Varian 356-LC RI Detector

Transcription

1 Varian 356-LC RI Detector Operation Manual Installation Category II Pollution Degree 2 Safety Class 1 (EN ) PL July 2007

2 Varian, Inc. offices Varian, Inc. has offices in most countries. The major offices for chromatography products are listed below: Polymer Laboratories Ltd, now a part of Varian, Inc. (Manufacturing site) Essex Road, Church Stretton, Shropshire SY6 6AX, UK International telephone: International fax: Varian, Inc. Scientific Instruments 2700 Mitchell Dr Walnut Creek, CA Telephone: International telephone: International fax: Varian B.V. Postbus EA Middelburg, The Netherlands International telephone: International fax: Varian Australia Pty. Ltd 679 Springvale Rd, Mulgrave, Victoria 3170, Australia International telephone: International fax: Varian Industria e Comercio Ltda. Avenida Dr. Cardoso de Mello 1644 Sao Paulo , Brazil International telephone: International fax: Internet The Internet home pages for Polymer Laboratories and Varian can be found respectively at: and Polymer Laboratories, now a part of Varian, Inc., is the owner of copyright on this document and any associated software. Under law, the written permission of Varian, Inc. must be obtained before either the documentation or the software is copied, reproduced, translated or converted to electronic or other machine-readable form, in whole, or in part. 2 Publication date: July 2007

3 First published July 2007 in Church Stretton, UK. Comments about this manual should be directed to the MarCom Manager, Polymer Laboratories at the factory address above or by to: Polymer Laboratories, now a part of Varian, Inc. is ISO9001:2000 certified Polymer Laboratories, now a part of Varian, Inc. All product and company names herein may be the trademarks of their respective owners. All rights reserved. Publication date: July

4 DECLARATION OF CONFORMITY We, Polymer Laboratories Ltd, now a part of Varian, Inc. Essex Road Church Stretton Shropshire SY6 6AX UK declare that the product: Varian 356-LC RI Detector Varian 356-LC Part number PL conforms with the requirements of the EU EMC Directive 2004/108/EC & Low Voltage Directive 2006/95/EC by complying with the following Harmonised European Standards: Safety: EN : 2001 Class I Installation category II Pollution degree 2 REINFORCED INSULATION EMC: EN55011: 1998 Conducted emissions: Class B Radiated emissions: Class B* EN : 1995, EN : 1996 ESD, Radiated immunity, EN : 1995, EN : 1995 Fast transient burst, Surge immunity, EN : 1996, EN : 1994 Conducted immunity, Voltage dips, EN : 2000, EN : 1995 Current harmonics, Fluctuations & flicker. No: 011, Church Stretton, 10 June 2007 Dr. S.O Donohue, Technical Director Instrumentation Publication date: July

5 Contents Safety Practices and Hazards... 9 General 9 Electrical Hazards 10 High Pressure Hazards 11 Cleaning 12 Other Precautions 12 Warning and Caution Messages 13 Warning Symbols 14 Colour Coding 15 US FCC Advisory Statement 15 Spurious and Harmonic Radiation Compliance 15 CE Compliance 16 Local Varian Offices or Distributors General Information Introduction Pre-installation Requirements User Documentation Conventions Notes and hot tips Specifications Unpacking and Installation Packing List Site Preparation Checklist Location of the Detector Module Do s and Don ts of RI Detectors Installation Site Requirements Pumping systems 23 Publication date: July

6 Mobile phase priming RI detector pressure and outlet tubing Installation Installation Checklist Power Connections Other Connections Digital output Analogue output Connection to a Varian Star 800 Module Interface Box Control I/O connector Fluid Connection Inlet Outlet Connecting the Detector Installing the Door Installing the door cap Installing the Varian 356-LC RI Detector Driver Software Reinstalling or upgrading drivers Configuring the communication server Configuring the Varian 356-LC RI Detector System Description and General Operation Basic Principles of Operation Internal flow path of Varian 356-LC RI Detector Instrument Controls Indicator lights Error conditions Operational Parameters Purge Autozero Sensitivity 41 6 Publication date: July 2007

7 3.3.4 Temperature Response time Polarity Baseline offset Galaxie Chromatography Software Control Controlling the Varian 356-LC RI Detector Status overview Automating the Varian 356-LC RI Detector General Considerations Solvent recommendations Solvent limitations Sample preparation Routine Maintenance Maintaining and Cleaning the Flow Cell Cell cleaning procedure Preparation for cell cleaning Cell cleaning hints Acid cleaning Other cell cleaning procedures Shutdown Procedures Corrosive solvents No flow versus reduced flow Continuous power on versus power off Long-term storage Flow Cell Integrity Checking the drain tube Replacement of Flow Cell Assembly Upgrading the Firmware Tools required Firmware upgrade procedure 54 Publication date: July

8 5. Troubleshooting Troubleshooting an HPLC System Instrument Errors General Problems 59 APPENDIX 1 Miscibility Chart APPENDIX 2 Polarity Index APPENDIX 3 Varian 356-LC RI Detector Test Procedure APPENDIX 4 Spare Parts Listing APPENDIX 5 Cleaning and Decontamination Procedure Cleaning 73 Decontamination 73 8 Publication date: July 2007

9 Safety Practices and Hazards General Your Varian instrument and accessories have been carefully designed so that when used properly you have an accurate, fast, flexible and safe analytical system. Information about safety practices appears throughout the documentation (both hard copy and online) provided with your instrument and accessories. Before using the instrument or accessories, you must thoroughly read these safety practices. Observe all relevant safety practices at all times. This instrument is designed for chromatographic analysis of appropriately prepared samples. It must be operated using appropriate gases and/or solvents and within specified maximum ranges for pressure, flows, and temperatures as described in this manual. If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Varian assumes no liability for the customer s failure to comply with these requirements. It is the responsibility of the customer to inform Varian customer support representatives if the instrument has been used for the analysis of hazardous biological, radioactive, or toxic samples, prior to any instrument service being performed or when an instrument is being returned to the service center for repair. Operation of a Varian 356-LC RI Detector involves the use of pressurized liquid and hazardous materials including corrosive fluids and flammable liquids. Careless, improper or unskilled use of this RI Detector can cause death or serious injury to personnel, and/or severe damage to equipment and property. The Varian 356-LC RI Detector incorporates covers that are designed to prevent inadvertent contact with any potential hazards. If the instrument is used in any manner not specified by Varian, this protection may be impaired. It is essential that no cover is bypassed, damaged or removed. Publication date: July

10 The safety practices described below are provided to help the user operate the instrument safely. Read each safety topic thoroughly before attempting to operate the instrument and ALWAYS operate the RI Detector in accordance with these safety practices. Follow these safety practices to ensure safe equipment operation. Perform periodic leak checks on all supply lines and pneumatic plumbing. Do not allow gas lines to become kinked or punctured. Place lines away from foot traffic and extreme heat or cold. Store organic solvents in fireproof, vented and clearly labeled cabinets so they are easily identified as toxic and/or flammable materials. Do not accumulate waste solvents. Dispose of such materials through a regulated disposal program and not through municipal sewage lines. This instrument has been tested per applicable requirements of EMC Directive as required to carry the European Union CE Mark. This equipment may be susceptible to radiation/interference levels or frequencies that are not within the tested limits. Electrical Hazards Exposure to high voltages can cause severe skin damage and cataracts of the eyes, while close contact with the electrical components can result in severe heat burns to the skin, and an electrical discharge which may cause death, severe electric shock or sub-surface skin burns. The RI Detector has been carefully designed to operate safely and effectively when using components that conform to Varian s design criteria. Use of non-approved components in the detector may render the system inoperative and/or hazardous. It may also invalidate the warranty on the instrument. Use only related components supplied or authorized by Varian. Voltages above 110 V AC are present within the instrument. Disconnect the instrument from all power sources before removing protective panels to avoid exposure to potentially dangerous voltages. Panels or covers which are retained by screws on the detector may be opened ONLY by Varian-trained, Varian-qualified, or Varian-approved Customer Service Representatives. Consult the manuals or product labels supplied with your instrument to determine which parts are operator-accessible. 10 Publication date: July 2007

11 Whenever it is likely that the protection has been impaired, the instrument must be made inoperative and be secured against any unintended operation. When it is necessary to use a non-original power cord plug, make sure the replacement cord adheres to the colour coding and polarity described in the manual and all local building safety codes. Good grounding/earthing is essential to avoid a potentially serious electric shock hazard. Consult the manuals or product labels supplied with your instrument for the relevant grounding requirements. Replace blown fuses with fuses of the size and rating shown on the fuse panel or in the manual. Replace faulty or frayed power cords immediately with the same type and rating. Ensure the power switch and appliance coupler at the rear of the instrument remain easily accessible at all times. Make sure that voltage sources and line voltage match the value for which the instrument is wired. Avoid using power supplies from a source that may be subject to electrical or RF interference from other services (for example, large electrical motors, elevators and welders). High Pressure Hazards If a line ruptures, a relief device opens, or a valve opens accidentally under pressure, potentially hazardous high liquid pressures can be generated by the pump causing a high velocity stream of volatile and/or toxic liquids. Wear face protection when you inject samples or perform routine maintenance. Never open a solvent line or valve under pressure. Stop the pump first and let the pressure drop to zero. Use shatterproof reservoirs capable of operating at 50/60 psi. Keep the reservoir enclosure closed when the reservoir is under pressure. Read and adhere to all NOTES, CAUTIONS, and WARNINGS in the manual. Publication date: July

12 Cleaning The individual or group responsible for the use and maintenance of this equipment must ensure that appropriate decontamination is carried out if hazardous material is spilt on or inside the instrument. Before using any cleaning or decontamination method except those recommended by Varian, users should check with Varian that the proposed method will not damage the equipment. See Appendix 5 for detailed recommended procedures. Other Precautions Use of the RI detector and accessories may involve materials, solvents and solutions that are flammable, corrosive, toxic or otherwise hazardous. Careless, improper or unskilled use of such materials, solvents and solutions can create explosion hazards, fire hazards, toxicity and other hazards that can result in death, serious personal injury or damage to equipment. Always ensure that laboratory safety practices governing the use, handling and disposal of hazardous materials are strictly observed. These safety practices should include wearing appropriate safety clothing and safety glasses. Air flow to the cooling fans of the RI detector and accessories must be unobstructed. Do not block the ventilation grills on the RI detector and accessories. Consult the manuals supplied with your PC and monitor for their specific ventilation requirements. Keep solvents from direct contact with polyurethane gas supply tubing as certain solvents will cause weakening and leaks with possible bursting. All components of the system should be connected to a common power supply and common ground. This ground must be a true ground rather than a floating ground. Non-polar solvents can develop a static charge when pumped through the system. All vessels that contain mobile phase (including tubing and collection vessels) must be grounded to dissipate static electricity. Employ static measuring and static discharge devices (e.g., air ionizers) to safeguard against the buildup of static electricity. The detector weighs approximately 11.0 kg (24.2 lb). To avoid injury to personnel or damage to the instrument or property, always use suitable lifting procedures to move the instrument. Use only Varian-supplied spares with your instrument. 12 Publication date: July 2007

13 Warning and Caution Messages A Warning message is used in the text when failure to observe instructions or precautions could result in death or injury. The list of symbols that appear in conjunction with warnings are detailed in the next section. A Caution message is used when failure to observe instructions could result in damage to equipment (Varian supplied and/or other associated equipment). Publication date: July

14 Warning Symbols The following is a list of symbols that appear in conjunction with warnings in this manual or on the RI Detector. The hazard they describe is also shown. A triangular symbol indicates a warning. The meanings of the symbols that may appear alongside warnings in the documentation or on the instrument itself are as follows: Corrosive liquid Electrical shock Explosion hazard Eye hazard Fire hazard Heavy weight (danger to feet) Heavy weight (danger to hands) Hot surface Moving parts The following symbol may be used on warning labels attached to the instrument. When you see this symbol, refer to the relevant operation or service manual for the correct procedure referred to by that warning label. The following symbols appear on the instrument for your information. I Mains power on 0 Mains power off Fuse Single phase alternating current 14 Publication date: July 2007

15 Direct current When attached to the rear of the instrument, indicates that the product complies with the requirements of one or more EU directives. When attached to the rear of the product, indicates that the product has been certified (evaluated) to CSA and UL Colour Coding The various indicator lights appearing on Varian instruments and associated accessories are color coded to represent the status of the instrument or accessory. A green light indicates the instrument is in normal/standby mode. An orange light indicates that a potential hazard or the instrument is under going change and not stable. A blue light indicates that operator intervention is required. A red light warns of danger or an emergency. US FCC Advisory Statement This equipment generates, uses and can radiate radio frequency energy, and if not installed and operated in accordance with the instruction manual may cause interference to radio communications. It has been tested and found to comply with the limits for a Class A computing device pursuant to Subpart J of Part 15 of Federal Communications Commission (FCC) Rules, which are designed to provide reasonable protection against such interference when operated in a commercial environment. Operation of this equipment in a residential area may cause interference, in which case the user will be required to take whatever measures may be necessary to correct the interference at his or her expense. Spurious and Harmonic Radiation Compliance Your Varian 356-LC RI Detector complies with the requirements of FCC rules and Regulations part 18, subpart H Miscellaneous Equipment, at the date of manufacture. Publication date: July

16 CE Compliance Your Varian 356-LC RI Detector has been designed to comply with the requirements of the Electromagnetic Compatibility (EMC) Directive and the Low Voltage (electrical safety) Directive (commonly referred to as the LVD) of the European Union. Varian has confirmed that each product complies with the relevant Directives by testing a prototype against the prescribed EN (European Norm) standards. Proof that a product complies with these directives is indicated by: the CE Marking appearing on the rear of the product, and the documentation package that accompanies the product containing a copy of the Declaration of Conformity. The Declaration of Conformity is the legal declaration by Varian that the product complies with the directives listed above, and shows the EN standards to which the product was tested to demonstrate compliance. It is also signed by Varian Authorized Representative in the EU, and by the representative of the manufacturing plant. Local Varian Offices or Distributors We stress the importance of standard laboratory safe practice (e.g., COSHH regulations) for dealing with electronic laboratory equipment, solvents, etc., in preventing accidents, fires, or potentially hazardous conditions. If in any doubt about the use of the instrument contact your local Varian office or local distributor. The Varian, Inc. Internet home page can be found at: 16 Publication date: July 2007

17 1. General Information 1.1 Introduction The Varian 356-LC differential refractometer is a universal detector designed for high-performance analyses where the refractive index of a flowing liquid with respect to a reference is required. Its small cell volume, high sensitivity, and accurate temperature control make it wellsuited for use as a detector in automated and manual high performance liquid chromatography. The Varian 356-LC RI Detector can be operated as a stand-alone HPLC detector or can be integrated into a Liquid Chromatography System. The detector is controlled remotely by Galaxie Chromatography software, through serial communications This manual instructs the user in the installation and operation of the Varian 356-LC RI Detector for stand-alone applications. For information on operating the detector using a desktop PC, refer to the Galaxie Chromatography software manual. 1.2 Pre-installation Requirements 1.3 User Documentation The Varian 356-LC RI Detector operation manual describes the environmental and operating requirements of the RI detector. You must prepare your laboratory according to these instructions before the RI detector can be installed. If you have misplaced your copy, you can obtain a free replacement from your local Varian office. Alternatively, download a PDF from the Varian, Inc. Web site, You have been provided with this operation manual to help you set up and operate your Varian 356-LC RI Detector. This operation manual contains safety practices and hazards information, where to find information about installing and maintaining instrument components and a brief operation overview. Publication date: July

18 1.3.1 Conventions The following conventions have been used throughout the documentation: Menus, menu options and field names (e.g., choose Copy from the Edit menu) have been typed in bold. Bold is also used to signify the buttons appearing throughout the software (e.g., click OK). ALL CAPITALS indicate keyboard commands (e.g., press the F2 key) and text you must type in from the keyboard (e.g., type SETUP at the prompt) Notes and hot tips 1.4 Specifications A Note is used to give advice or information. A Note is denoted by the following symbol: A Hot Tip is used to give practical hints to help you achieve the best possible performance from your instrument. A Hot Tip is denoted by the following symbol: The instrument is suitable for indoor use only and is classified suitable under the following categories (EN ): Installation category II Pollution degree 2 Safety class 1 Performance specifications: 18 Publication date: July 2007

19 RI range Range Linearity Sensitivity Short-term noise 1 Drift Response time Temperature control Cell volume 6 µl Light source Analogue output Digital output Polarity External communication Flow rate range Pressure rating 1.0~1.75 RIU x10-6 /FS RIU 600 µriu LOW (2), STD (4), HIGH (8) mv/µriu <5.0x10-9 RIU <2.5x10-7 RIU/hour sec OFF, C (1 C increments) LED 880 nm 1 V FSD 24 bit (10 Hz) via serial port Positive/Negative RS ml/min 100 kpa (15 psi) Total internal volume Normal operation 474 µl Purge mode 491 µl Wetted material Power requirements Power consumption Dimensions (wxdxh) (unpacked) Weight (unpacked) PC control (software) Remote operation Safety features 316 SST, Quartz Glass, PTFE, Perfluoroelastomer AC 100~240 V 50/60 Hz 150 W (max) 296 x 475 x 212 mm 11 kg Galaxie Chromatography Software Remote purge & autozero Error and leak detection Table 1. Performance Specification of the Varian 356-LC RI Detector 1 According to ASTM method E Practice for Refractive Index Detectors used in Liquid Chromatography. Detector conditions; temperature 35 C, response time 4 sec. Publication date: July

20 1.5 Unpacking and Installation 1.6 Packing List Care has been taken to ensure that the instrument should be received in proper condition. The packing and protection are designed for normal hazards of road, rail or air transit. Any damage to the container or instrument should be reported immediately to your local distributor, or to Varian. It is recommended that the shipping container be kept, if possible, for re-shipment or return to a service centre. Examine the shipping carton for visible signs of exterior damage. Unpack the instrument and examine it for transit damage. Check that all items on the packing list are included. Notify your local distributor or Varian of any damage or missing items. Standard items Varian 356-LC RI Detector Manual for Varian 356-LC RI Detector Varian 356-LC RI Detector Galaxie Driver software Mains leads (UK, EUR, USA) RS232 data cable Analog Output Cable 1.5 m PTFE outlet tubing (1.0 mm/0.040 ID) 65 cm Stainless Steel inlet tubing (1/16th OD/ ID) Valco Nut (1/16 ) Valco Ferrule (1/16 ) Half-moon door (grey) Door cap 20 Publication date: July 2007

21 1.7 Site Preparation Checklist Environmental conditions Power Bench space Extraction requirements Temperature 15 to 35 C (59 to 86 F) At constant temperature Avoid positioning in direct sunlight Humidity 40-80% V (AC), 1 A max, 50/60 Hz single phase with protective earth connection The mains supply should be fitted with an RCD Note: Ensure the power switch and appliance coupler remain accessible at all times. The standard instrument will require the following bench space: width 42 cm (16.5 ) depth 60.5 cm (23.8 ) height 31.2 cm (12.3 ) Note: The instrument is computer controlled and additional bench space will be required for the computer. The instrument is not fitted with extraction, but depending on the solvents used in the equipment it might be necessary to place the unit under an extraction hood or install in a fume cupboard. Note: Housing the unit in a fume cupboard may compromise the performance. Table 1. Site preparation checklist Warning The RI Detector weighs approximately 11.0 kg (24.2 lb). To avoid injury to personnel or damage to equipment, always follow all precautions for lifting heavy objects when moving the instrument. It is advisable to locate the system on a sturdy bench. Publication date: July

22 1.8 Location of the Detector Module Place the detector conveniently near your HPLC system. The modular design of the Varian 356-LC RI Detector enables you to locate it anywhere within the limitations imposed by the length of the power cord, fluid lines and signal cables. In order to keep liquid dead volume as low as possible and to minimize peak broadening in the lines, the distance between the column outlet and the flow cell inlet should be kept to a minimum. Provide approximately four inches of space behind the unit so that the cooling fan intake is not impeded, and to allow easy access to the rear panel. 1.9 Do s and Don ts of RI Detectors 1. NEVER subject the reference and sample cells to back pressures greater than 100 kpa (15 psi). High pressures can cause internal fluid leakage or break the cells, which are difficult to replace. 2. NEVER place a back-pressure regulator (such as Part No ) on an RI detector. 3. When placing more than one HPLC detector in series, always place the RI detector last. 4. Use tubing of 1.0 mm ID (0.040 ) or greater on the waste outlet of the Varian 356-LC RI Detector for less back-pressure. 5. Locate the Varian 356-LC RI Detector in an area that does not experience large temperature changes. The Varian 356-LC RI Detector has a sophisticated temperature control system but care in choosing its operating environment will ensure better operation. See Installation Site Requirements, page Always pre-mix and de-gas the mobile phase. Do not allow pumps to mix solvents for the mobile phase. Use only isocratic conditions. 7. Keep the RI cell clean. See page 49 for the cell cleaning procedure. 8. When filling the reference cell, ALWAYS use mobile phase that has eluted through the analytical column. Allow mobile phase to flow through the column for 10 minutes prior to filling the reference cell. 22 Publication date: July 2007

23 1.10 Installation Site Requirements Select the installation site in accordance with the Do s and Don ts in the previous section. Temperature fluctuation and airflow will affect the performance, causing drift, noise, unstable baseline, etc. The following locations should be avoided: Near an air conditioner Near heating equipment; especially cycling ovens Areas exposed to direct sunlight Near a ventilation fan Near vibration sources Near open windows and doors Near sources of electrical noise Areas where corrosive gases exist Dusty areas Allow sufficient bench space for peripheral instrumentation and to satisfy the following additional installation site requirements Pumping systems System pressure fluctuations will be measured as density variations by the Varian 356-LC RI Detector. Use a high-performance pumping system with no flow pulses to minimize the baseline noise level. For Varian Star 9000 series pumps and Varian 5000/5500 pumps or ProStar 210/215 pumps, make sure the pump is operating at 10 MPa (1500 psi) of backpressure and that the pulse damper is functioning properly. The backpressure can be achieved either by the column itself or a coil of ID tubing placed between the pump and the column. If pulse damping is needed, use the Varian Pulse Reduction Accessory (Part No ) or the Ultra High Pulse Reduction Accessory (Part No ). For the PrepStar SD1 pumps, no special operating pressure is required Mobile phase priming The entire flow system, consisting of pump, damper, injector, column, Publication date: July

24 sample and reference cells, etc., must be primed with the mobile phase. Flush the entire system with 5 10 times the system volume until a steady baseline is obtained. The mobile phase should be pre-mixed and degassed before use. Alternately, the mobile phase can be degassed online by a solvent degassing system. Note Make sure that the solvent within the detector is completely miscible with the intended solvent to be used. If this is not the case, the cell must be rinsed through with an intermediate solvent (e.g., acetone), which is miscible with both solvents RI detector pressure and outlet tubing Note If you are using more than one detector in series with the Varian 356-LC, the RI detector must always be the last component. The cell pressure upper limit is 100 kpa (15 psi). Use tubing of 1.0 mm ID (0.040 ) or greater on the waste outlet to protect the cells from elevated pressure, which could break them. Should the cells be damaged, the eluent will flow from the DRAIN on the side panel. The cell assembly should only be replaced by a Varian customer support representative. Warning Do not use a backpressure regulator on the waste outlet. In order to minimize equilibration time at the HIGH sensitivity setting, leave the system powered continuously for daily operation. 24 Publication date: July 2007

25 2. Installation 2.1 Installation Checklist The Varian 356-LC RI Detector must be installed by a Varian-trained, Varian-qualified or Varian-authorized customer support representative. Details for unpacking the instrument and what to do in case it has been damaged in transit are outlined in this operation manual in Chapter 1, sections 1.5 through This chapter describes how to install the Varian 356-LC RI Detector. Your CSR will: Unpack the RI detector Remove all packing list items that are shipped with the instrument (including this operation manual) and present them to you for review. Unpack the accessories supplied with the instrument and check that you have received everything on the packing lists (enclosed in the packing crate and inside each accessory box). Install your RI detector. Note One of the items you will receive is the Varian Quality card. Please fill in this card and return it to Varian Australia. Although the Varian-trained, Varian-qualified or Varian-authorized customer support representative will install the hardware and Galaxie software for you, you may need to install hardware or software yourself at some later stage, for example if you move the RI detector or change the PC. To install the Refractive Index detector: Connect the power and other cables Connect the fluid tubing Connect the detector Publication date: July

26 Install the door 2.2 Power Connections Install the Varian 356-LC RI Detector driver on the PC Configure the Varian 356-LC RI Detector driver. Before connecting the power cable, ensure the instrument voltage rating matches your local power supply. Use only a supply with protective grounding. The correct fuses should be installed. Note This unit is double-fused. For 115V (AC) or 230V (AC) use two 250V H 2A T 20x5 mm fuses Warning Risk of fire, replace fuses as marked. 2.3 Other Connections The Varian 356-LC RI Detector can communicate with other modules and devices in the entire HPLC system. All power and signal connections are made on the rear panel of the Varian 356-LC RI Detector. See Figure 2. The connectors on the rear of the panel support communication configurations to a wide range of Varian modules and non-varian devices, such as autosamplers, pumps, valves and injectors. 26 Publication date: July 2007

27 Figure 1. Front view of the Varian 356-LC RI Detector 1. Indicator lights 2. Power switch 3. Eluent outlet 4. Eluent inlet 5. Drain port Publication date: July

28 Figure 2. Rear view of the Varian 356-LC RI Detector 1. Serial RS232 connector 24 bit digital output 2. Control firmware flash upgrade switch 3. Connector control I/O 15 pin D type female 4. Analogue output - ±1 V 5. Mains switch 6. Mains input Digital output The Varian 356-LC RI Detector is fitted with a standard RS232 (DTE- DCE) 3-wire serial connector that provides a 24 bit (10 Hz) digital output for connection to a chromatographic acquisition device. 28 Publication date: July 2007

29 For digital output signals, attach the supplied serial cable (Part No. PL ) to the rear of the detector and the other end to a PC. The Varian 356-LC RI Detector is controlled from a PC using the Varian Galaxie Chromatography Data System software Analogue output For analogue output signals, connect the supplied analogue output cable (Part No. PL ) to the rear of the module. The open end of the analogue output cable has one positive (red) and one negative (black) wire, for connection into a Star MIB 800 module Connection to a Varian Star 800 Module Interface Box The Star 800 Module Interface Box (MIB) provides analogue-to-digital signal conversion (ADC). Connections are made by connecting the analogue output cable to one of the analogue signal input ports on the middle right side of the Star 800 MIB. For more information about connecting your Varian 356-LC RI Detector to a Star 800 MIB, refer to your Star 800 MIB documentation Control I/O connector The Varian 356-LC RI Detector can be connected to auxiliary equipment to pause or stop the operation of a pump or autosampler if the Varian 356-LC RI Detector reports an error condition. The Varian 356-LC RI Detector is equipped with one contact closure, which is normally open, and two TTL logic inputs, both active-low (with internal pull-up resistors to 5 V). These inputs can be used to remotely autozero and purge the detector by connecting them to ground, as described in Table 2. Note Pump stop facility must be employed if the instrument is to be left unattended, or if units are stacked. Publication date: July

30 I/O description Pin number Input Remote A/Z TTL Active Low 7 & ground Input Remote Purge TTL Active Low 14 & ground Output Pump stop contact closure normally open 3 & 10 Ground (to case) 1, 5, 6, 11 Table 2. Control I/O connector The instrument is not supplied with a connector for the I/O socket in order to make appropriate connections; however, a cable can be purchased for this from Varian (Part No. PL ). 2.4 Fluid Connection Inlet The eluent from the chromatography system is connected to the LOWER front port of the Varian 356-LC RI Detector via a low dead volume connector Outlet The eluent from the detector to the waste container should be connected to the UPPER front port of the Varian 356-LC RI Detector, using tubing of 1.0 mm ID (0.040 ), or greater, to prevent back-pressure. Caution Check all connection carefully for leaks before replacing the front panel. Warning Do not exceed a back-pressure of 15 psi (100 kpa) on the flow cell. The Varian 356-LC RI Detector must be the last component in any LC system. No detector or back-pressure regulator can follow it. Do not use narrow-bore tubing on the outlet of the Varian 356-LC RI Detector. 30 Publication date: July 2007

31 2.5 Connecting the Detector To connect the detector: 1. Connect the power cord to the IEC inlet at the rear of the unit. 2. Connect the column outlet to the bottom inlet at the front of the detector, using the minimum length of tubing (1/16 OD, ID) and a 1/16 Valco fitting. 3. Connect the waste tube (1/16 OD, ID) to the top outlet at the front of the detector and position the other end in a waste collection bottle. 4. Connect the instrument to the PC using the serial cable provided. 5. Turn on the eluent flow and allow the system to stabilize for a minimum of 1 hour. For optimum performance it is recommended that the Varian 356-LC RI Detector be allowed to equilibrate with the laboratory environment for a period of 24 hours. 6. Purge the detector for a period of 5 minutes to flush the solvent through the cell and flow lines. All analyses should be performed with purge turned OFF. 7. Allow a further 15 minutes for the baseline to stabilize before autozeroing. If an analytical column(s) is connected to the Varian 356-LC RI Detector, allow at least 3 column volumes for the baseline to stabilize. Note Make sure that the solvent within the detector is completely miscible with the intended solvent to be used. If this is not the case, the cell must be rinsed through with an intermediate solvent (e.g., acetone), which is miscible with both solvents. 2.6 Installing the Door The module door may be attached to the front of the Varian 356-LC RI Detector to cover the tubing connections to the flow cell Installing the door cap If the Varian 356-LC RI Detector is the top module in the stack, the door cap should be installed before installing the door on the module. If the Varian 356-LC RI Detector is not at the top of the stack, do not install the cap. Publication date: July

32 To install the door cap: 1. Stand the door upside down on a flat surface. See Figure Take the cap and remove the protective paper exposing the adhesive that will attach the cap to the door. 3. Insert the cap into the door and press the adhesive onto the inside of the door lip. Be sure to keep the door edges and the cap edges flush. See Figure 3. Magnetic latch Ribs Door cap Figure 3. Inserting the door cap onto the Varian 356-LC RI Detector door To install the door: 1. Insert the top hinge pin into the top hinge. See Figure Gently press down the top of the door and slide the lower hinge pin into the lower hinge. The door should now pivot on the pins and close. The magnetic door latch should stick to the instrument. 32 Publication date: July 2007

33 Figure 4. Installing the Varian 356-LC RI Detector door To remove the door: 1. Gently press down on the door and slide the lower hinge pin out of the lower hinge. 2. Lift and slide out the top of the door. 2.7 Installing the Varian 356-LC RI Detector Driver Software The Varian 356-LC RI Detector driver software is installed as part of the Varian LC drivers package. To install the Varian LC drivers, Varian Galaxie software must already be installed and you must have administrator rights for the PC. The Varian drivers must be installed only on the server. 1. When you insert the Galaxie software CD into your CD drive, the CD browser will automatically start. If it does not, you can launch the browser by running Install.exe from the root directory of the CD. 2. From the CD browser, click the Install Varian Drivers button. 3. Click the Install Varian LC button. The Welcome to the Publication date: July

34 InstallShield Wizard screen will appear. 4. Click the Next button, read and accept the License Agreement and enter your driver serial number in the corresponding field. Note The serial number is provided on your driver serial number card is different from the Galaxie serial number and is case sensitive. 5. Click the Next button and click Finish at the end of the installation Reinstalling or upgrading drivers If you want to reinstall or upgrade your drivers, you will need to: 1. Run the installation (steps 1, 2 and 3) again. 2. Click Next at the Welcome to Maintenance mode screen. The drivers will be automatically reinstalled or upgraded. 3. Click Finish at the end of the installation to exit the maintenance mode. To see which drivers are installed, select Options from the Galaxie Configuration Manager main menu and select Devices. The Add device from disk button can be used for installing the individual Varian LC-356 RI Detector driver. Use this button only when directed by a Varian technical support representative Configuring the communication server To communicate with the Varian 356-LC RI Detector an RS232 Interface or a RS232 PC communication bus must be configured on the acquisition server, using Galaxie Configuration Manager. The parameters of the bus to communicate with the Varian 356-LC RI Detector must be the following: Stops 1 Stop Baud Parity None Bits 8 Bits Flow Control None Buffer Tx 4096 Buffer Rx 4096 Table 3. Bus communication parameters 34 Publication date: July 2007

35 Note For more details about the configuration of a RS232 Interface or a RS232 PC bus, refer to the Galaxie Software Configuration Manager Operation manual Configuring the Varian 356-LC RI Detector The Varian 356-LC RI Detector driver software configuration is performed in Galaxie Configuration Manager. Please refer to the Galaxie Configuration Manager Operation manual for further information on how to use Galaxie Configuration Manager. 1. Click the button corresponding to the Varian 356-LC RI Detector driver. 2. In the Name field, select the name of the communication bus previously declared and configured with the Communication Server, as described in section In the Device Identifier field, enter the device identifier of the Varian 356-LC RI Detector. For example, if just one RI detector is connected to the system select 01. The device identifier must be unique for each RI detector connected to the system (The device ID can be set between 1-254). Note For more details about the Bus configuration, refer to Configuring the Communication Server in the Galaxie Configuration Manager Operation manual. Publication date: July

36 36 Publication date: July 2007

37 3. System Description and General Operation 2.1 Basic Principles of Operation In a deflection type refractive index detector a light beam is focused towards a flow cell containing a reference and sample compartment. See Figure 5. As the light beam passes through both the sample and reference compartments of the cell, the light undergoes a deflection in proportion to the difference in refractive index between the two liquids. The amount of deflection is measured using a photodiode, which is subsequently converted to an electrical signal at the output. Figure 5. Principle of RI detection When both cells contain pure solvent, the system is calibrated to zero by means of a parallel zero-plate that positions the beam on the two detector diodes I 1 and I 2 in such a manner that the measured light intensities (I 1 and I 2 ) of the two diodes are virtually identical. When the sample cell contains a solution with a different refractive index to that of the solvent, the light beam is geometrically proportionally deflected depending on the relative change of the refractive index Publication date: July

38 (according to Snell s refraction law). This results in a change of the light intensities I 1 and I 2, proportional to concentration and refractive index of the sample solution. From these intensity changes the signal value is calculated and indicated on the output display. I 1 = I 2 Figure 6. Zero Refraction I 1 > I 2 Figure 7. Refracted light beam Figure 8. Example of light beam shift on detector diodes Refractive index is a bulk property of the mobile phase and is a dimensionless constant that normally decreases with increasing temperature. Consequently RI detectors are very sensitive to changes in ambient temperature and need accurate control over their thermal surroundings. RI detectors are also sensitive to flow-rate changes and cannot be used for gradient elution. Note The refractive index is highly temperature dependent. The change per degree K for pure water is ~ 1x10-4 RIU and ~ 5x10-4 RIU for typical organic solvents Internal flow path of Varian 356-LC RI Detector The output from the column should be connected to the inlet of the RI detector using small bore tubing, typically 0.25 mm ID stainless steel or PTFE tubing. The detector outlet should be connected to stainless steel or PTFE capillary with a minimum internal diameter of 1.0 mm and go directly to the waste reservoir. It is advisable not to connect any additional detectors after the RI detector to minimize back-pressure on the measurement cell, which will result in pressure pulsation on the baseline and in the worse case crack the cell. 38 Publication date: July 2007

39 Key 1. Cell 2. T-piece 3. Solenoid Valve a. Normally closed b. Normally open c. Common Ref 1 Sample Purge flow path Normal flow path 2 3 a b c Waste Column Figure 9. Flow scheme of Varian 356-LC RI Detector The maximum recommended flow rate permitted through the Varian 356- LC RI Detector is 10 ml/min. For more viscous solvents (e.g., 2- propanol) it is not recommended to work at the maximum flow rate. Before running solvent through the device make sure that the solvent within the detector is completely miscible with the intended solvent to be used. If this is not the case, the cell must be rinsed through with an intermediate solvent (e.g., acetone), which is miscible with both solvents. Note The Varian 356-LC RI Detector is shipped in isopropanol. Publication date: July

40 3.2 Instrument Controls Indicator lights Three indicator lights are located at the top left of the front panel of the module. They are: Ready/Run Auto-Zero Purge The following table defines each status: LED Status Colour Ready/Run Auto-zero Purge Ready/Run Not Ready (i.e., changing temp) Error Auto-zero on Auto-zero off Purge on Purge off Green Orange Flashing Green/Orange Green Off Green Off Table 4. LED indicator lights Error conditions 3.3 Operational Parameters Purge The Varian 356-LC RI Detector is equipped with a number of sensors and error checking facilities to ensure safe operation. If an error is detected the instrument gives an audible warning and the Ready/Run light will flash green and orange. A complete list of instrument errors and remedial actions are given in the troubleshooting section of this manual. When Purge is selected, the purge indicator light is highlighted, and solvent flow is diverted through the reference side of the RI cell, instead of the sample side; before going to waste. See Figure 9. The purge time can be adjusted to compensate for different solvents and eluent flow rates. 40 Publication date: July 2007

41 Note Ensure the purge valve is closed before making a sample injection Autozero Sensitivity The detector output is re-set to zero when the autozero function is selected. An error will occur if the autozero process takes longer than 5 minutes. The sensitivity of the Varian 356-LC RI Detector can be adjusted to provide greater flexibility. The three possible sensitivity settings are described below: Sensitivity Low Standard High Application Suitable for highly concentrated samples (e.g., Prep LC) Default setting, ideal for the majority of LC applications Suitable for samples of low concentration in order to maximise signal-to-noise. Table 5. Sensitivity settings Changing the detector s sensitivity will affect the instruments dynamic range; for example, changing the detector from Low to High sensitivity alters the dynamic range from 600 µriu/fs to 150 µriu/fs. Note Longer equilibration times maybe required at the high sensitivity setting Temperature The temperature of the Varian 356-LC RI Detector can be set between C (1 C increments) or turned off. The default value is 35 C. It is recommended that the temperature should be set at least 5 C above ambient temperature in order to ensure a stable thermal environment. The Ready/Run indicator light on the front of the detector turns green when the detector has reached temperature. If the detector temperature is not equilibrated then the Ready/Run light will turn orange. For best operation it is recommended that the system be powered continuously for daily operation. Publication date: July

42 3.3.5 Response time The Varian 356-LC RI Detector has a default response time of 1.0 second, with a settable range of seconds. Lower response time settings remove less baseline noise and produce narrow peaks with minimal peak distortion and time delay. Lower settings also make very small peaks harder to discriminate from baseline noise. Higher response time settings can reduce baseline noise, but will shorten and broaden peaks Polarity Baseline offset The output of the Varian 356-LC RI Detector can be either positive or negative; therefore, it is essential that the polarity of the detector signal be configured. The polarity parameter is used to invert the sign of the refractive index data. A positive polarity leaves the data unaffected, whilst a negative polarity essentially inverts the chromatogram. The baseline offset of the Varian 356-LC RI Detector can be set between the ranges 100 to +100 µriu. 3.4 Galaxie Chromatography Software Control The Varian 356-LC RI Detector requires Galaxie Chromatography Software and a free Serial (RS-232) communications port (1 to 255) on your PC in order to operate Controlling the Varian 356-LC RI Detector Within Varian Galaxie s main menu screen, direct control of the instrument can be accessed via the Varian 356-LC RI Detector button Status window In the status window, the detector s output, status and control parameters are shown with their current values. When the Autozero button is selected, the detector will begin an autozero operation. When this function is active, the autozero indicator light turns solid green. When the Purge button is selected, the detector will begin purging. When this function is active, the purge indicator light turns solid green. The 42 Publication date: July 2007

43 purge time can be set in the configuration menu Parameter configuration menu To set the detector parameters, click the Configure button to access the Parameter Configuration menu. In the Parameter Configuration menu, the following detector parameters can be set and downloaded to the detector. Parameter Sensitivity Operational Range (1) Low (2) Standard (3) High Temperature OFF, C Response Time Baseline Offset ±100 µriu Polarity Normal (+), Inverted (-) Purge Time Off, 30, 60, 120, 300 seconds Table 6. Detector parameters Once the detector parameters are set, click the Send button to download the values to the detector. The Send option is disabled during an injection Connection status When the Varian 356-LC RI Detector is connected to Galaxie Chromatography Software, the detector s firmware version (e.g., 1.2.1) is shown at the top left corner of the configuration screen. If communication is lost, or no RI detector is connected, offline is shown Status overview A general overview of the HPLC system and current status of the Varian 356-LC RI Detector can be accessed via the Overview button, within Galaxie s main menu screen. The overview screen displays a graphical representation of the Varian 356-LC RI Detector, along with the detector s key parameters. The status of the detector is indicated by the Ready message and status light, shown at the top of the detector icon. The Varian 356-LC RI Detector has 3 status modes, which are indicated with the following Publication date: July