2.9 Key Points for Analysis
2.9.3 Precautions When Replacing the Mobile Phase
2.3 Placing Mobile Phases and Autosampler Rinse Solutions
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1 If a column is connected, disconnect the column and replace it with the coupling (B) that is included with the tubing kit.
The process is unnecessary if the column and mobile phase are not changed.
2 Click [AutoPurge] in the LabSolutions [Realtime Analysis] window.
If the column is disconnected, clear the [Warm up] and [Init conc-replacement] checkboxes on the [AutoPurge] tab page in method settings.
"2.2.1 Editing Methods" P.15
3 When a window appears to confirm executing auto-purging, click [OK].
Auto-purging is executed based on method settings.
2.3 Placing Mobile Phases and Autosampler Rinse Solutions
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2.3.3 Manual Purging the Solvent Delivery Pump
If adequate purging cannot be achieved with auto-purging or a suction tube does not contain liquid, for example, use the following procedure to manually purge with a syringe.
1 Insert the suction tube for the flow channel to be purged into the reservoir bottle containing the mobile phase and place the bottle on the reservoir tray.
2 Pull the solvent delivery pump drain
tube out from the waste tank and
insert a syringe, with a needle
attached, into the end of the drain
tube.
3 Open the right panel on the solvent delivery pump and open the drain valve (A) by turning it 90 degrees counterclockwise.
4 Suction mobile phase into the syringe.
In general, suction about 10 mL.
5 To manually purge other mobile phase ports as well, switch to a different mobile phase port and repeat the same process to suction mobile phase with the syringe.
6 When finished manually purging all flow channels, close drain valve (A) by turning it clockwise all the way until it stops.
7 Close the right panel on the solvent delivery pump.
8 Remove the syringe.
2.3 Placing Mobile Phases and Autosampler Rinse Solutions
2
9 Perform each auto-purging step for at least three minutes.
Continue auto-purging each manually purged mobile phase port until all bubbles disappear from the flow channel.
"2.3.2 Performing Auto-Purging" P.24
• Manual purging does not purge the flow channels downstream from the solvent delivery pump. To purge the flow channels downstream from the solvent delivery pump, perform auto-purging for at least three minutes on each channel, until bubbles disappear from the flow channel.
• To manually purge , do not leave the column disconnected but connect the tubing with the coupling. Because the mobile phase flows into the analysis flow channel, liquid leaks in the column oven when the tubing is not connected, causing a leak error.
4 Connect the tubing to the column.
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Connect the stainless steel tubing (B) from the autosampler to the inlet end (a) of the column (A).Refer to the table regarding the fittings to use.
Part Name P/N Remarks
Male Nut Fittings (set of
2) 228-45717-01
These are hand-tightened fittings. They can be used for analysis at pressures up to 35 MPa.
Male Nut, 1.6 MN 228-16001 Male nut and ferrule are made of stainless steel.
"Connecting Stainless Steel Ferrules" P.295
Ferrule, 1.6F 316L 228-16000-10 UHPLC Fitting
(set of 1) 228-56867-41 Fittings are reusable up to about 20 times at the 130 MPa pressure capacity.
For installation instructions, refer to the instruction manual included with the fittings.
"Connecting UHPLC Fittings"
P.296 UHPLC Fittings
(set of 10) 228-56867-43
Nexlock Fitting 228-62544-90
Dedicated Nexlock hand-tightened fitting that is reusable up to about 100 times at the 130 MPa pressure capacity.
For tightening instructions, refer to the Instruction Manual (228-92510)
included with the fittings.
In some cases, the detector cell temperature cannot be controlled, such as when both the column oven temperature and the solvent delivery pump flowrate are high (with a column oven temperature over 70 ℃ and the solvent delivery pump flowrate over 3 mL/min, for example), so that the cell temperature rises above 50 ℃. In such cases, switch OFF cell temperature control and wait for the temperature to stabilize before resuming analysis (about 60 minutes).
Switching the cell temperature control OFF can increase the baseline fluctuation range in response to room temperature fluctuations. Keep the room temperature constant to stabilize the baseline.
2
Use the male nut (b), included with the detector, to connect the tubing (B) from the detector cell inlet to the column (A) outlet."Connecting Tubing from the Column Outlet to the Detector Inlet" P.303
Attaching Multiple Columns
Multiple columns can be attached using optional column clamps. Attach columns using a number of column clamps appropriate for the length and number of columns.
Flow can be switched automatically between two to six columns by installing an optional FCV valve.
5 Close the column oven door.
Fully close the column oven door.
To make sure the door does not open accidentally during analysis or open from contact with tubing inside the column oven, close the door until it makes an audible locking click sound.
2.4 Attaching the Column
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2.4.2 Warming Up the System
Auto-purge and warm-up processes are performed automatically when the system is started up. The warm-up process ensures solvents can be pumped without applying undue pressures on the column.
"3.1 Startup and Shutdown Functions" P.70
1 Create a method that specifies warm-up parameter settings.
Confirm that the [Warm up] checkbox is selected on the [Advanced]-[Autopurge] tab page. If auto-purging was already performed, clear the checkbox.
User-specified parameter settings can be used for startup by editing the startup method.
"2.2.1 Editing Methods" P.15
2 Click [Startup] on the toolbar in the LabSolutions [Realtime Analysis] window.
3 When a window appears to confirm executing startup, specify settings and click [OK].
Specify the date and time to begin startup, select the created method file, and select the [Perform LC Autopurge] checkbox.
If the [Perform LC Autopurge] checkbox is selected, the auto-purge and warm-up actions specified in the selected method begin. If not selected, then [Pump] and [Oven] actions are started immediately.
2.4 Attaching the Column
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3 Fill the sample vial or well in a microplate or deep-well plate with the sample.
Use genuine Shimadzu brand or Shimadzu-recommended sample vials and microplates.
Using non-genuine or non-recommended vials or plates could cause flow channel blockage from septum debris or could prevent the needle from piercing the septum.
For sample vials with an independent septum and cap, attach the cap with the PTFE side of the silicone septum facing downward (toward the liquid).
• Installing the PTFE side in the opposite orientation might result in the sample solvent melting the silicone rubber.
• On a sample vial, attach cap (C) with the PTFE film side (a) (dark color) of septum (B) oriented downward.
If samples that contain a volatile acid are used, be sure to install caps on sample vials. Be sure to install a mat or sheet on microplates.
If not installed, volatile acids could corrode metal parts inside the instrument, resulting in an instrument failure.
Fill sample vials to the level shown below. Filling them to a higher level could prevent proper suctioning.
2.5 Placing Samples
2
Three different types of plates can be loaded on the sample rack. Available plates include sample vial plates (1, 1.5, 4, or 10 mL vials or microtubes), microtiter plates (MTP), or deep-well plates (DWP).
The top of each plate is marked with numbers for each sample vial fill position and well position. Those sample numbers are specified when specifying parameter settings.
Do NOT switch OFF the temperature control mode on models with temperature control functionality.
Due to the high airtightness of spaces inside the instrument, operating the system with temperature control OFF for a while could cause temperatures inside the instrument to exceed about 30 ℃, due to heat generation from drive motors, which could affect samples. Heating samples can be prevented by leaving the temperature control ON and specifying an appropriate temperature setting.
Be sure to place sample racks on the autosampler when cooling samples.
If the sample racks are left without being placed, they could generate
condensation water, which could damage the instrument or mix with samples.
An alarm is displayed after five minutes if the sample rack is left pulled out during temperature control. After ten minutes, the temperature control mode is forcibly shut OFF.
Plates for 1.5 mL sample vials are included as standard. To use other plates, be sure to specify the plate ID, specify the needle stroke distance, and adjust the plate position.
1 Pull out the sample rack (A).
Unlock the sample rack by pulling the lever (a) inside the handle.
Always pull the lever to unlock the sample rack before pulling out the rack.
Not doing so could cause part damage or equipment failure.
Always pull out the sample rack by pulling horizontally with one hand and supporting the rack with the other hand.
2.5 Placing Samples
2
2 Place the sample plates filled with samples on the sample rack.
• Sample vial plates: Place vial 1 in the far left position.
Microplates and deep-well plates: Place well A01 in the far left position.
• Without Plate Changer: The sample rack numbers 1 to 3 correspond to the front section, central section, and rear section of the sample rack.
With Plate Changer: For the sample rack numbers, the front section of the sample rack is assigned with No. 101 and the central section No.
102.
• To set the multiple vials on the sample plate, remove the sample plate from the sample rack and set them.
Place sample plates positioned toward the right-front corner of the instrument.
If the sample plate is shifted out of position, with respect to the sample rack, then the needle cannot be lowered properly into the specified vial or well, which could cause needle damage or other equipment failure.
3 Insert the sample rack horizontally all the way in along the sample rack guides.
Insert the sample rack all the way in until it contacts the far side.
If it is not inserted adequately, the needle could accidentally pierce the wrong position, which could damage equipment or jam the needle.
Also, the needle could pierce the sample cap and cause an error during sample suctioning.
For models that include temperature control functionality, it can increase the risk of forming condensation inside the unit, due to decreased airtightness of the seal.
If a microplate is placed on a sample rack, insert the rack horizontally, so that the samples do not spill.
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Removing a Sample Rack during Analysis
If the status indicator is illuminated yellow, without flashing, then the sample rack can be removed even during analysis.
Do NOT insert a hand inside the instrument during analysis.
The Z-mount and needle keep moving even when the sample rack is removed.
Inserting a hand inside the instrument during analysis is dangerous.
Do NOT remove the sample rack if the status indicator is flashing yellow.
Doing so could damage the needle. The status indicator on the operation panel flashes yellow during injection actions.
• Attempting to remove the sample rack when the status indicator is flashing yellow will pause injection actions. Injection actions will resume when the sample rack is returned to its proper position. Injection actions will not start if a sample plate of samples to be analyzed is not placed on the sample rack.
Injection actions will resume when a sample plate is placed on the sample rack.
• If the sample rack remains removed for five minute or more during temperature control actions, an alarm is displayed, followed by the
temperature control function automatically shutting OFF ten minutes later.
Temperature control function automatically restarts when the sample rack is returned to its proper position.
• If the main power switch is switched OFF on the back of the instrument during sample injection (while the status indicator is flashing yellow), the needle might stop in the lowered position in some cases. Pulling out the sample rack in that position could bend the needle.
2.5 Placing Samples
2
2.6.2 Performing a Single Run Analysis
Analysis is started using one method file.
n
Performing a Single Run Analysis in the LabSolutions [Realtime Analysis] Window
1 Specify analytical conditions.
"2.2 Specifying Analytical Conditions" P.15
2 In the LabSolutions [Realtime Analysis]
Window, click the (Single Run) icon.
The [Single Run] window is displayed.
3 Specify settings for a single run analysis.
[Single Run] Window
2.6 Performing a Single Run Analysis
2
No. Name Description
1 Sample Name Specifies the user-specified sample name.
2 Sample ID Specifies the user-specified sample ID.
3 Method file
Displays the name of the method file currently open in the [Data Acquisition] window. This is a read-only setting.
4
[Data File]-[Create into]
Specifies where to save data files. This field is not displayed in DB and CS versions of LabSolutions.
5 Data File Name
Specifies the data file name. If the [Auto-Increment]
checkbox is selected, a number is automatically added to data file names.
6 Report
If a report format file is specified and this checkbox is selected, a report of the acquired data is output when the analysis is finished.
7 Data Comment User-specified comments can be entered in this field.
8 Vial
Specifies the number that indicates the sample vial position and the number that indicates the position of wells filled with samples.
9 Injection
Volume Specifies the injection volume.
10 Tray Specifies the position of trays (or plates) where samples are placed.
4 Click [OK].
Return to the main analysis window and start the analysis.
“Running” is displayed at the top of the main analysis window so that the analysis status can be confirmed.
• To stop analysis in an emergency, click [Stop].
• If an emergency stop occurs during injection, the needle is moved to the drain port to discharge any suctioned sample remaining inside the needle and then the needle rinsing action starts.
The analysis is finished when “Ready” is displayed in the main analysis window.
The following describes the procedure for postrun analysis of the acquired data using LabSolutions.
Analysis parameter settings can also be specified before data acquisition.
1 Start LabSolutions and click [Postrun]
in the main window.
2 Double-click the [Postrun] icon.
In LabSolutions DB and CS versions, a window for selecting the project is displayed. Select the project where the data to be analyzed was saved.
2.6 Performing a Single Run Analysis
2
3 Select and double-click the data file to be analyzed.
• Data files can be listed by displaying Data Explorer and clicking the [Data] tab.
• In LabSolutions DB and CS versions, the files can be filtered based on file name, instrument type, analysis date, or other criteria.
4 Switch the Method View to the [Edit] mode and click the [Compound] tab.
5 Enter the [Name], [Ret. Time], and calibration point [Conc.] setting values.
• To change how the baseline is determined or if peaks were not detected correctly, click the [Integration] tab and correctly specify peak integration parameter settings or perform peak integration manually.
• Retention times can be entered automatically by clicking a peak in the [Chromatogam View] with the [Ret. Time] cell selected.
• To change the number of calibration curve concentration points, change the [# of Calib.
Levels] setting on the [Quantitative] tab page.
• Compound table setting parameters differ for quantitation methods other than [External Standard].
For more details about analysis parameters, refer to Help in LabSolutions or the LabSolutions Instruction Manual—Operators Guide.
6 When finished editing method parameters, save the data by overwriting.
2.6 Performing a Single Run Analysis
2
7 To apply the specified analysis
parameter settings to the method, click [Apply to Method] on the assistant bar.
8 Save the method by clicking [File]-[Save Data and Method File].
When the [Select Method Parameters]
window is displayed, select the checkboxes for parameters to apply to the method.
By using the method settings specified here for the remainder of the analysis, data analysis results can be checked immediately after the analysis is finished.
The following describes the procedure for batch analysis using LabSolutions, up to the point the batch analysis is started.
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Performing Realtime Batch Analysis in the LabSolutions [Realtime Analysis] Window
1 Specify analytical conditions.
"2.2 Specifying Analytical Conditions" P.15
2 Click [Quick Batch] in the LabSolutions [Realtime Analysis] window.
The [Quick Batch] window is displayed.
3 Specify the quick batch settings.
[Quick Batch] Window
No. Name Description
1 Method File Select the method file to use for batch analysis.
2 Data File Name
Specifies a file name for data acquired.
If [(Auto Filename)] is displayed, then the [Create filenames automatically with] checkbox is selected on the [Settings]-[Data File Name] tab page. Settings can be specified manually by clearing the checkbox.
3 Report
If a report format file is specified and this checkbox is selected, a report of the acquired data is output when the analysis is finished.
4 Sample Name Specifies the user-specified sample name.
5 Sample ID Specifies the user-specified sample ID.
6 Injection Specifies the number of injections and injection volume per sample.
7 Plate Diagram
Click the vial number for starting the analysis and drag the pointer to the vial number for ending the analysis to specify the starting vial number, ending vial number, and the number of samples. These settings link to the [Vial
Number]-[Start]-[End], and [Number of Sample] settings.
8 Plate Type Select the plate type.
9 Tray Name Select the tray number. This setting is linked to the [Tray name]
setting on the left side of the [Batch Setting] window.
10 Sample Type
Select the sample type. Available sample types are standard, unknown, and control samples. This setting is linked to the [Sample Type] setting on the left side of the [Batch Setting]
window.
2.7 Performing Realtime Batch (Sequence) Analysis
2
4 Check the batch table content and make any corrections necessary.
To add a batch row with no injection, set the [Vial Number] value to “-1”.
5 Click [Start].
If the batch file is not saved, the [Save Batch File As] window is displayed. If the batch file is saved, the realtime batch analysis is started.
4 Drag and drop the batch file that contains the data to be analyzed to the [Quantitative Results View].
• Data files can be listed by displaying Data Explorer and clicking the [Batch] tab.
• In LabSolutions DB and CS versions, the files can be filtered based on file name, instrument type, analysis date, or other criteria.
• To select and analyze multiple data files, drag and drop the data files to the [Quantitative Results View].
5 Files included in the batch file are displayed in the [Quantitative Results View].
To add parameters to be displayed, right-click on the [Quantitative Results View] and select [Table Style].
If method parameter settings need to be changed, switch the [Method View] to the [Edit]
mode and then change the setting values. Parameter setting changes are applied to all data displayed in the [Quantitative Results View].
6 Specify calibration curve [Level#] and [Cal. Point] settings and then confirm that the calibration curve is created accordingly.
Quantitation values for unknown samples are calculated based on the created calibration curve.
2.7.3 Outputting Summary Reports
1 Check the [Quantitative Results View] content and then click [Summary Report]
on the assistant bar.
2.7 Performing Realtime Batch (Sequence) Analysis