General Care to Use Packed Columns
Contents :
1. Name of column parts
2. Installation and start-up
1) Prior to connection of the column to the liquid chromatograph, replace
the solvent in the chromatograph with the solvent that is to be used as the
eluent.
Caution: |
In replacing water with, for instance, chloroform,
which is not soluble in water, first replace
the water with acetone and then replace acetone
with chloroform. |
Caution: |
In replacing a solvent containing a salt
with a solvent containing an organic solvent,
use intermediate solvents, first water and
next acetone, and, in replacing a solvent
containing an organic solvent with a solvent
containing a salt, first acetone and next
water. |
Caution: |
If the liquid chromatograph is equipped with
a device in which complete replacement of
the solvent is not possible, e.g., a Bourdon
pressure gauge, disassemble the device and
wash it with the solvent that is to be used
as the eluent. |
2) When a column is stored improperly or for a long time, there may be the air
at the inlet or outlet of the column. In such case, prior to connection of the
column to the liquid chromatograph, remove the air from the column.
Caution: |
Do not heat a column too rapidly or to the temperature higher than the
maximum of the usable temperature. |
Note: |
Remove the cap of column inlet, and heat
the column (usually warming by hands is enough)
until in-column solvent comes out from the
inlet endfitting. Then, connect the column
to the liquid chromatograph, remeve the cap
of column outlet, start the pump and check
that the in-column solvent comes out from
the outlet endfitting. The air in the column
can be completely eliminated by this procedure. |
3) Connect the column to the chromatograph
so that the arrow on the plate will face
downstream.
4) When the column is used at elevated temperature,
heat the column to the desired temperature
flowing the eluent at slow flow rate (0.2
to 0.3mL/min) and after reaching desired
temperature, gradually increase the flow
rate to the flow rate to be used.
5) Some columns need time for stabilization,
and, the first measurement after connecting
it to the chromatograph may be not so good.
3. Dismounting and safekeeping
1) After completing analysis, keep pumping
eluent into the column at a flow rate of
0.2mL/min until the column is cooled down
to room temperature.
2) After the column is cooled down to room
temperature, dissconnect the column from
the chromatograph and cap both ends of the
column to prevent the eluent from leaking
out.
Caution: |
When the in-column solvent is different from
the shipping-solvent from the manufacturer,
replace the solvent before dismounting. |
Caution: |
Cap both ends of the column tightly so that
the in-column solvent may not leak out. |
Note: |
The room temperature mentioned above means the temperature in the room
where the column is stored. |
3) Pack the column in the same way as when it was delivered from the manufacturer
and store it in a room where the temperature fluctuates little (preferably, in
a constant temperature room).
4) When GPC and GFC columns are stored for a long period of time, it is recommended
to store in wet. Connect a Teflon tube of 1/16 inch in outside diameter, 0.8mm
in inside diameter and 500mm in length to the column outlet.
Start pumping the eluent at a flow rate of
0.5mL/min and stop the pump as soon as it
begins to flow out from the free end of the
tube.
Put 80mL of toluene or water into a 100mL
bottle and soak the free end in the toluene
or water to prevent air from entering the
column.
Dismount the column from the chromatograph,
cap the column's inlet end and store it in
a room where the temperature fluctuate little.
The columns to be stored in wet and the solvents
to be used are as follows:
(a) The columns to be soaked in toluene:
GPC KF, K, KD, AT, HT, UT and HFIP.
(b) The columns to be soaked in purified
water (containing sodium azide).
OHpak SB-800 HQ and PRTEIN KW series.
Note: |
The solvent in the bottle should be replaced
once a month with a fresh solvent to maintain
the column performance as designated for
a long period of time. |
5) Stop the pump and leave the column on
the chromatograph, if it is to be reused
within three days.
Caution: |
In case of one or more days suspension of
chromatograph in which a solvent containing
salt was used as eluent, replace the eluent
with purified water, setting the flow rate
at 0.5mL/min. |
4. Use of guard column
1) Install a guard column immediately upstream
of the main column to protect it from cotamination
by the sample.
Note: |
The guard column is intended to maintain
the column performance as designated for
a long period of time and not to improve
its resolution. |
2) Replace the gurad column at regular intervals.
The intervals are different according to
sample property and injection volume.
5. Sample pretreatment
1) Dissolve the sample in the solvent that
is to be used as the eluent.
Note: |
When the eluent is not stable, dissolve the
sample in the eluent taken just before the
injection. This procedure is important to
have a smaller blank peak when RI detector
is used. |
Caution: |
When using gradient elution, dissolve the
sample in the starting eluent. |
Caution: |
Do not inject samples which are insoluble
in the eluent. |
2) Remove extraneous substances and insolubles
from the dissolved sample by passing it through
a 0.45micron filter. Use of disposable filter
is recommended.
3) The use of SPE filter is also useful to remove the extraneous substances in
the sample.
6. Eluent
1) Remove extraneous substances and insolubles
from the eluent by passing it through a 0.45micron
filter. Use of disposable filter is recommended.
2) Throughly degass the eluent. Apply ultrasonic
vivration and degas decreasing the pressure
using aspirator. When degassing of the eluent
is insufficient, stable baseline cannot be
obtained. Degassing is especially important
when RI detector is used for the detection.
The use of
Degasser (eluent degassing device), ERC-3000-alpha is recommended.
Caution: |
The degassing is important when the column
is used at elevated temperature to prevent
air bubble at inside and outlet of the column. |
7. Column testing method
The plate number in the inspection sheet
attached to the column was calculated by
the following formula:
The testing conditions to obtain the plate
number is shown in
Column Testing Conditions.
The chart speed should be 1.5 to 2.0 cm/min.
The sensitivity of the detector should be
adjusted to obtain peak height of about 10cm.
8. Other remark
Caution: |
Do not make an impact on the column because
the packing condition in the column may be
disturbed the the impact. For example, do
not drop a column from a high place. |
Caution: |
Do not bend the column or cause impact on
it. |
Caution: |
Under no circumstances should the end-fittings
of the column be removed as doing so will
cause deterioration of the column performance. |
Caution: |
Do not change the column pressure or flow
rate abruptly when a liquid chromatograoh
is in use. Use a damper-equipped or pulseless
pump to maintain the performance of the column
at the designated level for a long period
of time. |
Caution: |
Do not apply the pressure heigher than 20MPa,
or the column material of stainless steel
may be dtsfigured. |
9. Warranty
1) Showa Denko shall replace any Shodex column,
(a) If found damaged at the time of delivery.
(b) If the plate number obtained by the purchaser
as per the operation manual is significantly
smaller than the one given in the inspection
sheet attached to the column.
Claims must be filed with Showa Denko within
10 days following delivery.
2) The following shall not be subject to
warranty.
(a) Service life
(b) Deterioration of column performance resulting
from improper handling.