Contents:1. Column Components
2. Column Installation
3. Sample Preparation
5. Column Cleaning
6. Column Storage
7. Column Inspection
8. Column Disposal
9. Additional Warnings
2. Column Installation
(1) 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 (distilled water), first run methanol and then introduce water.
e.g. When replacing highly concentrated buffer solution or salt solution to water/acetonitrile, first run water and then replace it with the eluent.
|Attention!||If the eluent left in the system is not compatible with the column to be used, it may damage the column.|
|Attention!||A drastic change in the eluent compositions may remove substances adsorbed on the system and they may enter and deteriorate the column.|
(2) Connect the column to LC system by following the “flow direction arrow” (→) indicated on the column adhesive label. If guard column is used, position the guard column in front (before the inlet) of the analytical column.
(3) 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.
(4) Set the initial flow rate at half of the recommended flow rate 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.
|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.|
|Attention!||When restarting the system after column installation or after holding the eluent flow, start the system at half of the recommended flow rate. A rapid increase in pressure can damage the column.|
|Attention!||If the column was used at an elevated temperature, lower the flow rate to half of the recommended flow rate 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.|
3. 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. When gradient elution is used, it is recommended to use the initial eluent to prepare the sample.
(2) Filter the sample solution using disposable 0.45-μm filter to prevent the column from clogging or deteriorating.
(3) Use of guard column is recommended to protect the analytical column.
|Note||The purpose of the guard column is to maintain the performance of the analytical column for an extended time period and it is not intended to improve the separation ability.|
(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) 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.
(4) Start the system at half of the recommended flow rate when replacing the solvent.
(5) When using a gradient method, changes in the eluent compositions may increase the column backpressure. Adjust the flow rate and column temperature so that the column backpressure remains below the usable maximum pressure.
|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.|
|Attention!||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.|
|Attention!||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.|
|Note||Use of on-line degasser is recommended.|
|Note||It is recommended to set the pump limiter to avoid exceeding the maximum pressure.|
Problems in peak shapes and elution time 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 the washing solution flow from the column outlet go directly into the waste container and not let the solution go through the detector.
(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) Please refer to the individual products' operation manual for the product's-specific column cleaning method.
6. 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).
|Attention!||Never allow inside of the column to dry. It can damage the column.|