Shipping Solvent: Water/Acetonitrile = 70/30
Water (Asahipak GF-210 HQ)

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Product Code
Product Name
Target Molecular Weight
Range
Exclusion Limit
F7600000
Asahipak GF-210 HQ
300 –4,000
9,000
F7600001
Asahipak GF-310 HQ
300 - 30,000
40,000
F7600002
Asahipak GF-510 HQ
5,000 - 200,000
300,000
F7600003
Asahipak GF-710 HQ *
100,000 –(10,000,000)
(10,000,000)
F7600004
Asahipak GF-7M HQ
300 - (10,000,000)
(10,000,000)
Reference value only / Measured with pullulan
4. Usable Conditions
4.1 System Settings
Product Name
Flow Rate (mL/min)
Maximum Pressure
(MPa) (Per Column)
pH
Range
Temperature
Range (°C)
Recommended
Maximum
Asahipak GF-210 HQ
0.4 –0.6
1.0
9.0
2 - 9
4 - 60
Asahipak GF-310 HQ
0.4 –0.6
1.0
7.0
Asahipak GF-510 HQ
0.4 –0.6
1.0
6.5
Asahipak GF-710 HQ *
0.4 –0.6
1.0
2.5
Asahipak GF-7M HQ
0.4 –0.6
1.0
4.5
Asahipak GF-1G 7B
-
-
-
4.2 Usable Solvents
Solvent
Product Name Asahipak
GF-210 HQ
GF-310 HQ
GF-510 HQ
GF-710 HQ
GF-7M HQ
Water
O
O
O
O
O
Methanol
O
O
O
O
O
Ethanol
O
O
O
O
O
Acetonitrile
O
O
O
O
O
Tetrahydrofuran (THF)
O
O
O
O
O
Acetone
O
O
O
O
O
2-Propanol (IPA)
O
O
O
Ethyl acetate
O
O
O
O
O
N,N-Dimethylformamide (DMF)
O
O
O
O
O
Dioxane
O
O
O
Dimethyl sulfoxide (DMSO)
O
O
(0-50%)
O
(0-50%)
N,N-Dimethylacetamide (DMAc)
O
Chloroform
O
O
O
O
O
Hexane
X
X
O
O: Can be used X: Cannot be used Blank: No Data
(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, potassium
chloride, sodium sulfate, potassium sulfate, and ammonium sulfate. Please keep total concentration
of salts under 0.5 M. Their recommended concentration ranges are 0.05 - 0.3 M.
(2) Salts such as lithium bromide can be added to DMF and DMSO. Their recommended concentration
ranges are 10 - 50 mM.
(3) 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

Operation Manual Shodex Asahipak GF-HQ series Version 2106029E
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deterioration from the solvent replacement. It is recommended to dedicate the column for this specific
use.
(4) 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 lime than
replacement from general solvents. The replacement time can be shorten by using 30 - 50 % (v/v)
methanol.
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.
• 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 influences the repeatability of the analysis. If shear
degradation is suspected, use a lower flow rate.
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 0.45 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.
(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 50 to 100 μL per column.

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(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 filter is recommended to protect the analytical column.
7. Column Usage Procedure
7.1. 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/acetonitrile, first run methanol and then replace it with
water/acetonitrile.
e.g. When replacing highly concentrated buffer solution or 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.
7.2. 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 0.2 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.
(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. When connecting GF-7M HQ, use multiple number of the same columns in series. This is
because GF-7M HQ is prepared by using several different gels with different pore sizes, designed to

Operation Manual Shodex Asahipak GF-HQ series Version 2106029E
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provide linear calibration curves over a set molecular weight range. If a GF-7M HQ 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 0.2 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
0.2 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.
Note • It is recommended to set the pump limiter to avoid exceeding the maximum
pressure.
7.3. 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 limes of the column volume.
(2) Check miscibility/solubility of the desired new solvent and the solvent currently filled in the column.
(3) When replacing water with an organic solvent, first introduce methanol, and then replace it with
the new solvent. When replacing an organic solvent with water, first introduce methanol, and then
replace it with water.
(4) When replacing aqueous solution of salt with an organic solvent, first introduce water at the flow
rate at half of the recommended flow rate for 30 minutes. Follow step above (3), and then replace it
to the new solvent. When replacing organic solvent with aqueous solution of salt, replace with the
reverse procedure.
(5) When replacing an organic solvent with another organic solvent, introduce the new solvent directly.
(6) 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 water to hexane, first run methanol and acetone in that order, and then introduce
hexane.
e.g. When replacing THF to DMF with LiBr, first run DMF, and then introduce DMF with LiBr.
Attention! •Frequent solvent replacement deteriorates the column, and thus not recommended.
7.4. 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 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

Operation Manual Shodex Asahipak GF-HQ series Version 2106029E
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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 al less than 0.3 ml/min. In general, the solvent volume introduced
should be 5 to 10 times the column volume.
Method 1: Adsorption of hydrophilic 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.
Attention! • Keep the organic solvent and salt within the concentrations stated in 4.2 Usable
Solvents.
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 7.3 Solvent Exchange for how to replace the eluent.
Attention! • Never allow inside of the column to dry. It can damage the column.
9. 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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10. 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.
11. 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.