1. Purpose
Describes Standard Operating Procedures for Waters Validated Liquid Chromatography.
2. Scope of application
laboratory
3. Responsibility
Laboratory technicians should strictly follow the operating procedures, and the QC supervisor is responsible for supervising the implementation of the procedures.
4. Definition
HPLCHighPerformanceLiquidChromatography, effective liquid chromatography.
5. Safety Precautions
5.1 Organic solvents are harmful to health, and the mouths of each solvent bottle should be tightly sealed.
5.2 Pay attention to check that the waste liquid in the waste liquid bucket should be protected from light to prevent the waste liquid bucket from overflowing.
6. Procedures
6.1. Composition of WatersHPLC: liquid reservoir, infusion pump, automatic sampler, separation column (column thermostat), ultraviolet Detector control and data processing system (Millennium32 chemical workstation).
6.2. Opening and closing sequence of HPLC system
6.2.1 Open sequence
For the split type: please turn on the switch in the order of pump, sampler, column thermostat, and Detector, and turn on the Millennium32 ChemStation after each part shows that the initialization is complete.
Connection between the pump and the chromatographic workstation: For the 510 pump, because the pump is in one-way connection with the chromatographic workstation, if you want the pump to be controlled by the workstation in the Remote state, you must first press the Menu key on the keyboard of the pump until the display screen switches to In Mode state, its state is Loca, press Edit/Enter key, there is a flashing cursor display in front of L of Local, press key, Local is converted into Rem, then press Edit/Enter key, this command has been entered, and finally press Stop /Run key to make the pump in the Run state, then the connection between the pump and the workstation is successful. Any action of the pump can be controlled by the workstation. It should also be noted that this step must be performed every time the computer is turned on.
For the Alliance2690 system: first turn on the power switches of the Alliance2690 mainframe and the Detector, and then enter the Millennium32 chemical workstation after the initialization is completed.
Requirements for mobile phase: For liquid reagents, chromatographic pure reagents should be used as much as possible; for solid reagents used, analytical pure reagents should be used; for water, ultrapure water treated by a pure water machine should be used; mobile phase must be used before use. It can only be used after filtering through a 0.45m filter membrane (the filter membrane is divided into aqueous phase membrane and organic phase membrane, make sure to use the correct filter membrane, see the attached table) and ultrasonic vacuum degassing. It is strictly forbidden for the mobile phase containing salt to be unfiltered Use directly.
Good operating habits should be developed:
Standardize the operation and establish the concept of protecting the entire HPLC system, especially for infusion pumps, chromatographic columns, and Detectors, they should be washed in time after use to avoid contamination and shorten their life or damage, especially if the operator uses a mobile phase containing salt , more attention should be paid to the cleaning steps: first rinse with degassed pure water at a flow rate of about 1ml/min for 30 to 60 minutes, then rinse with an aqueous solution containing more than 50% methanol or acetonitrile at the same flow rate for about 20 to 30 minutes .
If you want to remove the column, after the above washing process, wash it with pure methanol or acetonitrile at a flow rate of 1ml/min for 20~30 minutes, and tighten the plug immediately after the column is removed.
The change of the flow rate should be in a stepwise increasing or decreasing manner. When the pump is started, it should be slowly increased from a small flow rate to the required flow rate within 2 minutes. The same is true when the pump is turned off (the flow rate decreases). Avoid sudden changes in flow rate.
6.2.2 Closing sequence
First exit Millennium32 ChemStation, and then close other parts of the system.
6.3.Millennium32 ChemStation
Millennium32 Chromatography Management System is a comprehensive system integrating data acquisition, processing, HPLC system control, result management and report printing. The following focuses on the basic operating knowledge of the system.
6.3.1 Entry of Millennium32 ChemStation
Open the chemical workstation, the system automatically enters the ProgramManager interface, double-click the portrait of Millennium, then enter the Millennium interface, double-click the SessionManager icon, the system will automatically initialize, so that the workstation and the entire HPLC system can be connected. After initialization, enter your UserName and Password according to the system requirements, and you have entered the system at this time.
6.3.2 Entry and establishment of Project
Project entry: In the SessionManager window, click the Project portrait (or select Projects from View), in which there are already built Projects, double-click the Project portrait you need, and then perform the work you want to do.
Project establishment
Select New from File to display the NewProject screen.
Enter your Project name in ProjectName.
Select filterandfields or Methods in Copy in the InitiallzeProject project.
Select an existing Project to copy in FromProject.
Choose Tablespacesize.
Select OK, save and exit.
When the Millennium software is creating a new Project, it will display the percentage of the new Project completed on the screen in time. When it reaches 100%, a new Project portrait will appear on the SessionManager screen.
6.3.3 General process for collecting data
The general process of using Millennium32 to collect data is as follows: establish the instrument method of the instrument required to collect data in the Project window; establish a method group including this instrument method; use this method group to collect data.
This section includes: Establishing an instrument method, establishing a method set, and collecting samples.
Create/modify instrument methods
From the SessoinManager window, open the desired Project.
Select the Method button and a portrait of the existing method appears on the screen.
Select File from the command menu, and then select NewInstrumentMethod among them, and the SystemOpen dialog box appears.
Select the name of the chromatographic hardware system that needs to be used to collect data in the catalog, such as: System_1. Press OK, then the InstrumentMethod window is displayed.
The upper part of the window is a portrait of all the instruments of the system. Use the mouse to activate the instrument components that need to set instrument parameters, then double-click on the instrument portrait, and the InstrumentDialogBox screen will appear.
Set operating conditions for individual components, or change previous parameters by entering new ones. Example: To define pump parameters, first double-click on the SIM/PCM portrait. Fill in the pump parameters and press OK.
When all parameters are set, press OK. That is, return to the InstrumentMethod window. Select SaveAs in File to save the instrument method file, if necessary, give a file name, and press OK.
Select Exit in File to return to the Project window. The new instrument method portrait appears under this window.
Establish method group (MethodSet)
After the instrument method is compiled, it needs to be stored in a method group before it can be called in QuickSetControl.
Select method portrait under the Project window.
Select NewMethodSet in File to display the MethodSet dialog box.
在此画面的InstrumentMethod一栏，选择已确定的仪器方法。其他项目可空着不填。然后选File，SaveAs。输入方法组名后，按OK。
选File，Exit。即退回到Project窗口。新设置的方法组肖像即出现在此窗口下。
收集样品：完成以上各步骤后，即可收集样品。
在Project窗口下选择QuickSetControl肖像。出现SystemOpen对话框。
选择建立仪器方法时确定的色谱硬件系统，按OK。即出现QuickSetControl窗口。
在此窗口下的SampleLoadingTable中用鼠标器按第一行，这时Millennium软件会自动填充各项初始参数。通过输入新值可更改表中的各项参数，表中的瓶号(对于有效液相色谱仪的进样表，除另有规定外，按括号法安排进样顺序。即第一针为标准品1作为系统适用性共进6针，要求RSD不大于2%，随后为标准品2，进样次数为2针，然后为样品1到样品3，各进2针。样品3后为标准品1，依次类推，直到将所有样品都安排在进样表中)进样次数，进样体积，运行时间等项目对自动进样器有控制作用。
在MethodSetReport一列中选择在前面刚刚建立的方法组。
在运行方式(mode)中选RunOnly。
选择进样方式：菜单mode下选单个样品或样品组进样。
将SampleLoadingTable中第一行点黑。击SetupInstrument按钮，启动系统。
系统启动后可击baselinemonitor图标，即可观察基线。
待基线平稳后，击绿灯或样品盘肖像准备进样。出现Injectorwait提示信息，随即进样。
在样品测试过程中，系统会在QuickSet窗口下显示瓶号和进样次数及运行时间。并可在此窗口下观察检测器的输出情况。屏幕上显示的检测器输出是自动满量程方式(即自动按最大信号满格输出)。
当收集完色谱数据后，选择File，Exit。退出QuickSet窗口。样品收集完成后，Millennuium32会自动存贮收集的数据。
6.3.4建立数据处理的方法
在Millennuium32中，完成不同的任务需编辑不同的方法，如仪器方法(收集数据)，处理方法(定性定量分析)，报告方法(打印实验报告)等。
本部分包括：
*重新调很好谱峰进行观察。
*谱图积分及确定积分表中的参数。
*输入组分表资料。
*存贮数据处理方法。
调阅色谱图
进入在其中收集样品的Project，在此窗口下选Channel肖像。
在ChannelViewTable中选择欲进行色谱分析的文件行并使其变黑。
在Tools下选择Review肖像，这时在ReviewData窗口下即显示出你调入的色谱图。
选File，NewMethod。在NewMethod对话框中指定数据处理的类型，如LC，选OK。
色谱图的积分
选View，IntegrationTable。在屏幕下部即出现IntegrationEventTable。
在PeakWidth，MinimumArea，MinimumHeight方框中输入适宜的数值。
按Integration按钮，观察积分情况，并适当进行积分参数的调整。
输入组分资料
选View，ComponentTable。即可见到空的组分表。
选Options，FillTableFromResult，即可自动填充积分完毕的色谱图的组分表。也可用手动方式修改表中的参数。
按Calibration/Quantitate后，选View，ResultTable。即可看到积分的结果。
存贮数据处理方法
在ReviewData窗口下，选File，SaveMethod。在ProcessingMethodSaveAs对话框的窗口下输入数据处理方法名称。
按OK，贮存ProcessingMethod。在组分表的上方可看到新的ProcessingMethod的名称。
选File，Exit。即可退出ReviewData窗口，回到Project窗口。
6.3.5重新观察色谱结果
进入你所需的Project窗口。击ResultView肖像。
在ResultViewTable中选择你想观察的色谱数据。
击Review肖像，你所选择的第一个色谱数据的谱图将在ReviewData窗口下，其结果将显示在ResultTable中的第一行。
按NextCh/Inj的方框即可看到下一针进样的有关资料。
选File，Exit。即可回到Project窗口。
6.3.6报告方法的编辑及试验报告的打印
启动报告出版系统
进入“PROJECT”窗口。
在Views下按一下方法肖像。
选择File，NewReportMethod。这时即可编辑新的报告方法。
在报告排版窗口中，右边为一张模拟的白纸，左上方的列表为排版时可放置在右边纸上的项目。如图谱，积分表，样品信息，仪器方法等，左下方为报告头标和脚标（如土〕。选择Individual为每个结果有一个报告，选SummaryofResultSetsAll方式为打一个集中的报告。按OK。
设计报告格式
按一下ChromatogramGroups肖像。在ChromatogramGroups列表中选择Auto-scaledChromatogram。并在报告页面上按一下，用鼠标器将此图形放在画面合适的位置上。
用鼠标器将箭头放在画面的边缘，当箭头变成双箭头时将图形放大或缩小。
选择Format，Landscape。可将图谱垂直放置。
按一下MethodGroup肖像，从MethodGroup的列表中选择InstrumentMethod，并在报告页面上的合适处按一下。这时InstrumentMethod方框将出现在报告页面上。
按一下TableGroups肖像，在TableGroups，并在报告格式上的合适位置上按一下。
选择Format，PageSize。可改变报告尺寸的大小。
选择Customize，Header。即出现Header窗口。在此窗口下选择Generalinformation肖像。在GeneralinformationField列表中按一下Date。这时在FieldName的方框中即出现Date方框。可用鼠标器将此方框移动到合适的位置。
可用文字，箭头，方框等工具在Header窗口下增加你感兴趣的内容。
选File，Save。输入Header的名称（如：My_Header〕，按OK，返回到Header窗口。
选File，Exit。即回到ReportPublisher窗口。
在Customize，Footer。即出现Footer窗口。
按一下Generalinformation肖像，在GeneralinformationField列表中按一下PageNumber的方框。将此方框放在报告页面的注脚位置上。
选File，Save。输入Footer的名称(如：My_Footer)，按OK，返回到Footer窗口。
选File，Exit。回到ReportPublish窗口。
在Footer列表中选刚编好的脚标。
存储报告方法
选File，Save。输入此报告的方法名(如：My_Report)，按OK，返回到ReportPublisher窗口。
选File，Exit。即回到PROJECT窗口。在此窗口下即可看到My_Report报告方法的肖像。
报告格式预览：报告格式设计好后，在打印之前，可在此预先看到打印效果，如需修改可随时切换到报告出版窗口，边看边修改，直到满意为止。
在PROJECT窗口下，选Result肖像，即出现ResultViewTable表。
在此表上选择需要报告的结果文件，使其变黑。
按一下Preview肖像，即出现ReportMethodSelection窗口。
从目录上选择刚才已建立好的报告方法，按OK。这时即出现刚才所选结果的报告。
在上画面上按NextPage或PrevPage即可看到报告的下一页或上一页。
按方法编辑肖像即可对当前方法进行编辑。
按打印肖像，即可将此报告打印出来。
选择File，Exit。即可退出报告画面，回到“PROJECT”窗口。
6.3.7批量进样结果的处理及打印报告
当已有确定的处理方法后，可用此方法处理数据。
在ChannelViewTable表中选择你要的样品行并将其按黑。
Click on the ProcessandReport icon under Tools. Then the ProcessandReportOptions dialog box appears.
In the processing method column, select UseSpecifiedProsessingmethod and specify the processing method, and remove the cross of the Print selection box. Press OK. At this time, the data you selected will be processed, and the ResultGenerator portrait will be displayed at the bottom of the screen.
Press the ResultView portrait, and the processed data will appear in the ResultViewTable.
Print report
Select the result row you want in the ResultViewTable table and press black.
Click the printer portrait under Tools, then the BackgroundReportGenerator dialog box appears, select the report method you want to use, and press OK. The chromatographic report can be printed out.
6.3.8 Use records should be filled in after use (see Appendix 1).
6.4. Routine maintenance
6.4.1 The following system maintenance needs to be done before each run of the sample group:
Check and replace the needle washing solution in time; flush the system with water, methanol-water (1:1) for 15-30 minutes, and then use mobile phase to balance the system; manually clean the injector and sampling needle on the autosampler 3-5 times; wash the plunger rod of the pump with a washing bottle.
6.4.2 The following system maintenance needs to be done after each run of the sample group:
Flush the system for 20-30 minutes with water, methanol-water or acetonitrile-water in a 1:1 ratio; on the autosampler. Clean the injector and sampling needle 3-5 times manually; clean the plunger rod of the pump with a washing bottle.
6.4.4. Overhaul is carried out by the maintenance engineer of Waters Company every year.
7. Reference
"Waters515 Infusion Pump Operation Manual"
"Waters717 Autosampler Operation Manual"
"Waters486 UV Detector Operation Manual"
"WatersMillennuium32 Workstation Operation Manual"
"Waters2690 System Operation Manual"
8. Distribution department
Quality Assurance Department
9. Revision history:
none
