HPLC Columns
MANUAL
Asahipak ODP-50
(C8P, C4P series)
Shodex HPLC Columns
Europe, Middle East, Africa, Russia
SHOWA DENKO EUROPE GmbH
Shodex Business
Konrad-Zuse-Platz 3
81829 Munich, Germany
E-mail: support@shodex.de
Phone: +49 (0)89 93 99 62 37
www.shodex.de
For technical support please use
contact details shown below:
Asahipak ODP-50 (C8P, C4P series)
Manual
Operation Manual
Shodex™ Asahipak
(Please read this manual carefully before using the column to keep its good performance and life.)
NOTE
: Although Shodex Asahipak Columns are designed to operate at extremes of pH, the user
should be aware that components of the HPLC system may be susceptible to acidic or basic
conditions.
Standard fittings on Asahipak HPLC Columns have deep Waters-type seats. Adjustable
connecting hardware such as the SLIPFREE are highly recommended to insure void-free
connection.
Caution! This manual describes procedures that should be followed for operator safety as well
as achieving the optimum performance and service life of the column. This manual should be
read before the column is placed in service.
™
ODP-50 (C8P, C4P series)
1. Introduction
Asahipak columns for reversed phase chromatography are packed with spherical porous
particles that have alkyl groups bonded to the surface. The underlying polymeric bead provides
a stable polar surface. Asahipak ODP columns are compatible with the commonly used
reversed-phase HPLC eluents between pH 2 to pH 13. The particles are available in two sizes.
The smallest has a diameter of five micron. These are identified by the suffix "-50" in the first
column of Table 1. The small particles give the highest column efficiency and resolution, but at
the expense of higher operating pressure. The Asahipak "-90 series", denote packings with nine
micron particles, Table 1. These columns are designed for preparative applications, where lower
operating pressure is more important.
Asahipak columns provide three different lengths of alkyl groups that modify the polar character
of the particle, giving a useful range of hydrophobicity. The least polar column is Asahipak ODP,
which has octadecyl groups bonded to the polymer. This is designed to give the strongest
reversed phase retention, with selectivity similar to octadecyl silane, "ODS," or "C18 ," packings.
The most polar is the C4P series of columns. These have short butyl groups bonded to the polar
polymeric surface. Asahipak C8P is intermediate in polarity with a octyl hydrocarbon covalently
bonded to the particle. This packing is designed to have a hydrophobic character similar to the
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Asahipak ODP-50 (C8P, C4P series)
Manual
popular "C8" or "octyl" columns. Thus Asahipak columns for reversed phase chromatography
combine a range of hydrophobic character commonly found in silica packings with the high
chemical and physical stability of polymer based particles.
The user should read this entire manual before using the column, especially for the first time.
2. Column
3. Specifications
The specifications for each column are in Table 1, below. The column tube and end fittings are
constructed from passivated 316 stainless steel. The column end fitting is designed to minimize
dead volume and band broadening. The depth of the fitting for the inlet and outlet tubes may
be different than those used on columns from some other manufacturers. If you use a tube with
a short nose extending from the fixed ferrule, there will be a cavity in the flow path that can
lower the column efficiency, (number of theoretical plates, NTP, see Section 12). For this reason,
users may want to reseat the ferrules to provide the correct depth or use adjustable nuts and
ferrules.
The mobile phase in the column at the time of shipment is listed on the Certificate of analysis
enclosed with the column.
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Asahipak ODP-50 (C8P, C4P series)
Column
Column
dimensions
(IDxL.mm)
Particle size
(µm)
C%
NTP*
Flow rate
Normal
(ml/min)
Maximum
(ml/min)
ODP-50
4E
4.6 x 250
5
17
>14000
0.5 – 0.8
1.5
4D
4.6 x 150
5
17
>9000
0.5 – 0.8
1.5
6E
6.0 x 250
5
17
>14000
0.5 – 1.0
2.5
6D
6.0 x 150
5
17
>9000
0.5 – 1.0
2.5
10E
10.0 x 250
5
17
>10000
1.5 – 2.0
3.0
C8P-50
4E
4.6 x 250
5
10
>11000
0.5 – 0.8
1.0
4D
4.6 x 150
5
10
>7000
0.5 – 0.8
1.0
10E
10.0 x 250
5
10
>8000
1.5 – 2.0
3.0
C4P-50
4E
4.6 x 250
5
6
>9000
0.5 – 0.8
1.0
4D
4.6 x 150
5
6
>6000
0.5 – 0.8
1.0
10E
10.0 x 250
5
6
>7000
1.5 – 2.0
3.0
ODP-90
20F
20 x 300
9
17
>9000
5 – 10
12
Grade
Column Dimensions
(IDxL.mm)
Columns guarded (IDxL.mm)
ODP-50G 4A
4.6 x 10
ODP-50 (4.6x150, 4.6x250)
ODP-50G 6A
6.0 x 10
ODP-50 (6.0x150, 6.0x250)
C8P-50G 4A
4.6 x 10
C8P-50 (4.6x150, 4.6x250)
C4P-50G 4A
4.6 x 10
C4P-50 (4.6x150, 4.6x250)
ODP-130G 7B
7.6 x 50
ODP-50 10E (10.0x250)
ODP-90 20F (20.0x300)
Manual
Table 1. Specifications of Asahipak ODP, C8P and C4P columns
Table 2. Guard columns
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Asahipak ODP-50 (C8P, C4P series)
Manual
4. Column installation
4.1 Direction of flow
Install and use the column with mobile phase flowing in the direction indicated on the column
tag.
If the column shows very high resistance to flow, it may be partially plugged up. This is probably
the end of the service life of the column. Some users have been successful in extending the life
somewhat by back flushing the column. The outlet of the column is connected to the injector so
the liquid flows into the normal outlet fitting. The other end, normally the column inlet is placed
into a beaker. The pump is then turned on at the minimum flow rate listed in Table 1. The
column is back flushed for about one hour to over night, or even longer, if the operating
pressure is declining. The column then should be retested according to the procedure on the
page "INSPECTION DATA" provided with each column. If the column is efficient, then one can
restore the column to operation in the instrument in the normal manner.
See also Section 5.1 on filtering and guard columns.
4.2 Connection tubes
For columns with diameters smaller than 10 mm id, use connection tubes from the injector and
to the detector that have the shortest practical length and internal diameter no larger than 0.3
mm, (0.010 inch) internal diameter. The outer diameter must be 1/16 inch, (0.0625 inch).
For preparative columns, (columns with internal diameters of 10 mm and larger), it is possible to
use connection tubes with internal diameter up to 0.5 mm, (0.020 inch). The length should be
kept as short as practical.
4.3 Pump
The use of pumps with strong pulsations in the output flow or pressure may shorten column life,
and should be avoided. Pulsation can often be reduced by using a pulse damper on the pump
outlet.
4.4 Preliminary purge
Before connecting the column to the system, purge the system and lines with liquid to remove
any air or particles.
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Asahipak ODP-50 (C8P, C4P series)
Manual
4.5 Connection of column to HPLC
Connect the column inlet to the tube from the injector. Do not turn the fitting tight. Turn on the
pump flow to one half the lower operating range in Table 1. Allow a small amount of the mobile
phase to leak out and displace any air bubbles from the cavities in the column inlet.
Tighten the inlet fitting. Then loosely connect the outlet fitting. After a few drops have formed,
tighten the fitting. This should displace any air from the system.
5. Mobile phase and sample preparation
5.1 Filtering
If particles are allowed to flow into the Asahipak column, the packed bed in the column will trap
the particles. Eventually the column will become plugged. To avoid this, the mobile phase and
sample should be passed through a 0.45 µm, or smaller, filter. For the mobile phase, the filter
should be installed between the pump and injector. The sample should be passed through any
one of the many different filters available for this purpose.
Filtering is not always effective in preventing plugging of the column. Experience shows that if
column plugging is a frequent problem, that, the plug is often located at the very top of the
column. Guard columns are short sacrificial columns that are installed ahead of the main
column to protect it. When a plug occurs, the guard column is replaced.
5.2 Degassing
Numerous practical problems are avoided when one uses mobile phases that have been treated
to remove, or least lower their content of dissolved gases, especially air. The most convenient
way to remove dissolved air is to gently bubble helium through the mobile phase reservoirs. If
one is using mobile phases containing a mixture with volatile components, then the helium may
slowly remove the volatile components, which can cause a systematic shift in the retention
times of the analyte. In this case, the use of a commercial on-line degasser is recommended.
6. Compatible mobile phases
Asahipak ODP, C8P and C4P columns can be used with mobile phases or eluents containing
water, methanol and acetonitrile. In general, water acetonitrile combinations provide greater
efficiency, especially for compounds containing an aromatic ring.
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Asahipak ODP-50 (C8P, C4P series)
Manual
For control of pH within the range of pH 2 to pH 13, phosphate, acetate, tris-HCl, NaCl, KCl,
Na2SO4, and trifluoroacetic acid buffers can be used. Please consult your Asahipak
representative if you have questions about buffering agents not listed above. It is important to
note that the mobile phase should be filtered before use as described in section 5.1.
Caution! Adding organic solvents to salt and buffer solutions can lead to precipitation of
the salt. Precipitation can occur in the column, which will often cause a catastrophic rise
in the pressure across the column. If this does occur, try washing out the precipitate by
reversing the flow on the column with distilled water. Use a slow flow rate initially to
keep the back pressure low, below 15MPa.
It is important to check the solubility of mixed solvent buffers before running a solvent
program. This can be done by making a quick scouting run in a test tube. Slowly add the
strong eluent to a small quantity of the starting eluent. If the solution turns cloudy or
white, this means a precipitate is forming. Other buffer combinations should be tried.
7. Elution modes
All Asahipak columns can be used in isocratic and gradient elution modes. Take care to avoid
precipitation of buffer components during the run, Section 6.
7.1 Asahipak ODP- 90, C8P-90 and C4P-90
With the columns with a "- 90" suffix, Table 1, avoid sharp changes in mobile phase composition
at high flow rates. Rapid changes can cause settling of the column bed. To minimize this risk,
changes in composition should be made at flow rates of 5mLimin or lower. Large changes in
solvent composition should be made gradually as shown in Figures 2 and 3 for both increasing
and decreasing concentration.
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Asahipak ODP-50 (C8P, C4P series)
Manual
7.2 Asahipak ODP-50, C8P-50 and C4P-50
There are no restrictions on the rate of change of mobile phase composition provided that the
column is operating within the specified range of flow rate and pressure, Section 3 and Table 1.
8. Flow rate
Operation of the column at flow rates higher than specified as normal in Table 1, will probably
shorten the useful life of the column. Exceeding the maximum flow rate specification in Table 1
will probably immediately destroy the column. For this reason, the pressure limit on your pump
should be adjusted to about 10% to 20% above the pressure normally found for your particular
column and mobile phase. This should provide quick protection should a high flow excursion
occur. Never allow the pressure limit be set above 15 MPa.
9. Operating temperature
Asahipak ODP, C8P and C4P columns can be operated at temperatures from 4°C to 60°C. At
elevated temperatures, degassing may be required to prevent formation of bubbles in the
detector. At low temperature, the viscosity of the mobile phase will increase. Care should be
taken to avoid exceeding the maximum pressure specification of the column. This may require
decreasing the flow rate. Flow rate is proportional to pressure, so cutting the flow in half will
decrease the pressure on the column by half.
10. Column handling and storage
The column bed should not be allowed to dry out. Retain the plugs used in shipping the column
and reinstall them tightly when the column is not being used.
During storage, the column should not be exposed to strong swings in temperature, vibration or
direct sunlight.
Asahipak ODP, C8P and C4P columns may be left connected in the HPLC system for several days
as long as the instrument is free from corrosive agents, buffers and organisms that could
support growth of material that plugs the column. If such agents are a potential problem, the
column can be flushed with first with water, then 50% methanol, and then left standing in the
instrument. Washing the column with water is important to remove buffer components that
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Asahipak ODP-50 (C8P, C4P series)
Manual
could precipitate during standing or by adding the 50% methanol, see Section 6. Also it is
important that the column not be permitted to dry out.
11. Sample preparation
In general, the sample should be dissolved in the mobile phase. Dissolving the sample in the
mobile phase often avoids large "system peaks" in the beginning of the chromatogram. For
gradient elution the sample should be in a solution corresponding to the initial mobile phase
composition. In some cases it is desirable to prepare the sample in a solution with an elution
strength slightly weaker than the mobile phase. Upon injection, the sample components will
tend to focus in a narrow band on top of the column. This can be improve detection limits.
12. Measurement of NTP
The measurement conditions employed in the determination of NTP are described in the
INSPECTION DATA sheet enclosed with Asahipak ODP, C8P and C4P columns.
Note that the use of different solutes or eluents will result in different NTP values. Any
substantial dead volume in the LC system flow line will also result in lower NTP values.
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Asahipak ODP-50 (C8P, C4P series)
Manual
*The information in this document is correct to the best of our knowledge. However, it is not
guaranteed to be so, and by publishing it we assume no responsibility.
*No licence or immunity under any patent is granted or implied by our sales of any material.
* Shodex Asahipak products in this document are for laboratory use only. They are not intended
for medicinal or food use. We assume no responsibility if these products are used for medicinal
or food purposes, or are misused in any way.
* Shodex Asahipak products are not intended for clinical use. While clinical applications may be
shown, these products are not tested for clinical applications.
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