- Name of column parts
- Installation and start-up
- Dismounting and safekeeping
- Use of guard column
- Sample pretreatment
- Column testing method
- Other remark
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.
|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.
|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.
|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.
|Do not heat a column too rapidly or to the temperature higher than the maximum of the usable temperature.
|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.
|When the in-column solvent is different from the shipping-solvent from the manufacturer, replace the solvent before dismounting.
|Cap both ends of the column tightly so that the in-column solvent may not leak out.
|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.
|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.
|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.
|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.
|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.
|When using gradient elution, dissolve the sample in the starting eluent.
|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.
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.
|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
|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.
|Do not bend the column or cause impact on it.
|Under no circumstances should the end-fittings of the column be removed as doing so will cause deterioration of the column performance.
|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.
|Do not apply the pressure heigher than 20MPa, or the column material of stainless steel may be dtsfigured.
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.