LC-2010AHT/LC-2010CHT

Transcription

1 Jan AHT/CHT FOR SHIMADZU HIGH PERFORMANCE LIQUID CHROMATOGRAPH Operation Manual Read the instruction manual thoroughly before use the product. Keep this instruction manual for future reference.

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3 Introduction Read this Documentation Before using the Instrument Congratulations on your purchase of the High Performance Liquid Chromatograph. This documentation describes the installation, operation, hardware validation, precautions for use, and details on the accessories and options. Read the manuals thoroughly before operating the instrument. Operate the instrument in accordance with the instructions contained therein. Keep these manuals for future reference. Important Do not operate this instrument before fully understanding the contents of this documentation. Provide this documentation to the next user in the event that the instrument is borrowed or sold. If this documentation or the warning labels on the instrument become lost or damaged, promptly obtain replacements from your Shimadzu representative. To ensure safe operation, read the Safety Instructions before operating the instrument. Safety of operation may be compromised if procedures in this manual are not followed. Copyright Copyright Shimadzu Corporation All rights reserved. This publication may not be reproduced in whole or part without written permission from Shimadzu Corporation. Since Shimadzu products are frequently upgraded and improved, information in this publication is subject to change without notice. We appreciate notification of any errors or omissions. Introduction INTR - i

4 Warranty and Post-sales Service Warranty 1. Validity Please consult your Shimadzu representative for information about the extent of the warranty. 2. Term The manufacturer will provide free replacement parts for, or repair free of charge, any instrument that fails during the warranty period, if the cause can be attributed to a defect in manufacturing. 3. Items Not Covered by the Warranty The warranty does not cover malfunctions that result from: a) misuse; b) repairs or modifications made by any company other than the manufacturer or an approved company; c) external factors; d) operation under severe conditions, such as environments with high temperature, high humidity, corrosive gas, vibration, etc.; e) fire, earthquake or other forces of nature; f) moving or transporting the unit after its initial installation; g) the consumption of items or parts that can be regarded as consumable. For example, the service life of an LCD display panel depends on actual operating conditions. If any problems occur with this instrument, inspect it and take appropriate corrective action as described in the Section 4 "Error Messages" p. MAINT and Section 5.1 "Hardware Problems" p. MAINT If the problem persists, or symptoms not covered in the Troubleshooting section occur, contact your Shimadzu representative. Replacement Parts Availability Replacement parts for this instrument will be available for a period of ten (10) years after the discontinuation of the product. Thereafter, such parts may cease to be available., however, that the availability of parts not manufactured by Shimadzu shall be determined by the relevant manufacturers. Hardware Validation The instrument and the systems should be checked periodically to ensure that they function normally, or the analytical data may not be reliable. To this end, it is necessary to carry out periodic hardware validations. Maintain the records of such validations. Before shipment from the factory, this instrument was rigorously inspected. The results are summarized in the Inspection Result Certificate accompanying the instrument. Detailed information on the hardware validation can be found in Section 3 "Hardware Validation" p. MAINT Service Contract A service contract is available, where a qualified Shimadzu-approved service person performs periodic inspection of the components and the system, and provides reports of the results. Details of the service contract can be obtained from your Shimadzu representative. INTR - ii

5 Guide to Documentation Following is a description of the documentation provided with this instrument. Please keep these manuals available for reference while working with the instrument. Installation and Maintenance Manuals These are separate documents provided in a single, bound volume. Installation Manual This manual describes the initial installation of the instrument, connection to a Chromatopac or PC, as well as all plumbing connections. Main Topics Instrument installation Connection of Chromatopac Connection of the LC Workstation Instrument plumbing Maintenance Manual Consult this manual to help determine the causes of instrument problems. The manual also provides detailed descriptions of HPLC performance and function verification procedures (validation). Main Topics Error messages and remedial actions Troubleshooting procedures Validation Parts lists Operation Manual This manual provides descriptions to enable basic operation of the instrument. Main Topics Basic operation procedures Reference Manual This manual can be printed out, as required. This manual provides a complete description of each instrument function, including detailed information about the various screens and their settings. The Reference Manual is provided in a PDF file format on the CD-ROM shipped with the instrument. Main Topics Soft switch option settings File-related information (Method file, Sequence file) Function menu list Settings used with external equipment Log file manager Specifications File operation procedures Utility Software Reference Manual (English Version) The Reference Manual is also available optionally as a bound volume. Contact your Shimadzu representative to order this manual. (Part Number ) Introduction INTR - iii

6 1 Precautions 1 Safety Instructions 1 for Safe Operation To ensure safe operation of the instrument, read these Safety Instructions carefully before use. Observe all of the WARNINGS and CAUTIONS described in this section. They are extremely important for safety.in this manual, warnings and cautions are indicated using the following conventions. WARNING Indicates a potentially hazardous situation which, if not avoided, could result in moderate to serious injury or possibly death. CAUTION Indicates a potentially hazardous situation which, if not avoided, may result in minor injury or equipment damage. Emphasizes additional information that is provided to ensure the proper use of this instrument. 1.1 Application Precautions WARNING This instrument is a high performance liquid chromatograph. Operate the instrument for its intended purposes only. Using the instrument for any other purpose could cause accidents. INTR - iv

7 Safety Instructions 1 Precautions for Safe Operation 1.2 Installation Site Precautions WARNING The solvents used in high performance liquid chromatography are flammable and toxic. The room where the equipment is installed should be well-ventilated, otherwise, solvent vapors could cause poisoning or ignite and cause a fire. High performance liquid chromatography uses large amounts of flammable organic solvents. Use of open flame in the vicinity of this unit must be strictly prohibited. Do not install in the same room with equipment that emits or could potentially emit sparks, since sparks could result in a fire. Provide fire extinguishers for use in case of fire. Provide protective equipment near the unit. If solvent gets into the eyes, or on the skin, it must be flushed away immediately. Provide equipment, such as eye wash stations and safety showers, as close to the unit as possible. WARNING The lab table or other surface on which this instrument is installed should be level, stable, sufficiently strong to support the units weight, and have a depth of at least 60 cm. Otherwise, the instrument could tip over or fall off the surface. Avoid installation sites that are exposed to corrosive gases or are excessively dusty. These adverse conditions may be detrimental to maintaining the instrument performance and may shorten its service life. 60 cm For details on installation site requirements, refer to the Installation Manual, Section "Installation Conditions" p. INST - 8. Introduction INTR - v

8 Safety Instructions 1 Precautions for Safe Operation 1.3 Installation Precautions WARNING Take measures to prevent the instrument from falling in the event of an earthquake or other disaster. Strong vibrations could cause the instrument to fall over, resulting in injury. The power supply voltages and power consumption of this instrument are listed below. The power supply voltage of the instrument is indicated by the voltage selector on its back, and on the label affixed to its right side. Connect the unit only to a power supply of the voltage denotes "fuse and "~" denotes AC current indicated, otherwise, fire or electric shock could result. Check that the power supply voltage is stable and its current capacity is sufficient to operate all the components of the system. If not, the unit will not operate at its rated performance. CHT AHT Part Number Power Supply Voltage Power Consumption Frequency AC100 V 700 VA 50/60 Hz AC115 V 700 VA 50/60 Hz AC230 V 700 VA 50/60 Hz Part Number Power Supply Voltage Power Consumption Frequency AC100 V 700 VA 50/60 Hz AC115 V 700 VA 50/60 Hz AC230 V 700 VA 50/60 Hz Ground the instrument. Grounding is necessary to prevent electrical shock in the event of an accidental electrical discharge, and important for ensuring stable operation. See Section "Connecting to the Power Outlet" p. INST Do not place heavy objects on the power cord, and keep any hot items away. It could be damaged, resulting in fire, electric shock or malfunction, if the cord becomes damaged, contact your Shimadzu representative immediately. Do not modify the cord in any way. Do not bend it excessively or pull on it. It could become damaged, resulting in fire or electric shock. If the cord becomes damaged, contact your Shimadzu representative. INTR - vi

9 Safety Instructions 1 Precautions for Safe Operation CAUTION When installing the instrument, be careful not to let your finger get caught in any small openings, as this could result in injury. 1.4 Operation Precautions WARNING Take thorough measures to prevent buildup of static electricity. "Static Electricity Precautions" p. ix. Static buildup could result in fires or explosions. Always wear goggles when handling solvents. If the solvent gets into the eyes, blindness could result. Should solvent get into the eyes, flush immediately with large amount of water and get medical attention. Do not use flammable sprays (hair sprays, insecticide sprays, etc.) near this instrument. They could ignite and cause a fire. Introduction INTR - vii

10 Safety Instructions 1 Precautions for Safe Operation 1.5 Precautions for Inspection, Maintenance, Adjustment, and Care of the Instrument WARNING Unplug the instrument before performing inspection, maintenance, or parts replacement. Otherwise, electrical shock or short circuit accidents could occur. Never remove the main cover. This may cause injury or malfunction of the instrument. The main cover does not need to be removed for routine maintenance, inspection and adjustment. Have your Shimadzu representative perform any repairs requiring removal of the main cover. Replace fuses only with fuses of the proper type and capacity. Any other fuses could cause a fire. If the power cord plug gets dusty, remove the plug from the power cord and wipe away the dust with a dry cloth. If dust is allowed to accumulate, fire could result. Replacement parts must be of the specifications given in Section 6 "Parts" p. MAINT If any other parts are used, they could become damaged and cause a malfunction. If any water gets into the instrument, wipe it away immediately to prevent rust. Never use alcohol or thinner solvents for cleaning this instrument. They could cause discoloration. INTR - viii

11 1 Static 2 Safety Instructions 2 Electricity Precautions WARNING Liquid chromatography (LC) uses flammable organic solvent(s) as the mobile phase. LC systems are also often used where large amount of flammable substances are present. Thus, an accident can produce large scale damage. Operators must be constantly on guard against accidents involving fire or explosion. The major cause of these accidents is static electricity. Devising preventative measures for static can be difficult, because the symptoms before an accident vary and can be hard to detect, since such accidents occur as a result of several simultaneous coincidences. Recommended methods for preventing static electricity accidents are provided below. Take thorough safety measures based on this information. 2.1 Typical Cause of Static Electricity Accidents Static electricity accidents are generally caused by this sequence of events: Generation of Static Electricity Accumulation of Static Electricity Release of Energy through Electrical Discharge Ignition of Flammable Substances When liquid is passed through thin tubing at high flow rates, as in liquid chromatography, the electrostatic charges of the flowing matter generate static electricity, as shown in the figure below. Generation of Static Electricity by a Liquid Flowing over a Solid Flowing liquid A:Charges move with flow of liquid B:Immobile charges, fixed to surface of solid If electrostatically charged liquid is allowed to accumulate in an electrically insulated container, the charge will gradually increase, and can eventually reach several thousand volts. If this happens and an electrical conductor is brought within a certain distance of the container, electrical discharge will occur, releasing thermal energy which will ignite any flammable gas in the vicinity. Solid Potential Accident Situation Spark Dry Air Liquid flowing through thin tubing at high flow rates *Air bubbles in liquid facilitate generation of static electricity Insulated containre of polyethylene, etc. Flammable gas present in container Spark Flammable organic solvent with large electro-static charge Floor covered with rubber, etc., cannot conduct electricity away Introduction INTR - ix

12 Safety Instructions 2 Static Electricity Precautions 2.2 Preventing Static Electricity Accidents The best way to prevent static electricity accidents is simply to prevent the occurrence and accumulation of electrostatic charges. Take two or more of the following preventative measures. If large amounts of flammable solvents are collected in a large container, implement preventative measures 1, 2, and 3 below. Preventative Measure 1 Use a metal container for the waste liquid, and ground the container. This will ensure that the electrical charges of the container and liquid pass to the ground. Accessories for this measure: (1) Grounding wire with clip... Part Number (2) 18 Liter metal container... Part Number (3) 4 Liter metal container... Part Number Be sure to ground the metal waste container properly. If the grounding wire is not properly attached or connected to the ground, static electricity can build up in the container. Some metal containers have surfaces that are laminated or oxidized, and therefore do not conduct electricity. After grounding the metal container, use a tester to verify that electricity is conducted to the ground. If the liquid to be drained into the waste container is virtually non-conductive (10-10 S/m or less), it will be necessary to add properly conductive, and therefore safe, liquid to the tank. This conductive liquid may be added beforehand. INTR - x

13 Safety Instructions 2 Static Electricity Precautions Preventative Measure 2 Cover the spaces between the tubing and the sides of the inlet and outlet openings of the waste container with caps or other protective covering. This will prevent any sparks generated outside the container from getting inside. Preventative Measures for Static Connect clip to metal parts. Reduce the opening with a cap. Connect to ground terminal of instrument or other grounding point, to ground container. 18 Liter metal can (preferably plated) Static of the liquid is conducted through container to ground. Accessories for this measure: (1) Caps for 18 liter or 4 liter containers...part Number (with three 3 mm diameter openings) Preventative Measure 3 Keep electrostatically charged objects, including the human body, away from the waste container. To prevent electrostatic charging of the human body, take the following precautions: Wear anti-static clothing and shoes. Ground the human body with anti-static wrist straps. For safety, the wrist strap should be connected to the ground using an intervening resistor of about 1 MΩ. Spread anti-static matting or the like on the floor, to make the floor conductive. Persons who have not taken anti-static precautions should touch a grounded metal object before coming near the waste container to drain static charges. Introduction INTR - xi

14 Safety Instructions 2 Static Electricity Precautions Preventative Measure 4 Use tubing with an inner diameter of at least 2 mm for drain lines with high flow rates. Air bubbles in liquid can multiply the electrostatic charge by a factor of 20, 30 or more. Periodically check the tubing connections for leaks. Preventative Measure 5 If it is not possible to use a conductive waste container, take the following precautions: Ensure that the end of the inflow tubing is always submerged inside the container. Place some type of grounded metal object, such as a ground wire connected to the instrument, into the liquid. The above precaution will be ineffective for low conductivity (less than S/m) liquids. Use as small a container as possible to minimize damage in the event of fire. Ambient humidity exceeding 65% will prevent static. Keep the room at a proper humidity. Anti-static equipment (anti-static clothing, shoes, and matting) and charge measurement equipment (potentiometer) are sold by speciality manufacturers. INTR - xii

15 1 Precautions 3 Safety Instructions 3 for Mobile Phase Selection and Use CAUTION PEEK resin possesses high mechanical strength, and among synthetic resins, it is the most suitable for use in high performance chromatographs. The following are safety-related considerations which require certain limitations in the use of PEEK, as follows. 1. The following mobile phases should not be used when PEEK resin parts are used in the plumbing. concentrated sulfuric acid concentrated nitric acid dichloroacetic acid acetone tetrahydrofuran (THF) dichloromethane chloroform dimethyl sulfoxide (DMSO) These mobile phases weaken the PEEK resin, which could result in cracked plumbing and mobile phase leaks. NOTE: Briefly using a weak solution of no more than 0.5% acetone in water (e.g. to check gradient performance) will present no problems. 2. PEEK Tubing 2-1. The resistance to pressure of PEEK tubing varies depending on the internal diameter, manufacturing process and solvents used. For this instrument, be sure to use the PEEK tubing having an internal diameter of 0.13 mm or 0.25 mm, provided standard with this instrument. Be advised that Shimadzu will assume no responsibility for any mishaps associated with the use of PEEK tubing provided by another manufacturer. For example, the PEEK tubing might rupture if acetonitrile is delivered at a pressure continuously exceeding 20 MPa (204 kgf/cm 2 ), posing considerable danger. When using an organic solvent as a mobile phase, be sure to use a pressure that does not exceed the above noted pressure. If delivery at a higher pressure is necessary, replace the tubing with stainless steel tubing If PEEK tubing is bent to a curvature with an excessively short radius, that portion of tubing will be weakened. Be sure that the curvature radius of the PEEK tubing is no shorter than 10 mm. Avoid forcibly bending and fixing the tubing, and strive to obtain as natural a curvature as possible. Radius of 10 mm or greater Introduction INTR - xiii

16 Safety Instructions 3 Precautions for Mobile Phase Selection and Use Use only HPLC grade or comparable mobile phase and filter with a filter of 0.45 µm mesh or finer before use to remove particulates and foreign matter. Halogen ions can corrode the stainless steel material (316L SS) used in the plumbing, so avoid mobile phases that contain halogen ions - such as KCl, NaCl, NH4Cl - mobile phases that generate halogen ions in certain reactions. If such mobile phases must b used, clean all lines thoroughly with distilled water immediately after analysis. Always degas the mobile phase, otherwise bubbles will form during mixing of solvents or when a change in temperature or pressure occurs. Air bubbles can cause pump malfunctions and detector signal noise. For boiling points, viscosities, and other data relating to the mobile phases used, refer to Reference Manual Section 10.3 "Mobile Phase Characteristics" p. REF INTR - xiv

17 1 Warning 4 Safety Instructions 4 Labels To help users operate the unit safely, labels are affixed to it at places where special attention is required. Should any of these labels peel off or become damaged, contact your Shimadzu representative to obtain replacements. Warning label (Part No ) CAUTION Always pull out the sample rack before installing or removing MTP or vials. Failure to pull out the sample rack may result in instrument malfunction or damage. WARNING During the teaching function, the Auto Sampler Unit operates even with the front door open. Do not touch moving parts. Cunsult the instruction manual before starting the teaching function. Warning label (Part No ) WARNING Warning label (Part No ) Heat-Block in this unit become hot o when temperature is 40 C or higher. Do not touch columns, pipings and Heat-Block until cooling down. BE SURE TO READ THE INSTRUCTION MANUAL BEFORE REPLACING THE LAMP. Warning label (Part No ) Introduction INTR - xv

18 Reading This Document This is a summary of the procedure, etc. to be explained. This shows the page being referenced. In PDF files, singleclick to jump to the page being referenced. Keys and the like shown at the end of a procedure, inside ( ) indicate keys that can be used in that procedure. Reference information for use in work. Tips on improving performance and supplemental explanations. See page iv. regarding s and Warnings. Page numbers are noted as follows. Page No. INST - 1-XX MAINT - 1-XX OP - 1-XX REF - 1-XX Manual Installation Maintenance Operation Reference Notation Conventions The following punctuation marks are used to describe specific indications on the display. Mark Description Example in the Above Figure " " Text Displayed on Screen "STEP" "RACK" etc. [ ] Function Displayed on Screen [PAGE.UP] indicates the icon is above the F3 key and the Page Up function is accessed by pressing that key. INTR - xvi

19 Table of Contents 1 Overview...OP Component Identification... OP File Types and Content... OP Operation Flow Charts...OP Files Necessary for Analysis... OP Creating a New Method File... OP Using an Existing Method File... OP Creating a New Method File...OP Turning ON the Instrument... OP Preparing the Mobile Phase... OP Purging the System... OP Auto Purge... OP Manual Prime... OP Setting Analysis Parameters (Normal Mode)... OP Description of the Method Screen... OP Creating a Method File (Normal Mode)... OP Pump Parameters... OP Column Oven and Peripheral Device Parameters... OP UV-VIS Detector Parameters... OP Time Program Screen... OP Creating a Time Program... OP Setting Analysis Parameters (Simple Mode)... OP Description of the Simple Mode Screen... OP Creating a Method File (Simple Mode)... OP Replacing the Mobile Phase... OP Installing the Column... OP Installing the Column... OP Starting Column Equilibration... OP Preparing Samples... OP Preparing the Samples... OP Using the Standard Sample Rack... OP Using the Microtiter Plate (MTP)... OP Setting Sample Injection Parameters and Starting Analysis (Test Run)... OP Description of "Single Run" Screen... OP Single Run Settings... OP - 50 Contents OP - I

20 Operation 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) OP Description of the Sequence Screen...OP Creating a Sequence File...OP Setting and Verifying the Sequence in the Method Screen...OP Shutting Down the System after Analysis...OP Operation with an Existing Method File...OP System Preparation File Parameters...OP Description of the System Prep Screen...OP Creating a System Preparation File...OP Shutdown File Parameters...OP Description of the Shutdown Screen...OP Creating a Shutdown File...OP Startup File Parameters...OP Description of the Startup Screen...OP Creating a Startup File...OP Settings for Auto Processing...OP Executing Auto Processing...OP LCD Screen Functions...OP Turning OFF the LCD...OP Using Keylock...OP Analysis Wizard...OP Using the Analysis Wizard...OP Instrument Parameters...OP Pump Parameters...OP Column Oven and Peripheral Device Parameters...OP UV-VIS Detector Parameters...OP Time Program Parameters...OP Analysis Sequence Parameters...OP - 85 Index...IX - 1 OP - II

21 1 Overview The, a compact, integrated, low-pressure gradient HPLC instrument, enables new users to easily perform gradient analysis. This Basic Operation Manual describes the fundamental procedures necessary for operating the instrument. Refer to the Installation Manual for information about preparing the instrument for operation, including installation and connections. For detailed operational information, please refer to the Reference Manual Component Identification This section identifies the various components of the instrument and describes the operation panel on the system controller. For detailed descriptions of the components and connections, refer to the Installation Manual. Front View Reservoir Box Autosampler System Controller Power Button Column Oven Pump Detector Operation Manual OP - 1

22 1 Overview 1.1 Component Identification Side View Clip CHT Condensation Water Drain (Left Port) Rinse Solution Drain Tube (Right Port) E D C B A Waste Fluid Container Mobile Phase DrainTube DrainTube (marked "P") } Leakage Drain Tube Waste Fluid Container OP - 2

23 1 Overview 1.1 Component Identification Interior View 1 Autosampler Measurinng Pump Panel (CHT) Low Pressure Value Sample Cooler (CHT) High Pressure Value Column Oven Drain Valve Column UV/VIS Detector D2 Lamp Cover Solvent Delivery Pump Seal Rinsing Kit Operation Manual OP - 3

24 1 Overview 1.1 Component Identification System Controller Operation Panel The operation panel on the system controller is used for all instrument operations except data processing. LCD (Screen Display) Method Screen Shown Function Menus Function Keys Help Display Area Status Display Area Indicator LED's Floppy Disk Eject Button Floppy Disk Drive Operation Keys Screen Selection Keys Floppy Disk Drive LED Editing Keys Function Keys The function keys are used to select functions and menus on the LCD and to switch between function menus. Used to select functions and menus from icons displayed on the LCD. Used to switch function menus. Up to three levels of function menus can be displayed. Operation Keys The operation keys control operation of the various instrument components. Starts and stops analysis. When the status is "READY", the Run key starts analysis, and the green indicator to the left illuminates. Pressing Run once more stops analysis, and turns off the green indicator. Switches the pump ON and OFF. Pressing Pump once turns the pump on, illuminating the green indicator to the left. Pressing Pump again turns the pump and the indicator lamp off. Switches the column oven ON and OFF. Pressing Oven once turns the oven on, illuminating the green indicator to the left. Pressing Oven again turns the oven and the indicator lamp off. Washes the pump mobile phase ports and injection rinse port. Pressing Purge displays the purge parameter screen. Rinses the autosampler needle. Corrects the 0 absorbance position of the UV-VIS detector baseline. OP - 4

25 1 Overview 1.1 Component Identification Screen Selection Keys A screen is associated with each function performed by this instrument. The screen selection keys switch between the screens. 1 Switches to the Sequence screen. Switches to the Method screen. Switches to the Validation/Maintenance screen. Switches to the System Preparation screen. Switches to the Shutdown screen. Refer to Section 3.11 "Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis)" p. OP - 51 of this manual. Refer to Section 3.4 "Setting Analysis Parameters (Normal Mode)" p. OP - 22 of this manual. Refer to the Maintenance Manual, Section 3.2 "Description of the Validation Screen" p. MAINT Refer to Section 4.1 "System Preparation File Parameters" p. OP - 59 of this manual. Refer to Section 4.2 "Shutdown File Parameters" p. OP - 63 of this manual. Editing Keys These are used to enter numbers and characters, and edit existing values. - Numeric keys; used to enter numbers and characters. Enters a decimal point. Enters a minus sign. Used to adjust the screen contrast. Inserts copied data or a blank row. Deletes selected data. Moves the cursor. When an arrow key remains pressed for longer than one second, the cursor speed increases. Clear key; used in the following situations: When entering numbers, clears previously entered values. When an error message is displayed, clears the display and resets the alarm. Used to set a value that has been entered or selected. Operation Manual OP - 5

26 1 Overview 1.1 Component Identification Indicator Lamps The indicator lamps illuminate when the following operations are in progress. Data Aquisition Solvent Delivery Pump Active Column Oven Temperature Regulation In Progress Sample Cooler Temperature Regulation In Progress (CHT only) UV/VIS Detector Lamp Lit Activate Injector Sample Cooler Detector Lamp Run Pump Oven Purge Rinse Zero OP - 6

27 Overview 1.2 File Types and Content To perform data acquisition and automatic processing with this instrument, first use the system controller to create the files that are associated with the various operations. The file types created and data stored in each type are described in this section. 1 File Setting Screens Various types of files are created by performing the settings indicated in the following screens. Seq Sequence Screen Sequence File [Settings] Analysis Sequence Table (Sequence Screen) Multi-row analysis sequence Repeat Inj. Table (Repeat Inj. Sequence Screen) Control Sample Analysis Sequence Table Analysis Sequence Sets sample injection parameters including settings for rack (RACK), sample number (SMPL#), number of repeat injections (#INJ), injection volume (VOL(ul), file number/analysisr un time (F#/RUN.T). Met Method Screen Simple Mode Screen Method File p. OP - 22 [Settings] Analysis Parameters (Method Sceen) Pump, column oven, UV detector parameters Solvent delivery mode One row of analysis sequence Time Program (Time Program Screen) Method File [Settings] Analysis Parameters (Simple Mode Screen) Pump, column oven, UV detector parameters Solvent delivery mode One row of analysis sequence Time Program (Binary Linear Gradient Only) Used tochange analysis parameters with respect to time Sys System Prep Screen System Preparation File [Settings] AutoPurge judgement Baseline stability auto judgement Post baseline stabilization processing Shutdown Screen F5 Start Up Screen Shutdown File [Settings] Cool down settings Settings for degasser and sample cooler condition during sleep state p. OP - 63 Start Up File p. OP - 66 [Settings] System startup settings Post startup settings Operation Manual OP - 7

28 1 Overview 1.2 File Types and Content File Use Flow Chart Files may be stored on the instrument or on a floppy disk. The following figure shows the sequence in which files are used during instrument operation. Start Up File System Preparation File Method File Parameter Setting File No.:Fn Test Run Method File STEP: SNGL-RUN Analysis Start Single Row of Analysis Sequence Set Method File Time Program STEP: 001/001 Contintuons Analysis Sequence File Repeat Inj.Table STEP: 1 : : : : STEP:202 Multiple Rows of Analysis Sequence Set Analysis Start Shutdown File Files Used for Analysis Files Used for Auutomatic Processing File Name Location Where Saved No. of Files (in unit) Description Floppy Disk Contents (*2) Start Up *1 All file System Preparation 10 All parameters are saved. All file Method 20 Method file includes Time All file or Method file (including Simple mode) Program Sequence 1 Sequence file corresponding to the file number in the Editing screen can be saved. All file or Sequence file Shutdown 2 All file System setting *1 All file *1 There is no file number. Files are saved and preserved in the instrument even when power is off. *2 For details on file types and the contents of the All file, refer to the Reference manual, Section 7.1 "Types of Files" p. REF OP - 8

29 2 Operation Flow Charts In order to start analysis, a Method file containing the parameter settings is required. This chapter describes two types of analysis procedures, one in which the Method file is newly created and the other where the Method file already exists Files Necessary for Analysis The files that are used during analysis include the Method and Sequence files. The specific screens and items which require setting are as follows. 2 Method File Creation Test Run Method Screen Parameter Setting [Settings] Pump Parameters p. OP - 24 Column Oven Parameters p. OP - 28 Peripherial Device Parameters p. OP - 28 UV Detector Parameters p. OP - 30 p. OP - 50 [Settings] Rack Sample No. Injection Volume Injection Time Interval/Method File No. Time Program Screen Time Program Creation p. OP - 33 Setting Injection Sample Analysis Parameters, such as Gradient Parameters Continuous Analysis Sequence File Creation p. OP - 53 Sequence Screen Analysis Sequence Table Settings p. OP - 55 [Settings] Rack Sample No. Number of Injections Injection Volume Injection Time Interval/Method File No. Repeat Inj. Sequence Screen Repeat Inj. Table Creation p. REF - 23 Method Screen Analysis Sequence Table Settings (1 row only) p. OP - 55 [Settings] Rack Sample No. Number of Injections Injection Volume Injection Time Interval/Method File No. Operation Manual OP - 9

30 Operation Flow Charts 2.2 Creating a New Method File This section charts how to create a new Method file and set analysis parameters. Parameters are set in either Normal Mode or Simple Mode. The Simple Mode is used for either isocratic or binary linear gradient analysis when there is only a single set of analysis parameters. Switch ON Power p. OP - 12 Enter Login Pass-ID-Number (User Name) p. OP - 12 Prepare Mobile Phase and Set Up Instrument p. OP - 14 Purge p. OP - 15 Simple Mode Set Analysis Parameters Enter System Pass-ID-Number p. OP - 12 Normal Mode Create Method File (Simple Mode) p. OP - 38 Create Method File p. OP - 23 Deliver Mobile Phase p. OP - 40 Mount Column p. OP - 41 Start Column Equilibration p. OP - 44 Load Sample p. OP - 44 Multi-Analysis Run Set Sample Injection Parameters Single Run Create Sequence File p. OP - 53 Set Single Run p. OP - 49 Analysis Starts On Analysis Completion, Confirm Oven Cool Down and Replace Solvents p. OP - 57 Switch Power OFF p. OP - 57 OP - 10

31 Operation Flow Charts 2.3 Using an Existing Method File After a Method file has been created, the instrument can be operated automatically. Example of Automatic Processing 2 Mobile Phase Purge System Preparation File Column Equilibration Analysis System Cool Down Shutdown File Sleep State System Startup Startup File Press Switch Power ON p. OP - 12 Enter Login Password/ID No. (and User Name) p. OP - 12 Prepare Mobile Phase Install Column and Load Sample p. OP - 14 Create System Preparation File p. OP - 59 Automates Purge Through Column Equilibration Create Shutdown File p. OP - 63 Automates System Cool Down Through Sleep State Procedures Create Startup File p. OP - 66 Automates Startup of System at a Specified Time Set up Auto Processing p. OP - 69 Set the System Preparation,Shutdown and Startup files to be used in the Sequence screen and set Sample Injection Parametersto include Auto Processing Press Operation Manual OP - 11

32 Creating a New Method File 3.1 Turning ON the Instrument When the instrument is turned ON, the user identification screen is displayed. The login password and user identification number must be entered in this screen. Verify with the system administrator that all users are registered. The default setting is "00000". If required, enter a User Name. When a user name is entered, it is displayed on the Monitor screen and printouts. 1. Press the power button. A popup screen requests entry of the login password and user ID number. Turning OFF the Instrument Press the power button again to turn OFF the instrument. Power Button Error Message If an air bubble is present in the detector flow cell when the instrument is turned ON, the following message may appear: Bubble may exist in the flow cell. Deliver solvent to flush bubble and perform wavelength accuracy test in VP menu. If this happens, start solvent flow to purge bubbles from flow cell and reperform the wavelength accuracy test. 2. Enter a login password and ID number at Login Pass-ID-No. (Numeric keys) F 1 F 2 F 3 F 4 F 5 FUNC 3. Press [OK]. The Menu screen is displayed. (see the following ) F 1 F 2 F 3 F 4 F 5 FUNC OP - 12

33 3 Creating a New Method File 3.1 Turning ON the Instrument About the Startup Screen By default, when the instrument is turned ON, the Menu screen is displayed, allowing access to the Sequence, Method and VP screens, among others. The screen displayed at startup can be changed in the System Preparation screen to provide quicker access. (See Installation Manual, Section 3.3 "Start Screen (Default Screen)" p. INST - 62.) ID No. User Name ID No. and User Name Objective Modification Procedure Comments To restrict users and maintain security. Allows user name to be included on printout of reports and instrument checks. Allows user name to be displayed on the Monitor screen. The default "00000" can be changed as desired. (See Installation Manual, Section "Canceling Login Pass- ID-No. at Startup" p. INST - 66.) The user name can be changed at instrument startup by entering a name in the user identification popup screen. When the instrument is started up, the previously entered User Name is displayed. Change or delete the displayed User Name as necessary. By default, the user must enter an ID No. to access the. However, as described under Modification Procedure, the ID No. can be eliminated. The does not require entry of a User Name to operate. Even if the Login Pass-ID-No. is cancelled, the User Name of the previous user is used for reports, lists and the Monitor screen. 3 Entering a User Name Before pressing [OK] in step 3 of the above procedure, perform the following steps: 1. Press to move the cursor. 2. Press [ABC]. This enables character entry using the numeric keys. (See Reference Manual, Section 7.6 "Entering Characters" p. REF ) 3. Enter the User Name, and return to step 3 above. Operation Manual OP - 13

34 Creating a New Method File 3.2 Preparing the Mobile Phase Prepare the mobile phases, and set the reservoir bottles in the reservoir box. About the Mobile Phase After preparation, the mobile phase should be filtered using a membrane filter with 0.5 µm diameter pores. Transfer the mobile phase to a reservoir bottle, and degas for 2 3 minutes using an ultrasonic bath. 1. Prepare the necessary mobile phases, and transfer them to reservoir bottles. 2. Insert the tubing from the appropriate mobile phase port into the corresponding reservoir bottle. Be sure the suction filter is attached to the end of the tubing. Marking on the Mobile Phase Intake Tubing The markings correspond to the ports as follows. Mark A B C D R Port Name Mobile phase A Mobile phase B Mobile phase C Mobile phase D Injection rinse solution Suction Filter Label:"A" Reservoir Bottle 3. Place the reservoir bottles in the reservoir box. Precautions When Replacing/Mixing Mobile Phases When the mobile phase is replaced, an intermediate solvent is sometimes used to prevent extraction, turbidity or reaction with the previous mobile phase. Likewise, certain solvents should not be mixed. Refer to the table below for examples of solvent combinations which must be avoided. Solvent Combinations Associated Problems Preventative Measure Water and low polarity solvents (hexane, chloroform, etc.) Buffer solution and organic solvent (methanol, acetonitrile, tetra hydrofuran, etc.) Turbidity, separation Extraction First replace with 2-propanol, acetone. First replace with water. Nitric acid and alcohol Reaction First replace with water. OP - 14

35 Creating a New Method File 3.3 Purging the System When the system is purged, the contents of the flow line from the suction filter through the column inlet, as well as the autosampler measuring pump, are rapidly replaced with mobile phase or rinse solution. A purge decreases the time required to replace solutions. The offers two purge options, Auto Purge and Manual Prime. In Auto Purge, a specific port is purged for a set time as defined by the Auto Purge parameters. In Manual Prime, a selected port is primed by manually starting and stopping the Manual Prime function. Purge Flow Lines Auto Purge Manual Praime :Suction Filter through Autosampler Injection Syringe :Suction Filter through Pump Head 3 Auto Purge Manual Prime Mobile Phase During Analysis Solvent Delivery Pump Autosampler Column Oven UV-VIS Detector Suction Filter During purge, column is replaced with couplings. Reservoir Drain Purging the Mobile Phase In Auto Purge, the mobile Phase is replaced in the flow line from the suction filter to the autosampler syringe, while in Manual Prime, the mobile phase is replaced from the suction filter to the pump head. Operation Manual OP - 15

36 3 Creating a New Method File 3.3 Purging the System Auto Purge This section explains how to automatically purge the mobile phase port or autosampler rinse port. The PEEK tubing and coupling should be installed, but not the column. The column is installed only after the mobile phase has been replaced in all the flow lines. Refer to Section 3.7 "Installing the Column" p. OP Unused Mobile Phase Ports Mobile phase ports which are not in use should be purged with the same solvent as the mobile phase or another solvent that will not affect analysis. Be careful not to allow any bubbles to remain in the tubing connected to unused mobile phase ports. 1. Install the standard accessory PEEK tubing and coupling in place of the column. 2. Press. A popup screen is displayed for setting the purge parameters. The pump indicator illuminates, and the pump starts operating. Coupling Heat Block 3. After purging, the solvent used in the last step of the purge will remain in the mixer and autosampler and will flow into the column when a run is started. To prevent this, press [IConc]. The InitConc-Replacement popup screen is displayed. InitConc-Replacement If the InitConc-Replacement function executed, the solvent of the mixture condition set in the method file flow at 2 ml/min into the drain of Autosampler for the set time. 4. Press to place a checkmark in the box as desired. Use the Right Cursor ( ) to move to the end and input a time value to be used. Press to exit the InitConc-Replacement popup. Buffer Solution In Column If the column contains a buffer solution, the solvent used for the last step of the purge (or InitConc- Replacement) should be an aqueous solution. Salt precipitation may occur in the column if a nonaqueous solution is used. OP - 16

37 3 Creating a New Method File 3.3 Purging the System 5. Move the cursor to the mobile phase port or rinse port to be purged. ( ) 6. Enter how long to purge the selected port. (Numeric keys, ) Purge Times The default purge times are as follows: Mobile Phase Port: 3 minutes Syringe Rinse Port: 10 minutes If bubbles are suspected in the flow line, extend the purge time by 2 5 minutes. F 1 F 2 F 3 F 4 F 5 FUNC 3 7. Repeat steps 3 and 4 to enter purge times for each applicable mobile phase port. 8. Press the function key corresponding to the mobile phase port to purge. If a purge is in progress for one mobile phase port, and a function key corresponding to a different mobile phase port is pressed, the latter purge is placed in a wait state. When the current purge is completed, the next purge is then executed. The following codes are displayed under the ports: exe req fin Purge in progress. Purge in wait state. Purge completed. F 1 F 2 F 3 F 4 F 5 FUNC Mobile Phase Port A: Purging Mobile Phase Ports B - D: Waiting The status display indicates the port that is currently being purged. "READY", is displayed in the status area when the purge is complete. Status Display PPURGEA PPURGEB PPURGEC PPURGED IPURGE Port Mobile Phase Port A Mobile Phase Port B Mobile Phase Port C Mobile Phase Port D Autosampler Rinse Port F 1 F 2 F 3 F 4 F 5 FUNC Mobile Phase Port A: Completed Mobile Phase Port B: Purging Mobile Phase Port C - D: Waiting Operation Manual OP - 17

38 3 Creating a New Method File 3.3 Purging the System Selecting a Port Select a port using the corresponding function key, as follows. Port Corresponding Function Key Remark Mobile Phase Port A Mobile Phase Port B Mobile Phase Port C Mobile Phase Port D Autosampler Rinse Port All Mobile Phase Ports (A - D) at one time GRAD. mode only Canceling a Purge Press the function key corresponding to the mobile phase port to cancel its purge. The code beneath the port changes from "req" to "fin". Press or [ALL] to cancel all purges. Performing Other Operations During a Purge Pressing [EXIT] returns to the previous screen, enabling other operations, such as Method development, to be performed. The purge continues uninterrupted. To redisplay the purge screen, press. Using Auto Purge While the Pump is Running If an automatic purge is performed while the pump is running, pump operation is interrupted, then resumes when the purge is completed. Excessive Bubbles in the Flow Line When the pump drain valve is opened, bubbles quickly exit the flow line. After completing the purge, close the drain valve. At this time, the flow line from the suction filter to the pump head is purged. Performing a Purge While the Column is Attached If the column contains buffer solution, ensure that an aqueous solution is used for the last step of the purge. If non-aqueous solvents are used for the last step of the purge, the amount remaining in the mixer will flow to the column and may cause crystallization in the column. OP - 18

39 3 Creating a New Method File 3.3 Purging the System Manual Prime Manually purge the instrument as described below. 1. Press. A popup screen is displayed for setting the purge parameters. The pump indicator lamp illuminates, and the pump starts operating Move the cursor to Manual Prime. F 1 F 2 F 3 F 4 F 5 FUNC (, ) A checkmark is placed in the Manual Prime checkbox, manually switching the to the purge flow line. 3. Verify whether the mobile phase drain tubing or rinse drain tubing is to be used, and insert the syringe needle into the appropriate tubing. F 1 F 2 F 3 F 4 F 5 FUNC Injecting into mobile phase drain tubing Mobile Phase Drain Tube When performing manual purge of a mobile phase port, inject into the mobile phase drain tubing marked "P", using the syringe. Rinse Solution Drain Tube Injecting into rinse solution tubing When manually purging the autosampler injection syringe, open the autosampler door and extract the tubing extension connected to the rinse drain tubing. Insert the syringe needle into the extension tubing. Purge After Manual Prime During Manual Prime, bubbles may be introduced in the autosampler flow line. Perform Auto-Purge of Auto sampler rinse port for at least 10 minutes following Manual Prime. Operation Manual OP - 19

40 3 Creating a New Method File 3.3 Purging the System 4. When purging a mobile phase port, open the drain valve. Open Drain Valve 5. Select a port using the corresponding function key as follows. Port Selecting a Port Corresponding Function Key Mobile Phase Port A Mobile Phase Port B Mobile Phase Port C F 1 F 2 F 3 F 4 F 5 FUNC Mobile Phase Port D Autosampler Rinse Port OP - 20

41 3 Creating a New Method File 3.3 Purging the System 6. Aspirate mobile phase or rinse solution into the syringe. For Mobile Phase Aspiration Volume Refer to the following for the volume to be aspirated. Mobile phase port - approximately 10 ml. Injection syringe rinse solution port - approximately 5 ml. 3 For Rinse Solution 7. Repeat steps 3-6 to perform a Manual Prime for another mobile phase port or the rinse port. 8. When the Manual Prime is completed, detach the syringe. 9. If a mobile phase port was purged, close the drain valve. Purge After Manual Prime Manual prime may cause the occurrence of air bubbles in the autosampler flow line. Perform Auto-Purge of Auto sampler rinse port for at least 10 minutes following a manual prime to prevent air bubbles from affecting analyses. Close Operation Manual OP - 21

42 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) After the mobile phase is placed in the reservoir box, the analysis parameters (separation and detection parameters) are set. The analysis parameters are set in the Method screen to create a Method file. When gradient parameters and other post-sample injection instrument parameters are to be set, a Time Program is created in the Time Program screen. To perform continuous analysis from multiple Method files, use the Sequence screen to make a Sequence file. Analysis from a single Method file can be initiated from the Method or Single Run screens. About Autosampler Injection Parameters For more information about setting the autosampler injection parameters, refer to the Reference Manual, Section 3.2 "Setting Autosampler Operation Parameters" p. REF Description of the Method Screen The items comprising the Method screen are described below. Pressing displays the Method screen. (1) File Number... Indicates the Method file number. For details on setting and selection of file number, refer to Reference Manual Section 7.5 "Method File Operations" p. REF (2) Mobile Phase Gradient Ratios... Defines mixing ratios for the mobile phase ports. (3) Mobile Phase Flow Rate... Sets the mobile phase flow rate. (4) Pressure Monitor Value... Displays the pressure monitor value. (5) Column Oven Temperature... Sets the column oven temperature. (6) Column Oven Internal Temperature... Displays the measured temperature inside the column oven. (7) Detection Wavelength... Sets UV-VIS detection wavelength. (8) Projected Analysis End Time... Displays time at which current analysis should end. (9) Current Sample Number... Displays the number of the sample currently being analyzed. OP - 22

43 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) (10) Current Injection Number... Displays the current injection number for the sample currently being analyzed. (11) Sequence Check/Setting Field... Displays the row of the Sequence file currently being processed. In addition, one row of the Sequence file can be set. (12) Help Display Area... Displays information and entry range pertaining to item at which cursor is positioned. (13) Status Display... Displays the operational status of the instrument Creating a Method File (Normal Mode) 3 Create a new Method file, entering the pump, UV-VIS detector and column oven parameters. 1. Press and [FILE] in the Method screen. The File screen is displayed. 2. Select a Method file number. ( ) F 1 F 2 F 3 F 4 F 5 FUNC Selecting a File Number To create a new Method file, be sure to select a file number that is not already in use. (See Reference Manual, Section "Selecting a Method file" p. REF ) F 1 F 2 F 3 F 4 F 5 FUNC 3. Press. The selected file number is now displayed on the Method screen. F 1 F 2 F 3 F 4 F 5 FUNC Operation Manual OP - 23

44 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) Pump Parameters Isocratic (ISO) Analysis The following mobile phase solvent delivery parameters are used to equilibrate the column for ISO analysis: Mobile phase flow rate (FLOW) Maximum pressure (P.MAX) and minimum pressure (P.MIN) Mobile phase port designation (PORT) Degasser ON/OFF control (DEGAS) Solvent Delivery Mode To switch to ISO mode from GRAD mode, press [PARAM], then [ISO]. The Gradient parameters are disabled. GRAD Mode ISO Mode 1. In the Method screen, move the cursor to the flow rate setting field. ( ) 2. Enter a mobile phase flow rate. (Numeric keys, ) F 1 F 2 F 3 F 4 F 5 FUNC OP - 24

45 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) 3. If other solvent delivery parameters are to be entered, return the cursor to the flow rate column, and press [PARAM]. (, ) The Pump Parameters screen is displayed. 4. Move the cursor to the desired pump parameter. ( ) F 1 F 2 F 3 F 4 F 5 FUNC 3 About the Pump Parameters To select a mobile phase port, enter the appropriate corresponding numerical value at "PORT". To specify Mobile Phase Port A, enter "1" for the PORT parameter. To use Mobile Phase Ports B D, enter 2 4, respectively. For information about the other parameters, refer to Section 6.1 "Pump Parameters" p. OP Enter a number value for the parameter. (Numeric keys, ) 6. Press [EXIT] when all parameters are entered. This completes the procedure for setting the pump parameters. Active Mobile Phase Port The mobile phase port currently in use can be verified in the Monitor screen ( [MONITOR]). For more information, refer to Reference Manual, Section "Monitor Display Description" p. REF Operation Manual OP - 25

46 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) Gradient (GRAD) Analysis The following mobile phase solvent delivery parameters are used to equilibrate the column for GRAD analysis: Mobile phase flow rate (FLOW) Mobile phase mixing ratio for each port (A D) Maximum pressure (P.MAX) and minimum pressure (P.MIN) Gradient curve value (B.CURV D.CURV) Degasser ON/OFF control (DEGAS) Solvent Delivery Mode To switch to GRAD mode from ISO mode, press [PARAM], then [GRAD]. The Gradient parameters are enabled. ISO Mode GRAD Mode 1. In the Method screen, move the cursor to the Gradient parameter. ( ) 2. To equilibrate the column, enter a mixing ratio (%) for each mobile phase port, A D. (Numeric keys, ) When the gradient parameters are completed, the cursor moves to the flow rate parameter. F 1 F 2 F 3 F 4 F 5 FUNC Entering Mobile Phase Mixing Ratios Enter values for the individual mobile phase mixing ratios for mobile phase ports A, B, C, and D, respectively, so that the total is 100%. Each time is pressed after entering a value, that value is set. If the total is not 100%, re-enter the values so that the total equals 100%. If a mixing ratios for any port is set to 100 (%), all other values are set to 0. If a mobile phase port is not to be used, enter "-1". (, ) OP - 26

47 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) 3. Enter a mobile phase flow rate. (Numeric keys, ) 4. If other solvent delivery parameters are to be entered, return the cursor to the mobile phase mixing ratio setting column, and press [PARAM]. (, ) The Pump Parameters screen is displayed. F 1 F 2 F 3 F 4 F 5 FUNC 3 F 1 F 2 F 3 F 4 F 5 FUNC 5. Move the cursor to the desired pump parameter. ( ) About the Pump Parameters For information about the specific parameters, refer to Section 6.1 "Pump Parameters" p. OP Enter a number value for the parameter. (Numeric keys, ) 7. Press [EXIT] when all parameters are entered. This completes the procedure for setting the pump parameters. Operation Manual OP - 27

48 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) Column Oven and Peripheral Device Parameters The following column oven and peripheral device parameters are used to equilibrate the column: Column oven temperature (OVEN.T) Maximum oven temperature (T.MAX) Event output terminal control (EVENT) Sample cooler ON/OFF (SMPL.C) Sample Cooler Temperature Control This parameter is displayed only on the CHT. To set the temperature, press [SIL] and enter a value at "Cooler Temp". See Reference Manual Section "Summary of Autosampler and Sample Cooler Parameters" p. REF In the Method screen, move the cursor to the column oven temperature parameter. (Cursor keys) F 1 F 2 F 3 F 4 F 5 FUNC 2. Enter a column oven temperature. (Numeric keys, ) 3. To enter the maximum column oven temperature and peripheral device parameters, return the cursor to the column oven temperature, parameter column, and press [PARAM]. (, ) A popup screen is displayed for entering the column oven maximum allowable temperature and the peripheral device parameters. F 1 F 2 F 3 F 4 F 5 FUNC OP - 28

49 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) 4. Move the cursor to the desired parameter. ( ) Column Oven and Peripheral Device Parameters For more information about the column oven and peripheral device parameters, refer to Section 6.2 "Column Oven and Peripheral Device Parameters" p. OP Enter a number value for the parameter. (Numeric keys, ) 6. Press [EXIT] when all parameters are entered. This completes the procedure for setting the column oven and peripheral device parameters. 3 Operation Manual OP - 29

50 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) UV-VIS Detector Parameters The following UV-VIS detector parameters are used to obtain a chromatogram and baseline: Detection wavelength (for 2 channels: WAVE 1, 2) Lamp control (LAMP) Dual detection mode (DUAL) AUX range (AUXRNG 1, 2) Response (RESP) Polarity (POL) UV-VIS flow cell temperature control (CELL.T) Setting Temperature for UV-VIS Detector Cell Perform setting based on the room temperature and column temperature, as indicated below. Room Temperature Column Temperature Up to 45 C C 4 C - 15 C Low (CELL. T=1) 15 C - 30 C High (CELL.T=2) 30 C - 35 C 1. In the Method screen, move the cursor to the UV-VIS detector wavelength parameter. (Cursor keys) F 1 F 2 F 3 F 4 F 5 FUNC 2. Enter a detection wavelength. (Numeric keys, ) OP - 30

51 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) 3. If other UV-VIS detector parameters are to be entered, return the cursor to the UV-VIS detector wavelength parameter column, and press [PARAM]. (Cursor keys, ) The UV-VIS Detector Parameters screen is displayed. Peak Width in Dual Wavelength Mode The Dual Wavelength Mode can be reliavly used for peaks with a half-height width of 5 seconds or more. (c. v. value less than 0.1%) Sharper peaks may show degradation in reproducability, due to the movement of the defraction grating from one wavelength position to another. F 1 F 2 F 3 F 4 F 5 FUNC 3 4. Move the cursor to the desired parameter. ( ) UV-VIS Detector Parameters For information about the UV-VIS detector parameters, refer to Section 6.3 "UV-VIS Detector Parameters" p. OP Enter a number value for the parameter. (Numeric keys, ) 6. Press [EXIT] after all parameters are entered. This completes the procedure for setting the UV-VIS detector parameters. Operation Manual OP - 31

52 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) Time Program Screen The items comprising the Time Program screen are described below. Press [T.PROG] to display the Time Program screen (1) Steps Left... Indicates the number of remaining steps available for the time programs. (2) File Number... Indicates the Method file number. The settings made in the Time Program screen are saved as part of the Method file indicated here. (3) Step Number (#)... Indicates the number of the Time Program step. (4) Elapsed Time (TIME)... Indicates the time elapsed since the start of the program. (5) Parameters (FUNC)... Indicates the parameter controlled by the Time Program. (6) Set Value (VALUE)... Indicates the value entered for the Time Program parameter. (7) Function Parameter List... Lists the parameters that are valid FUNC entries by component. (8) Function Parameter Classification... Selects the type of parameters to display in the function parameter list by component. (9) Help Display Area... When the cursor is at "VALUE", displays information, such as the range and step, pertaining to the parameter in that row. (10) Status Display... Displays the current operation status of the instrument. Switching Between the Editing and Time Program Screens A Method file that need not be executed immediately can be created in the Editing screen. (Reference Manual Section 2.4 "Creating and Editing a File during Analysis" p. REF - 14.) When the Editing screen is displayed, open the Time Program screen by pressing [T.PROG]. After editing is completed, exit the Time Program, and the display returns to the Editing screen. OP - 32

53 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) Creating a Time Program By creating a Time Program file in the Time Program screen, the analysis parameters can be automatically changed over time. The Time Program is saved as part of the Method file. 1. In the Time Program screen, enter the time at which to execute the step in the TIME column. (Numeric keys, ) Enter 3 Sorting TIME Values After all TIME values have been entered, pressing then [SORT] orders the program by time. 2. The function parameters displayed for selection differ according to the unit selected. Select the appropriate unit. (Function keys) The selected function parameters unit is highlighted at "FUNCTIONS". F 1 F 2 F 3 F 4 F 5 FUNC The function keys are selected according to the relevant unit, as follows. Parameter Classification Table Unit Function Key Remark PUMP [PUMP] Parameters related to pump operation. DET [DETECTOR] Parameters related to UV-VIS detector operation. OTHER [OTHERS] USER [USER] Parameters related to column oven, autosampler and other peripheral device operation. Functions that are used often can be assigned to userdefined function keys. For more information, refer to Reference Manual Section 2.1 "Customizing Parameter Tables" p. REF - 6 (USER). Operation Manual OP - 33

54 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) 3. Enter the function number from the Function Parameter List in the FUNC column. (Numeric keys, ) The function name corresponding to the entered number is displayed in "FUNC". Enter Function Parameters and Numbers For more information about function parameters and numbers, refer to Section 6.4 "Time Program Parameters" p. OP Entering Functions If an invalid number is keyed, the system beeps and the number is not entered. Enter a valid number. 4. Enter a number in the VALUE column. (Numeric keys, ) About the VALUE Parameter Some parameters do not require entry of a numerical value. (Refer to Section 6.4 "Time Program Parameters" p. OP - 82.) 5. Repeat steps 1 4, to finish creating the time program. Creating a Time Program If the key is pressed without having entered a numerical value for "TIME", "FUNC", or "VALUE", the number entered for the corresponding parameter in the previous row is automatically entered. This eliminates repeatedly entering the same value. To display the Gradient Monitor screen, press [SHOW.GRD]. Use the Gradient Monitor screen to verify the gradient curve. OP - 34

55 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) 6. Enter the [STOP] command ("50") in the FUNC column. (,, ) "STOP" ends analysis and completes entry of the Time Program. About the STOP Command If the STOP command is not entered, analysis cannot be completed with the Time Program. To manually stop analysis, press when the analysis resumes after the Time Program. 7. Press [EXIT]. The display returns to the Method screen. (If the Time Program was accessed from the Editing screen, the display returns to the Editing screen.) 3 Example: Creating a Time Program Mobile Phase Ports: Ports A & B Elapsed Time and Parameter Changes: After 10 minutes, the mixing port ratio of port B becomes 80%, where it is maintained until 15 minutes elapses. At minutes, the Port B mixing ratio is decreased to 0%. One cycle lasts 20 minutes. To create a Time Program from the conditions above, perform the settings as shown below: F 1 F 2 F 3 F 4 F 5 FUNC To verify the gradient curve, display the Gradient Monitor screen by pressing [SHOW.GRD]. (For more information, see Reference Manual Section 2.2 "Graphical Display of Pump Parameters" p. REF - 8.) F 1 F 2 F 3 F 4 F 5 FUNC Operation Manual OP - 35

56 3 Creating a New Method File 3.4 Setting Analysis Parameters (Normal Mode) Sorting of Time Program Steps Before analysis begins, the Time Program is sorted by start time of each step. If two steps are set to the same time, the Time Program may not run as expected. To prevent this, press [SORT] to sort the program steps by time. Examine the program and set the times of steps so that they vary by a small amount (0.01 min). Example: INCORRECT CORRECT B.CONC B.CONC B.CONC B.CONC B.CONC B.CONC STOP STOP OP - 36

57 Creating a New Method File 3.5 Setting Analysis Parameters (Simple Mode) Use the Simple mode for isocratic or binary linear gradient analysis if only one set of analysis parameters is necessary. In the Method screen, select "SIMPLE" and enter a system password and ID number to display the Simple Mode screen. In Simple mode, an isocratic or gradient delivery mode can be selected. When the Simple mode is selected, only the and keys can be used for switching screens Description of the Simple Mode Screen In the Method screen, press [SIMPLE], and then enter the System Pass-ID-No. By selecting the solvent delivery mode, the Simple Mode screen is displayed. In Simple mode, either the isocratic (ISO.) mode or gradient (GRAD.) mode is available as the solvent delivery mode. 3 ISO Simple Mode Screen (for isocratic analysis) 3 GRAD Simple Mode Screen (for gradient analysis) (1) Analysis Time (StopTime)... In ISO mode, sets the time of analysis. (2) Mobile Phase Gradient Parameters... In GRAD mode, sets the Mobile Phase B gradient maximum and minimum concentrations, the time at which Mobile Phase B reaches each concentration, and the end time. (3) Mobile Phase Flow Rate... Sets the flow rate. (4) Column Oven Temperature... Sets the column oven temperature. (5) UV-VIS Detection Wavelength... Sets the wavelength used for detection. (6) Analysis End Time... Displays the time at which the current analysis should end. (7) Current Sample Number... Displays the number of the sample currently being analyzed. (8) Current Injection Number... Displays the injection number of the current sample. (9) Sequence... Displays the row of the sequence file currently being executed. A sequence file consisting of a single row can be entered. (10) Help Display Area... Displays a description and setting range for the parameter at which the cursor is located. (11) Status Display... Displays the current operation status of the instrument. (12) [ADVANCE]... When returning to Standard mode, press [ADVANCE] and enter the System Pass-ID number. Operation Manual OP - 37

58 3 Creating a New Method File 3.5 Setting Analysis Parameters (Simple Mode) Creating a Method File (Simple Mode) Isocratic (ISO) Analysis 1. Press. The Method screen is displayed. 2. Press [SIMPLE]. 3. Enter a login password and ID number. (Numeric keys) 4. Press [OK]. A screen is displayed for selecting the solvent delivery mode. 5. Press [ISO.]. The ISO mode is selected. 6. Enter the time at which to end the analysis in the StopTime parameter. (Numeric keys, ) 7. Enter values for the flow rate, column oven temperature and F 1 F 2 F 3 F 4 F 5 FUNC UV-VIS detection wavelength. (Numeric keys, ) 8. There are two methods for setting the injection parameters If multiple samples are to be analyzed, set the sample injection parameters in the Sequence screen. See Section 3.11 "Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis)" p. OP If an injection is to be performed only once without modifying an existing Sequence file, set the injection parameters using Single Run. See Section 3.10 "Setting Sample Injection Parameters and Starting Analysis (Test Run)" p. OP This completes the procedure for setting the analysis parameters in the Simple Mode screen. Using a Sequence in the Simple Mode Screen The F#/RUN.T parameter cannot be set in the Sequence Check/Setting field of the Simple Mode screen or in the Sequence screen displayed from the Simple Mode screen. OP - 38

59 3 Creating a New Method File 3.5 Setting Analysis Parameters (Simple Mode) Gradient (GRAD) Analysis 1. Press. The Method screen is displayed. 2. Press [SIMPLE]. 3. Enter a login password and ID number. (Numeric keys) 4. Press [OK]. A screen is displayed for selecting the solvent delivery mode Press [GRADIENT]. The Gradient mode is selected. 6. Enter the Gradient program. (Numeric keys, ) Gradient for mobile phase por B. (Maximum and minimum concentration) Time for mobile phase port B to reach maximum and minimum concentrations. (Total time from start) End Time (Total time from start) F1 F2 F3 F4 F5 FU NC 7. Enter values for the pump, column oven and detector parameters, as well as for any peripheral devices. (Numeric keys, ) 8. There are two methods for setting the injection parameters If multiple samples are to be analyzed, set the sample injection parameters in the Sequence screen. See Section 3.11 "Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis)" p. OP If an injection is to be performed once without modifying an existing Sequence file, set the injection parameters using Single Run. See Section 3.10 "Setting Sample Injection Parameters and Starting Analysis (Test Run)" p. OP This completes the procedure for setting the analysis parameters in the Simple Mode screen. Using a Sequence in the Simple Mode Screen The F#/RUN.T parameter cannot be set in the Sequence Check/Setting field of the Simple Mode screen or in the Sequence screen displayed from the Simple Mode screen. Operation Manual OP - 39

60 Creating a New Method File 3.6 Replacing the Mobile Phase After performing a purge and prior to performing analysis, check for leaks and the absence or presence of bubbles. 1. Press. The Method screen is displayed. PUMP Key 2. Verify the flow rate of the mobile phase port flow to be used, and press. The Pump indicator illuminates, and solvent delivery begins. 3. Observe the liquid flowing from the end of the mobile phase drain tubing for approximately 10 seconds. The liquid should flow continuously with no bubbles observed. 4. Press again. Solvent delivery stops. 5. In the Method screen, verify that a pressure of MPa is displayed. If the pressure is not with the MPa range, press pressure to zero. [ZERO] to adjust the Eliminating Bubbles If the solvent flow starts and stops synchronously with the pumping sound, bubbles may be trapped in the pump head. Press to remove the bubbles from the pump head. No Solvent Flow If the solvent is not flowing through the pump, check for the following. Clogged tubing Leak Bubbles in pump head Refer to the Maintenance Manual, Section 5.1 "Hardware Problems" p. MAINT OP - 40

61 Creating a New Method File 3.7 Installing the Column Install the column in the upper portion of the column oven. CAUTION About the PEEK tubing The applicable pressure of AHT/CHT with the PEEK tubing is 20 MPa max. When AHT/CHT will be used in the condition the pressure is higher tan 20 MPa, exchange the PEEK tubing for the sus tubing. Contact your Shimadzu representative for exchanging the tubing, because the internal tubing of auto-sampler have to be changed. See, Introduction "Precautions for Mobile Phase Selection and Use" p. INTRO - xiii Installing the Column 1. Open the front door, then open the column cover. Column Cover 2. Detach the coupling. Heat Block Outlet Side UV-VIS Detection Inlet Side 3. Press to start solvent delivery, and visually verify that solvent is flowing from the tubing connected to the heat block. About the Heat Block There are two types of heat blocks, one for normal analysis (9 µl capacity) and another for semi-micro analysis (4 µl). Normal Analysis (OUT) UV-VIS Detection Inlet Side Semi-micro Analysis (OUT) Semi-micro Analysis (IN) Normal Analysis (IN) Operation Manual OP - 41

62 3 Creating a New Method File 3.7 Installing the Column 4. Connect the tubing to the column inlet. About the carryover For decreasing the carryover, connect the column and the high pressure valve directly with the attached PEEK tube (for semi-micro LC : ID 0.13 mm L 600 mm, for conventional LC : ID 0.25 mm L 600 mm). In this case, the temperature of the column is lower than the set temperature of column oven because the mobile phase is not preheated in the heat block. Heat Block Column 5. Connect the blue PEEK tubing from the UV-VIS detector inlet to the column outlet. Column Heat Block 6. Set the column blocks as shown in the figure. Column Blocks OP - 42

63 3 Creating a New Method File 3.7 Installing the Column 7. Insert the column between the column blocks and the retaining clips. Column Block Clips Column Block Column 3 8. Close the column cover, and close the door. Column Blocks The column blocks support the column and transfer heat from the heat block. Although there are 20 mm and 40 mm blocks, the 40 mm blocks should be used whenever possible. The 40 mm blocks enable better heat transfer from the column oven. Column Cover Guard Column Use If a guard column is used, install it in the lower position. The main column should be installed in the upper position for better temperature control. Guard Column Column Operation Manual OP - 43

64 Creating a New Method File 3.8 Starting Column Equilibration After installing the column, press and, and wait for the baseline to stabilize. Detecting Column Equilibration Refer to the documentation for the connected data processor (Chromatopac, LC Workstation, etc.). Automatically Detecting Column Equilibration By creating a Sequence file that includes a System Preparation file, column equilibration can be detected automatically. For more information, refer to Section 4.1 "System Preparation File Parameters" p. OP - 59, and Section 4.4 "Settings for Auto Processing" p. OP Press. The indicator illuminates, and adjustment of the column oven temperature begins. "WAIT CTO" is displayed in the status area while the column oven temperature is being adjusted. Once the column oven temperature reaches that set in the Method screen or Simple Mode screen, "READY" is displayed in the status area. OVEN Key OP - 44

65 Creating a New Method File 3.9 Preparing Samples This section describes the procedure for preparing samples and placing them in the instrument. The LC accommodates up to two vial racks or four microtiter plates at a time. About the Dummy Rack (standard accessory for the CHT) The CHT is provided with a dummy rack to prevent condensation. Be sure to place the dummy rack in the unused side of the sample chamber Preparing the Samples CAUTION Sample Precautions Filter all samples to eliminate debris and undissolved particles. Solid particles could clog the needle during sampling. Dilute highly viscous samples before use. High viscosity samples may not be aspirated successfully. 1. Completely dissolve the sample in a solvent with an identical or similar composition to the mobile phase. 2. Filter the sample. 3. Carefully load the sample(s) into a vial or microtiter plate well. 4. If using vials, position the silicon septum with the PTFE surface facing down (liquid side), and then cap the vial. Silicone (light color) Cap CAUTION Vial Precautions Only use vial septa supplied by Shimadzu. Septa supplied by other vendors may produce fragments that could clog the flow line or may prevent successful penetration by the needle. Shimadzu parts are listed in the Installation Manual, Section "Options" p. INST One side of the silicon septum is coated with PTFE. Place the septum on the vial so that the PTFE surface is facing down (liquid side). If the septum is positioned incorrectly, the solvent may dissolve the silicon rubber. PTFE (dark color) Septum Vial Operation Manual OP - 45

66 3 Creating a New Method File 3.9 Preparing Samples Using the Standard Sample Rack Sample racks for 1 ml, 1.5 ml and 4 ml vials are available. For more information, refer to the Installation Manual Section "Options" p. INST The upper surface of the rack is marked with numbers at the vial positions. Specify these numbers when setting sample number parameters. 1. Open the door. Door 2. Place the vial in the sample rack with the cap facing up. Vial Sample Rack 3. Slide the sample rack along the guides all the way to the back of the instrument. The sample rack clicks into place when it is inserted correctly. Sample Rack Manipulating the Sample Rack Be sure to move the rack horizontally when sliding it in and out of the instrument. Sample Rack Placement Ensure that the sample rack is fully inserted into the instrument. If the sample rack is not completely pushed to the back, the needle may strike the vial cap during sample aspiration. This will cause errors and may damage the needle. OP - 46

67 3 Creating a New Method File 3.9 Preparing Samples 4. Close the door. Door 3 About the Sample Vial Rack The installed sample vial rack is automatically detected, and the sample rack number and position are displayed in the Sequence screen. Select the sample rack position using the appropriate number, as shown in the figure at the right. Front of Instrument Using the Microtiter Plate (MTP) When a microtiter plate is used for the first time or replaced with a different type of microtiter plate (96- well, 384-well, deep well), the sampling position intervals must be calibrated. This is referred to as the "teaching" procedure. When the deep-well microtiter plate is used, the end position of the needle descent must be entered. See the Installation Manual Section 3.6 "Microtiter Plate (MTP) Rack Adjustment" p. INST - 68, for more information. 1. Open the door. Door Operation Manual OP - 47

68 3 Creating a New Method File 3.9 Preparing Samples 2. Set the microtiter plate(s) in the MTP rack. 3. Slide the MTP rack all the way to the back of the instrument. The MTP rack clicks into place when it is inserted correctly. Manipulating the MTP Rack Be sure to move the MTP rack horizontally when sliding it in and out of the instrument. MTP Rack Placement Ensure that the MTP rack is fully inserted into the instrument. If the MTP rack is not completely pushed to the back, the needle may strike the microtiter plate during sample aspiration. This will cause errors and may damage the needle. 4. Close the door. Door About the MTP Rack The installed MTP rack is automatically detected, and the MTP rack number and position are displayed in the Sequence screen. Select the microtiter plate position using the appropriate number, as shown in the figure at the right Front of Instrument OP - 48

69 Creating a New Method File 3.10 Setting Sample Injection Parameters and Starting Analysis (Test Run) The Single Run function can be used to verify that the instrument is stabilized for regular analysis by injecting a single sample Description of "Single Run" Screen In order to perform an analysis test run, parameters are entered for a single injection of one sample in the "Single Run" screen. The default values correspond to those in the Sequence Check/Setting Row of the Method screen. 3 [Display Method] Press [Single Run] to display the popup screen for entering the Single Run analysis parameters. [Screen Description] (1) Sample Rack Position (RACK)...Sets the position of the sample rack that the sample vial is placed in for the Single Run analysis. (2) Sample Number (SMPL#)...Displays position number where the sample vial is placed in the sample rack, or the well position number where sample is loaded in an MTP. (3) Number of Injections...In Single Run, the injection number can only be 1. (4) Injection Volume (VOL)...Sets the volume of sample to be injected. (5) File Number/Run Time (FILE#/RUN.T)...Enter the Method File number to be used for the gradient Time Program analysis. If a Time Program is not used, enter the analysis run time. Operation Manual OP - 49

70 3 Creating a New Method File 3.10 Setting Sample Injection Parameters and Starting Analysis (Test Run) Single Run Settings 1. Press [Single Run] in the Method screen. The Single Run screen is displayed. 2. For "RACK", enter the position number of the sample rack F 1 F 2 F 3 F 4 F 5 FUNC to be used for analysis. (Numeric keys, ) "RACK" Entry Referring to the diagram below, enter the corresponding number. (The bottom of the diagram corresponds to the front of the instrument.) Sample Vial Racks Microtiter Plates 3. Enter the sample number to be analyzed for "SMPL#". (Numeric keys, ) 4. Enter the volume of sample to be injected for "VOL". (Numeric keys, ) 5. For "F#/RUN.T", enter one of the following, depending on whether a Time Program is used. (Numeric keys, ) 5-1. If a gradient or other Time Program is to be used, press and enter a file number for "F#/RUN.T" If a Time Program will not be used, enter the analysis run time for "F#/RUN". 6. Press [OK]. Analysis begins. "PRETREAT" is displayed in the status area while the autosampler is preparing for injection. "RUN" is displayed in the status area while the sample is being analyzed. The analysis is complete when "READY" is displayed in the status area. OP - 50

71 Creating a New Method File 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) To use multiple Method files to perform continuous analysis, create a Sequence file in the Sequence screen. When the Analysis Sequence is Not Set If the analysis Sequence file containing the sample injection parameters is not selected and is pressed, only the Time Program is executed. If neither the analysis Sequence file or the Time Program is selected and is pressed, only the time countdown is performed Description of the Sequence Screen The items comprising the Sequence screen are described below. Press to display the Sequence screen (1) Sample Rack Type... Displays the name of the sample rack. For the sample rack types and codes, refer to the Installation Manual, Section "Options" p. INST (2) Required Volume of Mobile Phase... Press [CALC] to display the estimated volume of mobile phase required for the entire sequence. The estimated volume of mobile phase required may not be calculated correctly when performing gradient analysis. (3) Sequence End Time... Press [CALC] to display an estimated time of completion. During execution of the sequence, each time the operation moves to the next row, the end time is recalculated, Refer to TIP p. OP If a Startup file and System Preparation file are entered, the analysis end time is not calculated. For more information about the Startup and System Preparation files, refer to Section 4.4 "Settings for Auto Processing" p. OP (4) Current Sample Number... Displays the number of the sample being analyzed. Operation Manual OP - 51

72 3 Creating a New Method File 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) (5) Current Injection Number... Displays the number of injections completed in the current analysis. (6) Step Number (#)... Displays the step number of the analysis sequence. (7) Sample Rack Position (RACK)... Displays the position of the installed sample rack. (8) Sample Number (SMPL#)... Displays the position number where the sample vial is placed in the sample rack, or the well position number when the sample is loaded in an MTP. (9) Number of Injections (#INJ)... Displays the number of injections to be performed for a particular sample. (10) Injection Volume (VOL)... Displays the volume of sample per injection. (11) File Number/Run Time (F#/RUN.T)... Enter the Method File number to be used for the gradient Time Program analysis. If a Time Program is not used, enter the analysis run time. The System Preparation file, Shutdown file, and Startup file are also registered here. Refer to Section 4.4 "Settings for Auto Processing" p. OP (12) Help Display Area... Displays a description and setting range for the parameter at which the cursor is located. (13) Status Display... Displays the current operation status of the instrument. Calculating Required Volume of Mobile Phase and Sequence End Time If the Startup file, System Preparation file, and the Repeat Injection Sequence file are set in the Sequence file, the Required Volume of Mobile Phase and Sequence End Time are not calculated automatically. In addition, these are not calculated for priority (STAT) analyses. For details on the Startup file and Repeat Injection file, refer to Section 4.4 "Settings for Auto Processing" p. OP - 69 and for Repeat Injection Sequence file, refer to Reference Manual, Section 3.1 "Creating A Repeat Injection Table" p. REF Control Samples To prove that the analyses are valid, a control sample (standard solution) is periodically injected and the Repeat Injection Table is used to verify that the sample and instrument conditions are not changing over time. Refer to Reference Manual, Section 3.1 "Creating A Repeat Injection Table" p. REF OP - 52

73 3 Creating a New Method File 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) Creating a Sequence File In the Sequence screen, create a table with multiple rows of parameters, including sample rack (RACK), sample number (SMPL#), number of injections (#INJ), injection volume (VOL (µl)), and applied Method file or analysis run time (F#/RUN.T), to perform continuous analysis. 1. In the "RACK" column of the Sequence screen, enter the position of the sample rack used in the analysis. (Numeric keys, ) The default sequence values are displayed for one row. Entering a "RACK" Value Enter a numerical value as indicated in the following figure. that the sample vial racks and microtiter plates are viewed from above; the front of the instrument is at the bottom of the figure. F 1 F 2 F 3 F 4 F 5 FUNC Sample Vial Racks Microtiter Plates When Sample Injection is Unnecessary If a sample will not be injected, as when plotting the baseline, enter "-1" (, ) for "RACK" or for "SMPL#". 2. In the "SMPL#" column, enter the position number of the first sample to be analyzed. (Numeric keys, ) Entering Sample Numbers When the cursor is moved to the "SMPL#" column, the default parameters are displayed for that row. If a microtiter plate is used, enter a sample number as directed in the Reference Manual, Section 3.3 "Setting Sample Number when Using a Microtiter Plate" p. REF Operation Manual OP - 53

74 3 Creating a New Method File 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) 3. In the "SMPL#" column, enter the position number of the last sample to be analyzed. (Numeric keys, ) The sample vials or wells associated with the specified numbers will be injected in a continuous series. Entering a Single Sample Number ("SMPL#") If only one vial or sample well will be analyzed, enter the same number for the first and last sample numbers. 4. In the "#INJ" column, enter the number of injections. (Numeric keys, ) Sets the number of injections to be performed from one vial or well. 5. For "VOL", enter the volume of sample to inject. (Numeric keys, ) Sets the volume of sample for each injection. 6. For "F#/RUN.T", enter one of the following, depending on whether a Time Program is used. (Numeric keys, ) 6-1. If a gradient or other Time Program is to be used, press and enter a file number for "F#/ RUN.T". (Numeric keys, ) 6-2. If a Time Program is not to be used, enter the analysis run time for "F#/RUN.T". 7. Repeat steps 1-6 until the entire Sequence table is created. 8. Press. Analysis begins. Operations Possible During Analysis, Such as Pausing, Stopping Analysis, or Creating Files Refer to the Reference Manual, Section 3.4 "Emergency Operations During Analysis" p. REF OP - 54

75 3 Creating a New Method File 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) Setting and Verifying the Sequence in the Method Screen In the Method screen, a single row of the Sequence file which is currently being run can be verified and corrected. The displayed Sequence parameters include sample rack (RACK), sample number (SMPL#), number of injections (#INJ), injection volume (VOL (µl)), injection time interval and Method file to be used or analysis run time (F#/RUN.T). To set up a continuous analysis sequence, enter the parameters in the Sequence screen as directed in Section "Creating a Sequence File" p. OP In the Method screen, move the cursor to the Sequence area. (Cursor keys) 3 2. Verify that "001/000" is displayed in the "STEP" column. F 1 F 2 F 3 F 4 F 5 FUNC Verifying "STEP" A "STEP" setting of "001/000" indicates that an Analysis Sequence Table has not been entered in the Sequence screen. If a setting other than "001/000" is displayed, and you wish to modify the Analysis Sequence Table, the changes can be made directly from the sequence setting area of the Method screen. If you do not wish to modify the Analysis Sequence Table in the Sequence screen, perform analysis using the Single Run function. (For more information, see Section 3.11 "Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis)" p. OP - 51.) 3. In the "RACK" column of the Sequence screen, enter the position of the sample rack used for analysis. (Numeric keys, ) Entering a RACK Value Enter a numerical value as indicated in the following figure. that the sample vial racks and microtiter plates are viewed from above; the front of the instrument is at the bottom of the figure Sample Vial Racks Microtiter Plates When Sample Injection is Unnecessary If a sample is not to be injected, as when plotting the baseline, enter "-1" (, ) for "RACK" or for "SMPL#". Operation Manual OP - 55

76 3 Creating a New Method File 3.11 Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis) 4. In the "SMPL#" column, enter the position number of the first sample to be analyzed. (Numeric keys, ) Entering Sample Numbers If a microtiter plate is used, enter the sample numbers as described in the Reference Manual, Section 3.3 "Setting Sample Number when Using a Microtiter Plate" p. REF In the "SMPL#" column, enter the position number of the last sample to be analyzed. (Numeric keys, ) The sample vials or wells associated with the specified numbers will be injected in a continuous series. Sample Number Setting ("SMPL#") If only one vial or well will be analyzed, enter the same number for the first and last sample numbers. 6. In the "#INJ" column, enter the number of injections. (Numeric keys, ) Sets the number of injections performed from one vial or well. 7. For "VOL", enter the volume of sample to inject. (Numeric keys, ) Sets the volume of sample used for each injection. 8. For "F#/RUN.T", enter one of the following, depending on whether a Time Program is used: 8-1. To run a gradient or other Time Program, press and enter a file number for "F#/RUN.T". (Numeric keys, ) 8-2. If a Time Program is not used, enter the analysis run time for "F#/RUN.T". (Numeric keys, ) 9. Press. Analysis begins. Operations Possible During Analysis, Such as Pausing, Stopping Analysis, or Creating Files Refer to the Reference Manual, Section 3.4 "Emergency Operations During Analysis" p. REF OP - 56

77 Creating a New Method File 3.12 Shutting Down the System after Analysis This section describes the procedure for shutting down the instrument, from the time that analysis is completed to when power is turned OFF. When "READY" is displayed in the status area, the analysis is complete. After replacing the mobile phase in the flow lines with a storage solution and verifying that the column oven temperature has decreased sufficiently, the power is switched OFF. 1. Place a bottle containing column storage solution in the reservoir box. 2. Purge the instrument as directed in Section 3.3 "Purging the System" p. OP About Purge If the system has already been purged, it is unnecessary to purge it again. 3. Press. The Method screen is displayed. 4. Enter the mobile phase port to be used for delivering the storage solution, decrease the column oven temperature, and enter the flow rate. Selecting Mobile Phase Port D Storing the Column If the column will not be used for three or more days, detach the column and fill it with an appropriate storage solution. For more information about storage, refer to the documentation provided with the column or contact the column manufacturer. F 1 F 2 F 3 F 4 F 5 FUNC 5. Verify that the column temperature has dropped sufficiently, and press and. The Pump and Oven, as well as their indicator lamps, are turned OFF. 6. Turn OFF the instrument. Automatic Shutdown If the Sequence includes a Shutdown file, the shutdown procedure is performed automatically. For more information, refer to Section 4.2 "Shutdown File Parameters" p. OP - 63, and Section 4.4 "Settings for Auto Processing" p. OP Operation Manual OP - 57

78 4 Operation with an Existing Method File The can be run automatically with a Sequence file that contains the necessary files. Method file System Preparation file Shutdown file Startup file This section explains how to create and use these files. Operation Flow Chapter 3, "Creating a Method File", describes how to start the instrument and prepare it for analysis. Be sure to perform all procedures described through Section 3.11 "Setting Sample Injection Parameters and Starting Analysis (Continuous Analysis)" p. OP The files used by the are created in the screens listed below. File Type Creation Screen Description Max Num of Saved Files System Preparation System Prep Contains analysis start condition parameters. 10 Shutdown Shutdown Contains instrument shutdown parameters. 2 Startup Startup Contains instrument parameters used after startup. Sequence Sequence Contains the file information and order for the multiple Method files and System Preparation files used in continuous analysis. 1 Method Method Contains analysis parameters for the pump, UV-VIS detector and column oven (displayed in Editing screen). 20 File Limitations in Simple Mode System Preparation, Shutdown and Startup files cannot be created in Simple mode. (Section "Description of the Simple Mode Screen" p. OP - 37) OP - 58

79 Operation with an Existing Method File 4.1 System Preparation File Parameters This section describes the parameters used to automate the purge and column equilibration functions Description of the System Prep Screen The items comprising the System Prep screen are described below. Pressing displays the System Prep screen (1) File Number... Indicates the System Preparation file number (0-9). (2) Auto Purge Parameters... Selects the mobile phase port and the autosampler rinse port to be purged using AutoPurge, as well as the solvent delivery time (min). (3) Stabilization Parameters... Enables column equilibration detection and automatic baseline stability verification when selected. Time Baseline Check Used when baseline stability is verified according to elapsed time. Used when column equilibration is verified according to baseline stability. (4) Pause Parameter... Selection enables a wait time following detection of column equilibration before proceeding to analysis. (5) Help Display Area... Displays a description and setting range for the parameter at which the cursor is located. (6) Status Display... Displays the current operation status of the instrument. Operation Manual OP - 59

80 4 Operation with an Existing Method File 4.1 System Preparation File Parameters Creating a System Preparation File The following parameters determine the automatic startup process: Auto Purge parameters. Baseline Stabilization criteria for automatic judgement. Pause after baseline stabilization prior to any analysis. File Number Selection 1. In the System Prep screen, enter a file number for the System Preparation file. (Numeric keys, ) About the System Prep File Number Select a file number from Press. The cursor moves to "Auto Purge". F 1 F 2 F 3 F 4 F 5 FUNC Auto Purge Parameters 1. Select the appropriate mobile phase port. (, ) A checkmark is placed next to the port. Port Name Mobile phase A Mobile phase B Mobile phase C Mobile phase D Autosampler rinse solution Screen Indication A B C D INJ 2. Enter how long to purge the port. (Numeric keys, ) 3. Repeat steps 1 and 2 for each port to complete the Auto Purge settings. Canceling the Port Setting When a checkmark is placed next to a mobile phase port or rinse solution port, the cursor moves to the time setting field. To remove a checkmark, move the cursor to the checkbox using the cursor keys, and press once again. OP - 60

81 4 Operation with an Existing Method File 4.1 System Preparation File Parameters Stabilization 1. Press. The cursor moves to "Stabilization". 2. Select the verification method. ( ) Pressing changes which verification method is selected as follows: Time Baseline Check No checkmark 4 Method Description Entry Time The baseline is considered stable after the set time Elapsed Time: "Time (min)" has elapsed. Baseline Check The baseline is considered stable when measured "Drift" and "Noise" values are less than the specified values within a set time period. Drift: "Drift (mau/h)" Noise: "Noise (mau)" Observation time: "Time (min)" No checkmark Baseline stability is not verified. 3. Depending on the judgement method selected, one of the following is performed If either "Time" or "Baseline Check" is selected, move the cursor and enter a numerical value. (, Numeric keys, ) 3-2. If "Stabilization" is not to be enabled (checked), press ( ). The cursor moves to "Pause". Processing Following Baseline Stability Check To wait before executing analysis for the next row in the sequence, place a checkmark at "Pause". Operation Manual OP - 61

82 4 Operation with an Existing Method File 4.1 System Preparation File Parameters Post-Stabilization Pause To include a wait period after baseline stabilization detection and before executing the next row in the sequence, enable the "Pause" function. 1. Verify that the cursor is positioned at "Pause", and press. A checkmark is placed in the "Pause" checkbox. About the "Pause" Checkbox After the "Pause" checkbox is checked, the cursor moves to "Stabilization". To remove the checkmark, move the cursor to "Pause" using, and press again. After removing the checkmark, use the same procedure to place the checkmark again as necessary. About "Pause" When the "Pause" function is enabled, "Pause" is displayed in the status display area after baseline stabilization. Press to start analysis. This completes the procedure for setting the System Preparation parameters. Instrument settings are recorded and remain valid even if the screen is switched or the power is turned OFF. OP - 62

83 Operation with an Existing Method File 4.2 Shutdown File Parameters The parameters saved in the shutdown file, including the Cool Down and Sleep State parameters, control automatic shutdown after analysis Description of the Shutdown Screen The items comprising the Shutdown screen are described below. Press [Shutdown] to display the Shutdown screen (1) File Number... File number of Shutdown file (0-1) (2) Cool Down Parameters... Uses the following items to define the cool down process: Period Flow Conc. Oven Temp. Time during which solvent flow continues. Mobile phase flow rate. Sets solvent concentration (%) of mobile phase ports during cool down. Turns column oven temperature control ON and OFF. When column oven temperature control is on, sets cool down temperature. (3) Shutdown Unit Parameters... Determines the instrument status during the sleep state which follows cool down. Degasser Sample Cooler Check here to turn OFF the degasser. Check here to turn OFF the sample cooler. Operation Manual OP - 63

84 4 Operation with an Existing Method File 4.2 Shutdown File Parameters (4) Help Display Area... Displays a description and setting range for the parameter at which the cursor is located. (5) Status Display... Displays the current operation status of the instrument. Instrument Status During Sleep State During the Sleep State, the UV-VIS detector lamp is extinguished, and the pump and LCD display are automatically turned OFF Creating a Shutdown File The following parameters determine the automatic shutdown process: Cool Down parameters Settings for degasser and sample cooler operation during sleep state File Number Selection 1. In the Shutdown screen, enter a file number for the Shutdown file. (Numeric keys, ) About the Shutdown File Number Select either 0 or 1 as the Shutdown file number. 2. Press. The cursor moves to "Cool Down". F 1 F 2 F 3 F 4 F 5 FUNC Cool Down Parameters 1. For "Period", enter the time to continue solvent flow. (Numeric keys, ) 2. Enter a flow rate for "Flow". (Numeric keys, ) 3. Select a mobile phase port. ( ) OP - 64

85 4 Operation with an Existing Method File 4.2 Shutdown File Parameters 4. Enter a solvent concentration for the selected port. (Numeric keys, ) 5. Repeat steps 3 and 4 to set the solvent concentrations for the remaining ports. When concentrations have been set for all the ports, the cursor moves to "Oven". 6. Select OFF or ON for "Oven". ( ) When ON is selected, enter a numerical value for "Temp". (Numeric keys, moves to the Degasser checkbox. ) The cursor About the Oven Parameter After OFF or ON is selected for "Oven", the cursor moves to the next item. To switch between OFF and ON, move the cursor back to "Oven" using the cursor keys, and press. 4 Shutdown Unit Parameters 1. Place a checkmark in the Degasser checkbox. ( ) The degasser will be OFF during the sleep state. The cursor moves to "Sample Cooler". To Leave the "DEGASSER" on During Sleep State Move the cursor downward using and proceed to step Place a checkmark in the Sample Cooler checkbox. ( ) The sample cooler will be OFF during the sleep state. Degasser and Sample Cooler Checkmarks When either "Degasser" or "Sample Cooler" is checked, the cursor moves to the next item. To remove the checkmark, move the cursor back to the relevant item using the cursor keys and press again. This completes the procedure for setting the Shutdown parameters. Instrument settings are recorded and remain valid even if the screen is switched or the power is turned OFF. Operation Manual OP - 65

86 Operation with an Existing Method File 4.3 Startup File Parameters Set the Startup parameters to automatically start the instrument at a specified time Description of the Startup Screen The items comprising the Startup screen are described below. After pressing Shutdown to display the Shutdown screen, press [Startup] to display the Startup screen (1) Wake Up Parameters... Defines when the instrument starts automatically. (2) Warm Up Parameters... Defines the warm-up conditions. After warm-up is complete, operation proceeds to the next row in the sequence. Period Flow Conc. Time during which solvent flow continues. Mobile phase flow rate. Sets solvent concentration (%) of mobile phase ports during warm-up. (3) Help Display Area... Displays a description and setting range for the parameter at which cursor is located. (4) Status Display... Displays the current operation status of the instrument. Startup File Number There is no number associated with the Startup file. Only the currently entered parameters are saved. OP - 66

87 4 Operation with an Existing Method File 4.3 Startup File Parameters Creating a Startup File The following parameters determine the automatic Startup process: Wake Up parameters Warm Up parameters Wake Up Parameters 1. In the Startup screen, enter the date and time at which the instrument is to start. (Numeric keys,, ) Example: To startup on June 5th at 10:20, use the panel as follows: 4 F 1 F 2 F 3 F 4 F 5 FUNC The cursor moves to "Period". About the Time System The uses a 24-hour time system. Wake Up Date and Time Parameters If is entered for the date, startup is executed when the time parameter is met. For example, if the setting is "Date -/-, Time 08:00", startup is executed each day at 8:00 a.m. If the instrument is already running at the set date and time, the parameters are ignored. If the instrument is performing analysis at the set date and time, analysis continues uninterrupted. After completing the series of analyses, the Warm Up procedure is executed. Warm Up Parameters 1. For "Period", enter the time to continue solvent flow. (Numeric keys, ) 2. Enter a flow rate for "Flow". (Numeric keys, ) Operation Manual OP - 67

88 4 Operation with an Existing Method File 4.3 Startup File Parameters Flow Rate After Instrument Startup Immediately following startup, the flow rate is lowered to about one third that at measurement until the column oven reaches constant temperature. 3. Select a mobile phase port for which to set the "Conc." parameter. ( ) 4. Enter a solvent concentration for the selected port. (Numeric key, ) 5. Repeat steps 3 and 4 to set the solvent concentrations for the remaining ports. This completes the procedure for setting the Startup parameters. Instrument settings are recorded and remain valid even if the screen is switched or the power is turned OFF. Operation Following Warm Up If a Startup file is set in the Sequence file, enter a Method file number. After Warm Up is complete, column equilibration is conducted automatically according to the Method file settings. (Section 4.4 "Settings for Auto Processing" p. OP - 69) OP - 68

89 Operation with an Existing Method File 4.4 Settings for Auto Processing Automatic processing is performed in the Sequence screen. This section describes the procedure for performing automatic processing using the System Preparation, Shutdown and Startup files created. The flow chart below outlines a typical order of operations as defined by a Sequence file. Mobile Phase Port Purge Column Equilibration Analysis System Cool Down Sleep State System Preparation File Method File Shutdown File 4 System Startup Startup File Previously Created Files System Preparation File 0 Method File 1 Shutdown File 0 File Number Startup File 5 (Method File) Registering the System Preparation File 1. Press. The Sequence screen is displayed. 2. Enter "-" for "RACK" in the first row. (, ) The cursor moves to "#INJ". About the "#INJ" Value Although a value other than "1" may be displayed for "#INJ", the file is executed only once. Enter Operation Manual OP - 69