2.9 Key Points for Analysis
2.9.1 Before Analysis
If an HPLC system is used for analysis, sometimes unexpected results are obtained even if the instruments themselves are functioning normally. Problems can often be prevented by inspecting the instruments before starting analyses. The following are some simple routine precautions for that purpose.
Precaution Remarks
Return the mobile phase to room temperature before use.
If the mobile phase is at a different temperature from the room temperature, it could increase the chance of the baseline drifting or not stabilizing, bubbles forming, or other problems occurring until the mobile phase stabilizes at room temperature.
Note that if an organic solvent is mixed with water, the mixture temperature can deviate considerably from the room
temperature.
Use HPLC grade or better reagents for mobile phases.
If an absorbance detector is used with a lower grade mobile phase than HPLC grade, mobile phase absorbance can overlap with peaks at detected wavelengths, which can interfere with quantitation of target components.
Precaution Remarks
Filter mobile phases and samples.
Debris or other solid or insoluble substances in the flow channels could cause flow channel blockage or scratch stator and rotor friction surfaces in the autosampler high-pressure valve, which could cause premature leakage. Impurities in mobile phases can increase background signal levels, which can cause increased baseline noise, drift, or cause other phenomena, such as negative peaks or unknown peaks during gradient elution. It is recommended that mobile phases and samples with high purity levels are used, that preparations are mixed thoroughly, and then filtered.
Pump solvents at a rate that is within the optimal flowrate range for each column.
Pumping at flowrates outside the optimal flowrate ranges could prevent achieving good separation or it could shorten the column service life. To maintain column performance, it is recommended that the optimal flowrate indicated in the column instruction manual be confirmed and then solvent be pumped within that range. For additional information about using columns, refer to the column instruction manual.
Rinsing the column
If the column is new or if the column is contaminated in a previous analysis, rinsing it. If there is no need to rinse the column, omit rinsing and stabilize the column.
Select appropriate column fittings.
Using fittings not suited to the pressures and mobile phases used during analysis could cause joint leakage or increased pressure due to fitting wear debris.
For low-pressure gradient systems, fill the flow channels for all four solutions with mobile phase.
Bubbles inside the gradient valve or between the solvent delivery pump and the gradient valve can cause inaccuracy or instability in gradient concentrations. Such bubbles can also cause fluctuations or variability in retention times.
2.9 Key Points for Analysis
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Precaution Remarks
Precaut ions for purgin g
Purging with a compatible mobile phase
(1) Prepare a clean beaker and place the mobile phase to use for purging in the beaker.
(2) Pull the suction filter out of the reservoir bottle and insert it into the beaker from step (1). Then vibrate the filter section to rinse the suction filter.
(3) Purge the solvent delivery pump.
(4) Insert the suction filter into a new mobile phase reservoir bottle and then purge the solvent delivery pump. Next, purge the flow channels by pumping the solvent up to the column connection port.
(5) Lastly, connect the column and use the solvent delivery pump to purge all the flow channels.
Purging with an incompatible mobile phase
First purge the channels with an intermediate solution that is compatible with both the new and old mobile phases (with 2-propanol, for example), then purge the channels with the new mobile phase. In each case, follow steps (1) to (5) in Purging with a compatible mobile phase.
Purging with a buffer solution mobile phase
Contact between new and old mobile phases that contain a buffer solution could precipitate salts that cause flow channel blockage.
If a buffer solution is used for either or both of the new and old mobile phases, then first purge the old mobile phase with distilled water as the intermediate solution and then purge the intermediate solutions with the new mobile phase. After purging, pump the distilled water through the channels for a while (at least 20 minutes at 1 mL/min).
Be sure to also rinse the plunger seal using the automatic seal rinsing kit at that time.
Fill the flow channels with liquid.
If the suction tube is not filled with liquid or contains large bubbles, adequate purging may not be possible during auto-purging. If so, purge it manually.
Purge the instrument before use.
Bubbles can sometimes form in the solvent delivery pump head if the instrument has not been operated for several days or there is a large difference between day and night-time room temperatures. Purging the channels when there are bubbles in the pump head could cause a solvent delivery problem.
Purge the channels for about five minutes to flush out any bubbles before starting solvent delivery. Purging actions can be automated using the startup function.
When the high-pressure gradient mixer attached and only one liquid is used, the flow path on the non-used side is also filled with the same solvent.
If the flow path on the non-used side is filled with different solvent and it is connected to mixer, solvent on the non-liquid side may enter the liquid line due to the pressure shock at the time of sample injection, and the ghost peak may occur or cause baseline fluctuations.
Precaution Remarks
Check the automatic seal rinsing kit rinse solution.
If the rinse bottle itself in the automatic seal rinsing kit becomes white with contamination, rinse off the contamination.
If the 10 % aqueous 2-propanol solution in the rinse bottle becomes contaminated, replace it with new solution. If
contamination is significant, daily replacement is recommended.
Even if contamination is not noticeable, replace it with a new solution weekly.
"Replacing the Rinse Solution in the Solvent Delivery Pump Automatic Seal Rinsing Kit" P.61
Check the autosampler rinse solution.
Performing analysis without the rinse solution properly placed could decrease sample injection accuracy. Use an autosampler rinse solution that is the same as the mobile phase or has a similar composition. However, be sure to remove any salts from the rinse solution. Also, since the rinse solution contacts the mobile phase inside the high-pressure valve, select a rinse solution that does not form salt precipitates when exposed to the mobile phase solvent.
Purge autosampler measuring flow channels.
If the instrument has not been operated for several days or if there is a large difference between day and night-time room temperatures, bubbles can form inside measuring flow channels, which could decrease sample injection accuracy. Purge the measuring channels to eliminate the bubbles.
Check the column oven temperature setting.
Depending on the instrument operating environment, the temperature might not be controlled correctly to the specified temperature setting. Models CTO-40C CL is able to control temperature from 10 ℃ below room temperature. For example, if the room temperature is about 25 ℃, then temperature cannot be reliably controlled to a 10 ℃ temperature setting.
Check the wavelength accuracy of the detector.
If the wavelength accuracy decreases, it could decrease inter-day reproducibility. When UV detectors are switched ON, they automatically check the wavelength using the absorption line (240.4 to 241.7 nm) of the internal filter (holmium filter) and the bright line for the D2 lamp (deuterium lamp) at 656.1 nm.
If the wavelength has shifted, a warning message is displayed.
If a warning message is displayed, purge the flow cell with purified water and then check the wavelength again. If the wavelength is still out of position and the warning message remains displayed, it indicates that an instrument failure has occurred.
Check the tank for liquid waste and drain tube.
Check the available capacity of the tank to prevent the liquid waste from overflowing during analysis.
Do not allow excess slack of a U shape in the drain tube. Also, do not allow the drain tube to be below the waste liquid level in the tank.
n
Replacing the Rinse Solution in the Solvent Delivery Pump Automatic Seal Rinsing Kit
The automatic seal rinsing kit is used to automatically rinse the plunger seals and plungers in solvent delivery pumps. Select the rinse solution used for rinsing as described2.9 Key Points for Analysis
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Mobile Phases with Water, for Reversed Phase, Ion-Exchange, etc.
Use 10 % aqueous 2-propanol solution.
Mobile Phases without Water, for Anhydrous Normal Phase, etc.
Do not use a rinse solution.
If using the system for the first time or replacing the rinse solution, use the following procedure to suction the rinse solution into the flow channels from the rinse solution bottle. Use the same type of procedure even if rinse solution will not be pumped.
If 10 % aqueous 2-propanol solution in the rinse bottle becomes contaminated, replace it with new solution. If contamination is significant, daily replacement is recommended. Even if contamination is not noticeable, replace it with a new solution weekly.
1 If connected to a column, disconnect the column and replace it with the coupling (B). Connect the coupling to the detector inlet tubing (A).
2 Remove the cap (B) from the rinse
bottle (A) in the automatic seal rinsing
kit and fill the bottle with 100 to 200
mL of 10 % aqueous 2-propanol
solution.
3 Attach cap (B) from the automatic seal rinsing kit to rinse bottle (A) and remove the FEP tube (E)
(upward-facing U-shape) on the outlet end from the rinse bottle.
4 Attach a syringe needle (C) to the end of the FEP tube (E) (upward-facing U-shape) and use the syringe (D) to suction the 10 % aqueous 2-propanol solution into the rinse flow channels.
5 Disconnect the syringe needle (C) and the syringe (D) from the FEP tube (E).
6 Set the flowrate to 3 mL/min and start pumping the solution.
7 Verify that rinse solution is discharged from the end of the FEP tube on the outlet end of the automatic seal rinsing kit.
8 If rinse solution is discharged, stop pumping.
9 Return the automatic seal rinsing kit to its previous configuration.
2.9 Key Points for Analysis