Instructions for Use Laser Induced Fluorescence (LIF) Detector Manual LIF Instructions for Use PN 718113AF January 2014 Beckman Coulter, Inc. 250 S. Kraemer Blvd. Brea, CA 92821 U.S.A.
Laser Induced Fluorescence (LIF) Detector Manual LIF Instructions for Use PN 718113AF (January 2014) © 2012-2014 Beckman Coulter, Inc. All rights reserved. Made in U.S.A. No part of this document may be reproduced or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission from Beckman Coulter, Inc. Beckman Coulter and the stylized logo are trademarks of Beckman Coulter, Inc. and are registered in the USPTO.
Revision History This document applies to the latest software listed and higher versions. When a subsequent software version changes the information in this document, a new issue will be released.
Revision History iv PN 718113AF
Safety Notice Read all product manuals and consult with Beckman Coulter-trained personnel before attempting to operate instrument. Do not attempt to perform any procedure before carefully reading all instructions. Always follow product labeling and manufacturer’s recommendations. If in doubt as to how to proceed in any situation, contact your Beckman Coulter Representative.
Safety Notice Laser Light Laser Light This symbol indicates that a potential hazard to personal safety exists from a laser source. When this symbol is displayed in this manual, pay special attention to the specific safety information associated with the symbol.
Safety Notice FCC Class A Compliance CAUTION Do not attempt to load or remove labware manually while the instrument is in operation. WARNING Do not spill liquids on or around the instrument. Wipe up any spills immediately. WARNING To avoid serious damage to the instrument, make sure that the laboratory site voltage/frequency matches the voltage/frequency that was ordered for the instrument.
Safety Notice EMI Compliance viii PN 718113AF
Contents Revision History, iii Safety Notice, v Alerts for Danger, Warning, Caution, Important, and Note, v High Voltage Danger, v Laser Light, vi Laser Specifications, vi Cautions and Warning Found in this Manual, vi FCC Class A Compliance, vii EMI Compliance, vii Laser Induced Fluorescence Detection, 1 Specifications, 1 System Overview — LIF System, 2 The LIF Detector, 2 Component Description, 2 The Laser Modules, 3 488 nm Argon Ion Laser Module, 4 635 nm Laser Module, 5 Using Other Lasers with the LIF
Contents Preparation, 17 Configuring 32 Karat for LIF Operation, 18 Direct Control, 22 Laser Control, 22 Time Program, 23 Dynamic Range, 23 Calibration Correction Factor, 24 Calibration Wizard, 26 Calibration Wizard – Step 1, 27 Calibration Wizard – Step 2, 28 Calibration Wizard – Step 3, 30 Calibration Wizard – Step 4, 31 Manual Mode, 32 Care and Maintenance, 32 Introduction, 32 Inspecting the System, 32 Cleaning, 33 Storage, 33 x
Illustrations Illustrations 1 Instrument with LIF Detector, 3 2 488 nm Laser Module Back Panel, 5 3 LIF Optical System (Rotated 90° counterclockwise), 7 4 Optical Diagram, 8 5 Installing the Interconnect Module, 10 6 Installing Filters in the Filter Housing, 12 7 Installing the Filter housing into the LIF Detector, 13 8 Installing the LIF Plug and Probe Stabilizer, 15 9 Connecting the Probe to the LIF Cartridge, 16 10 Main Menu, 18 11 Configure Instrument menu command, 19 12 Choosing
Tables Tables 1 xii LIF Detection Specifications, 1
Laser Induced Fluorescence Detection Specifications Table 1 LIF Detection Specifications Specification Value Relative Fluorescence Units (RFU) range 0 to 1000 RFU Dynamic Range (at a dynamic range setting of 1000) > 104 Sensitivity 1 ´ 10-11M Sodium Fluorescein with a signal-to-noise ratio ≥ 2 Baseline Noise < 0.005 RFU peak to peaka Baseline Drift < 0.2 RFU per houra 488 nm Laser Module Dimensions Height 25.00 in. (63.5 cm) Width 10.25 in. (26.3 cm) Depth 14.25 in. (36.
Laser Induced Fluorescence Detection System Overview — LIF System Table 1 LIF Detection Specifications (Continued) Specification Value Power requirements 100/120V, 12A, 50/60 Hz or 220/240V, 6A, 50/60 Hz Ambient Temperature Operating Range 15 to 40°C (39 to 104°F) Recommended Best Performance Range 15 to 30°C (59 to 86°F) Relative Humidity <95% non-condensing @ 35°C (121°F) a. These specifications are for a 75 mm I.D. capillary.
Laser Induced Fluorescence Detection System Overview — LIF System Figure 1 Instrument with LIF Detector 1 2 3 4 PWP UV HV + HV - LASER ON CURRENT LIMIT I O 5 LASER ON CURRENT LIMIT I O BECKMAN P/ACE SYSTEM LASER MODULE 488 900708L.AI 1. Cartridge Cover 2. LIF Interconnect Module 3. 488 nm Laser Module 4. LIF Detector Module 5.
Laser Induced Fluorescence Detection System Overview — LIF System WARNING During normal operation of the LIF Detector, laser light is not accessible to the user. To prevent potentially harmful laser light from being emitted from the end of the fiber cable, an interlock mechanism turns off the laser if the laser fiber cable is disconnected from the interconnect module, or if the cartridge cover is opened. Always power off the laser module and instrument before removing any of the LIF system module(s).
Laser Induced Fluorescence Detection System Overview — LIF System Figure 2 488 nm Laser Module Back Panel 635 nm Laser Module IMPORTANT This laser is discontinued and is no longer supported by Beckman Coulter. The power switch is located on the back panel of the 635 nm laser housing. However, the laser will not light up unless the instrument is on, and all doors and interlocks are in place. A red light on the front panel of the 635 nm laser module indicates that the laser is on.
Laser Induced Fluorescence Detection System Overview — LIF System connected to your laser’s interlock circuit. The connectors and switches in the detector are rated for low voltage (TTL level) signals. CAUTION To avoid damaging the interlock connector and switches in the LIF Detector Module, do not connect interlock wires in the laser unit to a voltage greater than the rated voltage of the interlock connector and switches (5V, 0.1 A). U.S.
Laser Induced Fluorescence Detection System Overview — LIF System Figure 3 LIF Optical System (Rotated 90° counterclockwise) 7 9 1 8 2 5 6 10 3 4 900726L.AI PN 718113AF 1. Laser 1 6. Beam Splitter (50/50) 2. 3. 4. 5. 7. Photomultiplier Tube #2 (DETECTOR #2) 8. Laser Filters (Notch Filters) 9. Emission Filters 10.
Laser Induced Fluorescence Detection Installation Figure 4 Optical Diagram 1. Collimated Collected Beam To Detector 2. Modified 5mm Dia. Sapphire lens (R1 and R2 = 2.5 mm) 3. Cross Section of the Capillary 4. Spherical Reflector 5. 6. 7. 8. Excitation Fiber for Laser #1 Excitation Fiber for Laser #2 Plug Probe Installation WARNING Be sure to power down the instrument before proceeding with component installation.
Laser Induced Fluorescence Detection Installation Bench Space The instrument and the laser require an area of approximately four feet by two feet. Allow adequate space between instruments for air circulation (6 inch minimum). Additional space is required for a computer, printer, and other optional equipment, if applicable. Leave adequate space around the laser for air circulation. CAUTION Some laser modules produce significant quantities of heated air.
Laser Induced Fluorescence Detection Installation Figure 5 Installing the Interconnect Module 1. Probe Holder 2. Fiber Optic Connector 10 3. Interlock Connector 4.
Laser Induced Fluorescence Detection Installation Installing the LIF Detector Module Prior to installing the LIF Detector, install the filter(s) and filter housing. Installing Filters in the Filter Housing The filter housing, which is inserted in the right side of the LIF Detector, has two filter holders, one for each detector channel. Refer to Figure 6 for a detailed diagram of the Filter Housing.
Laser Induced Fluorescence Detection Installation 6 If the filter(s) are removed from a channel, re-install the beam blocker in its place to protect the photomultiplier tube. NOTE If there is no arrow on the edge of the filter you are using, look at both sides of the filter and determine which side has the more reflective surface. (Typically the more reflective side also has a wide edge rim that extends beyond the filter surface).
Laser Induced Fluorescence Detection Installation Installing the Filter Housing into the Detector Insert the filter housing into the opening in the right side of the detector. Refer to Figure 7 for more information. Make sure the “keyed” corner of the filter housing lines up with the “keyed” corner of the Detector Module. The housing will fit into the opening in this orientation only, so it cannot be installed incorrectly.
Laser Induced Fluorescence Detection Installation Installing the LIF Detector into the Instrument WARNING Be sure to power down the instrument before proceeding with component installation. Hold the LIF Detector level and place it into the instrument, as shown in Figure 7, keeping the two female connectors on the LIF Detector level to the two male connectors located on the instrument. Secure the Detector Module in place by fastening the captive screws to finger tightness.
Laser Induced Fluorescence Detection Installation Figure 8 Installing the LIF Plug and Probe Stabilizer 1 2 900808L.AI 1. LIF Plug 2. Probe Stabilizer Install the cartridge containing an LIF plug into the instrument. Refer to section 3 of the Installation and Maintenance Manual for more information on this procedure. Connecting the Probe to the Cartridge When connecting the probe to the LIF Cartridge, align the two pins on the left side of the probe to the corresponding openings on the clamping bar.
Laser Induced Fluorescence Detection Installation Figure 9 Connecting the Probe to the LIF Cartridge 1 PWP UV HV + HV - 2 900706L.AI 1. Probe Stabilizer 2.
Laser Induced Fluorescence Detection Operation Operation Preparation WARNING Make sure the laser fiber optic cable and interlock devices are connected to the LIF Detector before turning on power to the instrument or the laser. If the interlock is defeated, laser light could be accessible in the interior or emitted from the fiber optic cable. 1 Follow the instructions described in the Installation section before proceeding with step 2. 2 Turn on the instrument. 3 Turn on the laser.
Laser Induced Fluorescence Detection Operation Configuring 32 Karat for LIF Operation 1 The 32 Karat Enterprise screen is shown in Figure 10. Right-click in this screen to create a new instrument. Right-click, then click New > Instrument.
Laser Induced Fluorescence Detection Operation 2 Name the new instrument. Right-click the newly created instrument to configure, then click Configure > Instrument . . . See Figure 11 and Figure 12.
Laser Induced Fluorescence Detection Operation 3 Choose the type of instrument you want to control, P/ACE MDQ CE or ProteomeLab PA 800. A warning notice appears stating that changing the instrument type will erase ALL device settings. Since this is a new instrument configuration and there is no data to lose, choose OK to continue. Then click Configure. Figure 13 Confirmation There are two options for configuring an instrument, manual or auto configure.
Laser Induced Fluorescence Detection Operation The P/ACE System MDQ Instrument Configuration screen allows the user to define the types of trays installed, define the home position of each tray, identify the firmware version and access the LIF Calibration Wizard. Figure 15 Configuring the LIF Detector 5 PN 718113AF Choose OK to accept all the parameters set up in the configuration.
Laser Induced Fluorescence Detection Operation Direct Control Figure 16 shows the Direct Control screen for instruments configured with an LIF Detector. Figure 16 Direct Control Screen 1 Clicking the laser symbol on the Direct Control screen brings up the Laser Control dialog box, shown in Figure 17. Select ON to indicate that the lasers are on. NOTE The lasers cannot be controlled individually.
Laser Induced Fluorescence Detection Operation status on the Direct Control and Status screen reflects the position of this on/off switch and not the state of the laser. Time Program When programming a method with LIF Detection, select Method > Instrument Setup. The instrument setup window will appear. See Figure 18. Figure 18 LIF Detector Initial Conditions Screen NOTE Excitation and Emission Wavelength settings are for indication purposes only.
Laser Induced Fluorescence Detection Operation Figure 19 Electropherograms (A, B) with different Dynamic Ranges A. Electropherogram with Dynamic Range set to 100. B. Same Electropherogram with Dynamic Range set to 10. Note that the heights of all peaks have exceeded the dynamic range setting and were truncated. For optimal results, select a dynamic range slightly larger than the tallest peak in the electropherogram.
Laser Induced Fluorescence Detection Operation • Fluorescence yield of the molecule being measured • Background buffer composition and properties • Separation parameters (temperature, voltage, etc.) • Laser power To minimize variation caused by (small) changes to any of the parameters listed above, a correction factor can be used to normalize the detector response against a defined standard.
Laser Induced Fluorescence Detection Operation Once the CCF value is calculated and accepted by the user, all subsequent measurements will be multiplied by this value. As long as run conditions, capillary I.D. and chemistries remain unchanged, the CCF calculated should remain valid. It is recommended that the standard solution be measured (as a check) periodically or when a new capillary is installed. NOTE The Calibration Correction Factor determined in this process will affect all subsequent analysis.
Laser Induced Fluorescence Detection Operation 4 Click on the LIF Calibration Wizard. See Figure 20. Figure 20 Accessing the LIF Calibration Wizard 1. LIF Calibration Wizard button Calibration Wizard – Step 1 After starting the Calibration Wizard, the Calibration Wizard – Step 1 screen is displayed as shown in Figure 21.
Laser Induced Fluorescence Detection Operation Figure 21 Calibration Wizard – Step 1 Selecting Auto Mode will guide the user through a series of steps that will automatically determine the appropriate value for the calibration correction factor (CCF). Select Auto Mode, and click the Next button. The Calibration Wizard - Step 2 screen appears, as shown in Figure 22. NOTE Manual Calibration is discussed in the section Manual Mode.
Laser Induced Fluorescence Detection Operation 2 Click Next.
Laser Induced Fluorescence Detection Operation Calibration Wizard – Step 3 The following Calibration Wizard – Step 3 screen appears, as shown in Figure 23. 1 Load the buffer and calibration mix as shown in Figure 23. Figure 23 Calibration Wizard – Step 3 NOTE The Buffer and Calibration mixture must be appropriate for the analysis to be run. It is recommended to use 2.0 ml buffer vials with red caps for this procedure.
Laser Induced Fluorescence Detection Operation 2 After installing the vials in the tray, close the instrument lid and click Next. The screen shown in Figure 24 is displayed while the calibration is running. Please wait until the instrument has completed its calibration, which may take several minutes. WARNING DO NOT open the instrument doors during this step.
Laser Induced Fluorescence Detection Care and Maintenance Manual Mode The Manual Mode allows the user to enter values for the Calibration Correction Factor manually. When Manual Mode is selected in Step 1 (see Figure 21), the screen shown in Figure 26 is displayed. Enter the desired CCF values, and click Accept. These values will be used for all subsequent analysis. NOTE Running the Calibration Wizard in the Auto Mode is preferred for optimum performance.
Laser Induced Fluorescence Detection Care and Maintenance Cleaning Clean the exterior surfaces of the detector and laser module regularly by wiping with a clean, damp cloth. If necessary, a mild detergent can be used; finish by wiping with a soft, dry cloth. The filters are delicate optical components that must be protected from dirt, dust, and fingerprint contamination. Once installed in the filter housing, they are protected from contamination and normally do not require cleaning.
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