Möbiu Hardware User s Guide. (M3001 Rev. B)

Transcription

1 Möbiu Hardware User s Guide (M3001 Rev. B)

2 Copyright 2014, Wyatt Technology Corporation. All rights reserved. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of Wyatt Technology Corporation. WYATT TECHNOLOGY Corporation makes no warranties, either express or implied, regarding this instrument, computer software package, its merchantability or its fitness for any particular purpose. The software is provided as is, without warranty of any kind. Furthermore, Wyatt Technology does not warrant, guarantee, or make any representations regarding the use, or the results of the use, of the software or written materials in terms of correctness, accuracy, reliability, currentness, or otherwise. The exclusion of implied warranties is not permitted by some states, so the above exclusion may not apply to you. DAWN, HELEOS, TREOS, Optilab, ViscoStar, NanoStar, Calypso (hardware), Mobius, Möbiu, ASTRA, DynaPro, DYNAMICS, AURORA, International Light Scattering Colloquium, Light Scattering University, Light Scattering for the Masses, Protein Solutions, Wyatt Technology, and the Wyatt Technology legacy logo are registered trademarks of Wyatt Technology Corporation. minidawn, T-rEX, UT-rEX, WyattQELS, Plate Reader, Eclipse (hardware and software), Comet, Atlas, DNDC Kit, Orbit, CALYPSO (software) and Solaris are trademarks of Wyatt Technology Corporation. A variety of U.S. and foreign patents have been issued and/or are pending on various aspects of the apparatus and methodology implemented by this instrumentation. This equipment must be disposed of as electronic waste. Contact your nearest Wyatt Technology Corporation representative for instructions on how to return the product to Wyatt Technology Corporation for proper disposal.

3 Table of Contents Chapter 1: Introduction... 7 Overview...8 The Instrument...8 The Software...10 About This Manual...10 How the Manual Is Organized...10 How to Contact Wyatt Technology...11 Corporate Headquarters Sales Department Technical Support Where to Go from Here...12 Chapter 2: Safety, Warnings, and Cautions Warnings...14 Warnings on the Display Panel...15 Labels on the Chassis...16 Laser Specifications and Safety Notes...17 Chapter 3: Installing the Instrument Unpacking the Instrument...19 Installing DYNAMICS Software...19 System Requirements...19 User Accounts with Restricted Privileges...19 CD Contents...20 Installing the Software...20 Installing the Instrument...21 Connecting Auxiliary Devices...23 Chapter 4: Instrument Components Front Panel View...25 Back Panel View...27 Top Cover View...28 Laser...29 Laser Beam Warning...29 Laser Monitors...29 Alarms...30 Vapor Sensor...30 Liquid Level Leak Sensor...30 Turning Off the Alarm...30 Möbiu User s Guide (M3001 Rev. B) 3

4 Contents Chapter 5: Using the Instrument Display Navigating the Display Panels...32 Front Panel Buttons...32 Warning Lights and Alarms...33 Hazards...33 Audio Alarm...33 Main Panel...34 Selecting Display Settings for the X and Y Axes...35 Adjusting the Display Range...36 Setting the Cell Temperature...37 Laser...37 Mobility Panel...38 QELS Panel...39 Alarm Panel...42 System Panel...45 Comm Panel...47 Chapter 6: Plumbing and Introducing Sample Sample Preparation...50 Fluid Cells...51 Flow Cell...51 Optional Dip Cell Assembly...52 Disposable Cuvette (DLS Only)...52 Measuring Single Samples...53 Performing Manual Injections into the Flow Cell...53 Using an HPLC Pump and Manual Injector Valve...57 Using the Quartz Cuvette and Dip Cell...58 Making DLS-Only Measurements with Disposable Cuvettes...61 Automated Injections with an Autosampler...62 Terminology...62 Autosampler Sequence...63 Example Event Schedule and Data...64 Chapter 7: Maintenance Procedures General Maintenance...68 Outer Case Maintenance...68 Air Filter Maintenance...68 Changing a Fuse...69 Flow Cell Basic Maintenance...70 Regular Maintenance...70 On-line Cleaning...70 Protease Cocktail Rinse Möbiu User s Guide (M3001 Rev. B)

5 Contents Disassembling and Cleaning the Flow Cell...73 Preparation...74 Part List...75 Removing the Flow Cell...77 Disassembling the Flow Cell...77 Cleaning the Flow Cell Housing...80 Cleaning and Reinstalling the Electrodes...80 Cleaning and Reinstalling the Cell Windows...82 Cleaning and Reinstalling Inlet and Outlet Tubing...84 Reinstalling the Flow Cell...84 Chapter 8: Mobility Theory and Calculations Electrophoresis and Electrophoretic Mobility...86 Introduction to Mobility Measurement with Light Scattering...88 Massively-Parallel Phase Analysis Light Scattering (MP-PALS)...92 Parameters Derived from the Mobility...93 Net Charge...93 Zeta Potential...93 Appendix A: Connecting to a Network or PC Components...97 Instrument Connections...97 LAN Connection...98 Computer Connections...99 Crossover Cable Ethernet Cable Ethernet to USB Adapter Ethernet Switch Connecting to a LAN Instrument to LAN Instrument and Computer to LAN Connecting via USB Connecting via Ethernet Without a LAN Instrument Network Settings Troubleshooting and Diagnostics Verifying Instrument Connections Appendix B: Using an Alternative Autosampler Overview Terminology Autosampler Sequence Sample Injection Wash Buffer Injection Appendix C: Adding DLS to Instruments Requirements Connecting to a Wyatt HELEOS or TREOS Möbiu User s Guide (M3001 Rev. B) 5

6 Contents Appendix D: Troubleshooting Low Forward Laser Monitor Voltage Case A: Sample Blocks Laser Transmission Case B: A Setting on the Instrument is Incorrect Appendix E: Instrument Specifications Electrical and Optical Specifications Environmental Specifications Laser Safety Notes Index Möbiu User s Guide (M3001 Rev. B)

7 1 Introduction When you can measure what you are speaking about, and express it in numbers, you know something about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meager and unsatisfactory kind: it may be the beginning of knowledge, but you have scarcely, in your thoughts, advanced to the stage of science. Lord Kelvin William Thomson Kelvin, the 19th century physicist and mathematician who wrote that paragraph, would have been very comfortable with the Möbiu system. The Möbiu system is designed with the goal of measuring the electrophoretic mobility and the hydrodynamic radius of precious samples, with minimal perturbation of these fragile species. It is the first and only light-scattering instrument that makes reliable, reproducible, and non-destructive electrophoretic mobility measurements of macromolecules as small as 1 nm under dilute solution conditions. Simultaneous dynamic light scattering (DLS) detection performed with the optional, embedded WyattQELS hardware enables simultaneous measurement of the molecule's hydrodynamic radius (R h ). Together, the electrophoretic mobility and R h can be used to calculate zeta potential and net charge. The Wyatt Atlas accessory allows measurements to be taken in high-conductivity solutions, such as physiological saline, without the risk of bubble formation due to the electrolysis of water. CONTENTS PAGE Overview...8 The Instrument 8 The Software 10 About This Manual...10 How the Manual Is Organized 10 How to Contact Wyatt Technology...11 Corporate Headquarters 11 Sales Department 11 Technical Support 11 Where to Go from Here...12 Möbiu User s Guide (M3001 Rev. B) 7

8 Chapter 1: Introduction Overview The Möbiu makes fast and reliable measurements of macromolecular electrophoretic mobilities. Besides being capable of measuring mobilities of large particles, such as liposomes and virus-like particles (VLPs), the Möbiu is the only laser-based instrument that achieves reproducible measurements of traditionally very challenging protein samples, including antibody formulations. Adding dynamic light scattering (DLS) provides simultaneous measurement of the macromolecular hydrodynamic radius, which allows for the calculation of zeta potential and net molecular charge. The Möbiu is equipped with a low-volume reusable flow cell for measuring mobility. Samples can be introduced by manual injection, an auto-sampler, syringe pump, or an auto-titrator. The Möbiu is temperature controlled (4 70 C) for automated temperature studies. The Instrument The Möbiu implements massively-parallel phase analysis light scattering (MP-PALS) as shown in Figure 1-1. Sample is introduced in the flow cell and the flow is stopped. An oscillating electric field is applied to the cell, and the sample moves in the field according to its charge. The light scattered by the moving particles is quantified to determine their velocity, which is directly proportional to the electrophoretic mobility. Piezo modulator Beamsplitter DLS Photodiode Array Laser Cell +V -V Data Processing Figure 1-1: Optical path diagram. To measure the velocity of the particles moving in the flow cell, the laser beam is first split into separate sample and reference beams. The sample beam is scattered by the moving particles, collimated, and then recombined with the reference beam onto an array of 31 photodetectors. 8 Möbiu User s Guide (M3001 Rev. B)

9 Overview This technique is called Massively-Parallel Phase Analysis Light Scattering (MP-PALS). The signals from the array elements are processed in real time with a proprietary demodulation algorithm. The photodetector array has an extraordinary dynamic range, which allows it to measure the zeta potential of molecules as small as 1 nm. The multiple detectors used in the Möbiu efficiently reveal the electrophoretic velocity, even with low applied voltages. The Möbiu can accurately measure mobility with an applied voltage fewer than 3 V, although it is also capable of generating high fields (up to 600 V/cm) suitable for samples in (non-conducting) organic solvents. The Möbiu system uses a 50 mw diode pumped solid state (DPSS) singlelongitudinal-mode laser operating at 532 nm. The Möbiu also includes a state-of-the-art electronics package with an embedded microprocessor and a graphical user interface. The laser system, optics and piezo modulator, flow cell assembly, and multi-channel photodiode array are all anchored to the base plate to provide a single, stable optical bench. All functions are controlled by the microprocessor. Instrument Options and Accessories Dynamic Light Scattering (DLS): This is an internally installed option that measures time-dependent fluctuations in the back scattered light using a fast photon counter. DLS measurements can determine the hydrodynamic radius of macromolecules or particles. Disposable DLS-only cuvettes: The Möbiu is shipped with an adapter for disposable semi-micro cuvettes. This accessory allows you to measure the hydrodynamic radius of a sample but cannot be used for mobility measurements. Dip cell assembly: This optional assembly enables low-volume (45 L) mobility and DLS measurements in a reusable quartz cuvette. Atlas hardware accessory: Applying an electric field to conducting, aqueous samples often results in bubble formation due to the electrolysis of water. The optional Atlas accessory enables mobility measurements of highly conductive samples, including physiological saline, by pressurizing the Möbiu flow cell and preventing bubble formation. Removable door kit: This optional kit allows you to collect data with the top panel door open. Wyatt Injection System: This injection valve with a 1 ml injection loop allows you to make manual injections with a closed fluid path, using your HPLC pump to deliver solvent and sample. See Using an HPLC Pump and Manual Injector Valve on page 57. Möbiu User s Guide (M3001 Rev. B) 9

10 Chapter 1: Introduction Table 1-1: Accessories and part numbers The Software Item P/N Quartz Cuvette Kit (cuvette + adaptor) WMQC-00 Spare Quartz Cuvette WNQC01-00 Disposable Cuvette Kit Package of 100 Disposable Cuvettes P Dip Cell Kit WMDC-00 Removable Door Kit WMRD-01 Wyatt Injection System WISH Data collection and analysis are performed with the DYNAMICS software. DYNAMICS analyzes the electrophoretic mobility data from the Möbiu to calculate parameters such as zeta potential and net charge based on models selected by users. For Möbiu instruments with the DLS option, DYNAMICS also analyzes the light scattering data to determine hydrodynamic radius. Please see the DYNAMICS User s Guide for information about the software and details on data analysis. About This Manual This manual describes how to set up and use the Möbiu laser photometer. Please see the DYNAMICS User s Guide for details on data analysis. How the Manual Is Organized The chapters and appendices in this manual are organized as follows: Chapter 1, Introduction introduces the Möbiu and this manual, and describes the support options available from Wyatt Technology. Chapter 2, Safety, Warnings, and Cautions lists safety information you need to know when using the Möbiu. Chapter 3, Installing the Instrument takes you through the necessary first steps for unpacking, connecting, and testing the instrument. Chapter 6, Plumbing and Introducing Sample describes the different ways that sample can be introduced to the instrument and how to perform measurements. Chapter 4, Instrument Components takes you on a guided tour of the instrument. Chapter 5, Using the Instrument Display shows you how to navigate and change settings in the Möbiu front panel display. 10 Möbiu User s Guide (M3001 Rev. B)

11 How to Contact Wyatt Technology Chapter 7, Maintenance Procedures has procedures for keeping the instrument in good working order. It includes flow and batch cell cleaning instructions. Chapter 8, Mobility Theory and Calculations describes how the Möbiu works and how values can be calculated using the data it collects. Appendix A, Connecting to a Network or PC covers connecting the Möbiu to either a network through the Ethernet, or to a host PC through the Ethernet-to-USB converter. Appendix B, Using an Alternative Autosampler describes how to use a non-supported autosampler and HPLC pump to automate alternate injections of buffer and sample. Appendix C, Adding DLS to Instruments describes procedures for using the Möbiu DLS option with other Wyatt detectors. Appendix D, Troubleshooting provides ways to correct problems you may encounter. Appendix E, Instrument Specifications supplies the electrical, optical, and environmental specifications for the laser head. How to Contact Wyatt Technology If you have a question about your Möbiu, look in this manual or consult the online help that comes with the DYNAMICS software. If you cannot find an answer, please contact Wyatt Technology Technical Support. Corporate Headquarters Wyatt Technology Corporation 6300 Hollister Ave. Santa Barbara, CA, USA Sales Department Wyatt Technology Corporation Sales Hours are 8:30 A.M. to 5:00 P.M. Pacific Time. Sales Phone: (805) Sales Fax: (805) Technical Support Wyatt Technology Corporation offers a variety of support options to help you get the most from your Möbiu. You can also contact the Wyatt Technology Distributor in the country where you bought your product. Möbiu User s Guide (M3001 Rev. B) 11

12 Chapter 1: Introduction Before contacting technical support, try to resolve any problems through the DYNAMICS on-line help system and this manual. World Wide Web URL: Electronic Mail Address: Technical Support Phone: (805) Technical Support Fax: (805) To speak to our support personnel directly, please call between 8:30 A.M. and 5:00 P.M. Pacific Time, Monday through Friday. When you call you should be at your instrument and have the documentation at hand. Please be prepared to provide the following information: Instrument serial number (located on the back panel). Microsoft Windows version number; DYNAMICS version number; exact wording of any messages that appear. The software version number is located on the original distribution diskette(s), or you can view it by selecting About from the Help menu. The type of computer hardware you are using. What you were doing when the problem occurred. How you tried to solve the problem. Where to Go from Here First, review Chapter 2, Safety, Warnings, and Cautions for important safety notices. Then, continue to Chapter 3, Installing the Instrument to check out your shipment and make necessary initial checks and adjustments. If you have purchased the DLS option and would like to use your Möbiu to make DLS measurements in other Wyatt instruments, you will also want to read Appendix C, Adding DLS to Instruments. 12 Möbiu User s Guide (M3001 Rev. B)

13 2 Safety, Warnings, and Cautions As recommended by the American National Standards Institute, we have used icons along with messages to especially alert the user to potential hazards involved in this technology, following this standard: DANGER! Failure to follow will result in injury or DEATH. WARNING! Failure to follow may result in injury. Caution! Failure to follow may result in damage to equipment. Note: Additional information which may be of interest. For best results and user safety, the following warnings and cautions should always be followed when handling and operating your Möbiu. Möbiu User s Guide (M3001 Rev. B) 13

14 Chapter 2: Safety, Warnings, and Cautions Warnings DANGER! DO NOT CLEAN WITH FLAMMABLE SOLVENTS! Using volatile solvents to clean the Temperature Controlled Möbiu can create deadly vapors, fire, and explosions. Toluene and THF are permitted in the flow cell. DANGER! The power supply provided with your Möbiu is designed to convert high voltage to the level required for operation. Do not open the power supply or defeat any of its safety interlocks. Serious injury or Death may result. DANGER! The Möbiu has no user serviceable parts. For your safety, do not dismantle internal assemblies except for the flow cell assembly. Do not bypass any of the safety systems and interlocks that are in place for your health. If the instrument is not functioning properly, do not apply power. WARNING! The Möbiu read head, flow cell, cuvette, and other components can reach temperatures hot enough to cause serious burns. Wear heat resistant gloves and exercise extreme caution when handling heated components. 14 Möbiu User s Guide (M3001 Rev. B)

15 Warnings on the Display Panel Warnings on the Display Panel The Möbiu instrument actively controls the temperature within the sample cell. The range of temperature control for the Möbiu is from +4 C to +70 C. The Möbiu can reach temperatures hot enough to cause serious burns. The following caution is shown on the display any time the door is opened while the Möbiu is heated. Current Cell Temperature Under all operating conditions the laser beam is entirely contained within the read head. When the top door is opened, the laser is either blocked or attenuated depending on the model (see Laser Beam Warning on page 29), and the following warning is displayed. Möbiu User s Guide (M3001 Rev. B) 15

16 Chapter 2: Safety, Warnings, and Cautions Labels on the Chassis Möbiu Identification Plate Möbiu Standards Certification 16 Möbiu User s Guide (M3001 Rev. B)

17 Laser Specifications and Safety Notes Laser Specifications and Safety Notes The laser used in the Möbiu is a Class IIIb laser. However the Möbiu itself is classified as a Class 1 Laser Product according to IEC :1993+A1+A2 and CFR Title 21 Subchapter J. Note these environmental specifications apply to the laser subsystem and not to the instrument as a whole. This means that under normal operation, no laser radiation should escape from the instrument, and no protective equipment must be worn. However the following warning applies: WARNING! Use of controls or adjustment or performance of procedures other than specified herein may result in hazardous radiation exposure. The instrument also bears the following warning label affixed to the back panel. Note: All safety labels are in English. If you need safety labels in a language other than English, please contact Wyatt Technology. Möbiu User s Guide (M3001 Rev. B) 17

18 3 Installing the Instrument This chapter helps you get the Möbiu unpacked, tested, and connected. You will also make some first time adjustments. CONTENTS PAGE Unpacking the Instrument...19 Installing DYNAMICS Software...19 System Requirements 19 User Accounts with Restricted Privileges 19 CD Contents 20 Installing the Software 20 Installing the Instrument...21 Connecting Auxiliary Devices...23 Möbiu User s Guide (M3001 Rev. B) 18

19 Unpacking the Instrument Unpacking the Instrument Please read the shipping parts list (packing slip) included with your instrument shipment and check that everything arrived in good condition. 1. Carefully examine the shipping container. If it is damaged or shows signs of mishandling, contact Wyatt Technical Support immediately. 2. Unpack the instrument. 3. Place the Möbiu on a level surface and inspect the exterior of the instrument for damage. If you see any damage, contact Wyatt Technical Support immediately. 4. Check that the boxes contain all of the items listed as included with your instrument shipment in addition to the instrument (the packing slip sent with the instrument contains the most up-to-date list). Installing DYNAMICS Software The DYNAMICS software must be installed prior to connecting the Möbiu to your PC. You can read more about DYNAMICS in your DYNAMICS User s Guide. System Requirements As of the date of publication of this manual (April 7, 2014), the minimum system resources DYNAMICS requires are as listed below. For current DYNAMICS system requirements please refer to our website: DYNAMICS 7 requires either a 32-bit or 64-bit edition of Windows 8 (Pro or Enterprise), Windows 7 (Professional or Ultimate), or Windows Vista (Business, Enterprise, or Ultimate) or Windows XP Professional 32-bit edition Intel Core 2 Duo processor or better 4 GB of RAM or better At least 1 GB of available hard-disk space CD-ROM Drive (optional: for installation) Ethernet port or USB 2.0 port for instrument connections User Accounts with Restricted Privileges If DYNAMICS is to be run from a user account with restricted privileges, it is necessary to install DYNAMICS under the account to be used. If DYNAMICS is installed globally, you must have Administrator or Windows Power User privileges to run DYNAMICS. Möbiu User s Guide (M3001 Rev. B) 19

20 Chapter 3: Installing the Instrument CD Contents Your installation CD includes the following items required to control the Möbiu. DYNAMICS software files PDF file of the DYNAMICS User s Guide PDF file of this manual, the Möbiu Hardware User s Guide Software drivers related to the USB interface Additional software utilities Installing the Software Install the software as follows: 1. Restart your computer to ensure that no other programs are running and that any previously installed DYNAMICS components are not running. 2. Insert the DYNAMICS CD into your CD drive. On most systems, the DYNAMICS setup procedure will start automatically. If the setup procedure does not start automatically, use Windows Explorer or the Run dialog to run the setup.exe file in the DYNAMICS folder on the CD. 3. Answer the prompts in the setup procedure. 4. To verify the installation of DYNAMICS, open the Windows Start menu and look for All Programs Wyatt Technology DYNAMICS 7.x.x. 20 Möbiu User s Guide (M3001 Rev. B)

21 Installing the Instrument Installing the Instrument The installation procedure for the Möbiu involves some initial tests to see that everything is working properly. To install the Möbiu, do the following: 1. Chose a location for the Möbiu. Consider the following when making this choice: Place the instrument on a flat, clean surface, standing on its feet and positioned to allow air flow through the back to keep its electronics cool. While the Möbiu can be stacked above or below other Wyatt instruments, such as the DAWN or Optilab T-rEX, it cannot be used in online mode in conjunction with a continuous-flow process, like chromatography, and should not be plumbed to instruments running in this mode. Note that the drain system is designed to cascade, so only a single drain tube needs to be connected at the bottom of a Wyatt instrument stack. The Möbiu should be installed on top of all other instruments in the stack. This allows easy access to the flow cell through the top cover for cleaning or for using the quartz cuvette or dip cell. Make sure the Möbiu is not located in direct sunlight. The best results will be achieved if the environment is stable to ± 1º C or less and the ambient temperature is 15º C to 30º C. The humidity must be low enough that condensation will not occur at the ambient temperature. 2. Plug the instrument in. Make sure the supplied power plug is correct for the local power outlet. The Möbiu is equipped with a universal power supply, which operates anywhere in the world. It accepts inlet voltages between V and line frequencies from Hz. Möbiu User s Guide (M3001 Rev. B) 21

22 Chapter 3: Installing the Instrument DLS Connections 3. Connect one end of the supplied Ethernet cable to the Ethernet port on the back of the Möbiu and the other end to your local area network or the host computer. Alternatively, you can use the supplied USB-to- Ethernet converter and connect to the USB port on the host computer. See Appendix A, Connecting to a Network or PC for details. When the Möbiu is on the local area network, it may be accessed and controlled from any machine on the network. When connected directly to the host computer via Ethernet or the USB-to-Ethernet converter, the Möbiu can be accessed only by the host computer. See Instrument Network Settings on page 106 for network security information regarding these two configurations. 4. Switch on the instrument and let it warm up for 30 minutes. The power switch is on the front panel. 5. Nitrogen Purge Option (for instruments cooled below ambient temperatures): While the instrument is warming up, attach a filtered dry air or chromatographic-grade nitrogen line to the Nitrogen Purge connector on the back of the Möbiu. Use the 90-degree fitting and the 10-inch Polyethylene tubing provided. The dry gas will flow into the cell cavity and will minimize the amount of dust in the cell cavity. The pressure in the dry air or nitrogen line should be between 20 psi and 80 psi. If you are operating below ambient temperature, it is particularly important to use the nitrogen purge line to prevent condensation. Fan Nitrogen Purge Fuse Holder Power Plug Aux In Ethernet 1&2 USB Alarm In Alarm Out Auto Inject Out Auto Inject In 22 Möbiu User s Guide (M3001 Rev. B)

23 Connecting Auxiliary Devices 6. Plumb the Möbiu as described in Chapter 6, Plumbing and Introducing Sample and connect the instrument to any other devices as described in Connecting Auxiliary Devices on page 23. Connecting Auxiliary Devices You can connect the Möbiu to other devices using the connectors on the back panel. All auxiliary device connectors on the back panel of the Möbiu are current limited to protect the internal circuitry. The auxiliary device connectors on the back of the Möbiu are: Aux 1 and Aux 2: You can connect the Möbiu to up to two external detectors. The AUX input signals can accept an input range of -10 V to 10 V with a 0.3 mv resolution. These connectors are also intended for future use by WTC accessories for the Möbiu. Alarm In/Alarm Out: Selected on the instrument display Alarm panel. Note that TTL voltage levels are +5 V (logic 1) or 0 V (logic 0). Auto Inject In/Auto Inject Out: You can use this connector to sense or drive an injection from an auto injector. This signal is also monitored by the DYNAMICS software. Connecting to the Auto-Inject In port is not required if you are using an Agilent HPLC pump and autosampler (see Automated Injections with an Autosampler on page 62), but it may be useful for troubleshooting. Connecting to the Auto-Inject In port is required for automated sample analysis using a non-agilent HPLC pumps and autosamplers (see Appendix B, Using an Alternative Autosampler ). Möbiu User s Guide (M3001 Rev. B) 23

24 4 Instrument Components This chapter gives you a guided tour of the Möbiu components. If you have just installed the Möbiu, read this chapter to become familiar with the various instrument parts and their functions. CONTENTS PAGE Front Panel View...25 Back Panel View...27 Top Cover View...28 Laser...29 Laser Beam Warning 29 Laser Monitors 29 Alarms...30 Vapor Sensor 30 Liquid Level Leak Sensor 30 Turning Off the Alarm 30 Möbiu User s Guide (M3001 Rev. B) 24

25 Front Panel View Front Panel View The front panel (see Figure 4-1) contains the main power switch (On/Off), provides fluid connections for the Möbiu, along with the display window and display controls for operating the instrument and monitoring data. Fluid Connector Panel LCD Display Keypad On/Off switch Figure 4-1: Möbiu Front Panel LCD Display: The LCD display allows you to monitor, control, and configure the Möbiu. Chapter 5, Using the Instrument Display describes the functions of the tabs available on the LCD display. Keypad: The keypad allows you to control the LCD display. Navigating the Display Panels on page 32 describes how to use the keypad. IN/OUT Fluid Connectors: Fluid ports are behind the fluid connector panel door (see Figure 4-2). Fluid comes into the Möbiu through the IN port, and exits through the OUT port. If the Möbiu is stacked on top of the Atlas or another Wyatt detector, the drain system is designed to cascade so that only a single drain tube needs to be connected at the bottom of the instrument stack. Note: The fittings used by Wyatt instruments are standard chromatography fittings as supplied by Parker, IDEX Health & Science, or Valco. Fittings supplied by Waters Corporation will seal but may cause a gap within the fitting, giving rise to excessive mixing. Waters fittings are not recommended. Möbiu User s Guide (M3001 Rev. B) 25

26 Chapter 4: Instrument Components LEAK SENSOR RINSE: Use the leak sensor rinse port to empty the leak sensor reservoir after a leak alarm. Connect a luer-lock syringe to the LEAK SENSOR RINSE port and draw out the fluid. If you are using a mobile phase with salt, the salt can dry on the leak sensor causing it to malfunction by reporting a leak when no leak is present. In that case, several ml of water can be injected in and out of the leak sensor reservoir through the LEAK SENSOR RINSE port. After several rinse cycles, any dried salt should be removed. IN/OUT Fluid Connectors Leak Sensor Rinse Figure 4-2: Fluid Connectors 26 Möbiu User s Guide (M3001 Rev. B)

27 Back Panel View Back Panel View The back panel contains the AC power module, data connectors, nitrogen purge connector, correlator input for the DLS option, and the cooling fan. The main power fuses are located in the AC power module. DLS Connections Fan Nitrogen Purge Fuse Holder Power Plug Aux In Ethernet 1&2 USB Alarm In Alarm Out Auto Inject Out Auto Inject In Figure 4-3: Back panel The following sections of this manual provide information about the back panel connectors: Fuse holder: Changing a Fuse on page 69. Power plug: Installing the Instrument on page 21 Ethernet and USB: Appendix A, Connecting to a Network or PC Aux In 1 & 2: Connecting Auxiliary Devices on page 23 Alarm In and Out: Alarm Panel on page 42 Auto Inject In and Out: Connecting Auxiliary Devices on page 23 Nitrogen Purge: Installing the Instrument on page 21 Fan and Air Filter: General Maintenance on page 68 DLS Connections: Appendix C, Adding DLS to Instruments Möbiu User s Guide (M3001 Rev. B) 27

28 Chapter 4: Instrument Components Top Cover View The standard cover comes with a popup door to allow access to the flow cell and for introducing the batch cuvette when in batch mode. The drain ports are located on the top and bottom covers. Popup Door Drain Port In Drain Port Out Figure 4-4: Möbiu Top Cover 28 Möbiu User s Guide (M3001 Rev. B)

29 Laser Laser The 50 mw diode pumped solid state (DPSS) single-longitudinal-mode laser provides the light source for the system. The laser system provides very high power density at the illuminated sample by means of a narrow beam diameter (the 1 e 2 diameter of the Gaussian beam profile is 0.08 mm). This small beam diameter also helps reduce the noise contributions of larger particulate contaminants (such as dust). The laser is oriented so that the incident beam is vertically polarized. A beam monitor (laser monitor) is incorporated into the laser assembly. The output of this monitor can be displayed on the Main panel in the display window. Laser Beam Warning See Laser Specifications and Safety Notes on page 17 for details and warnings about the Class IIIb laser used in the Möbiu. Under all operating conditions the laser beam is entirely contained within the read head. The behavior of the laser when the door is open is dependent on the model number, which is shown on the back panel label and on the Certificate of Performance. Möbiu WMOB-01 (model 1): The laser beam is blocked when the popup door is open. Möbiu WMOB-02 (model 2) and higher: The laser beam is attenuated to Class 1 laser power when the popup door is open. It is good laboratory practice with any laser source, irrespective of its power, to AVOID LOOKING INTO THE BEAM. Figure 4-5 shows the warning label on the read head. Appendix E gives the laser specifications. DANGER LASER RADIATION WHEN OPEN AVOID DIRECT EXPOSURE TO BEAM. Figure 4-5: Laser beam warning label Laser Monitors The Main panel on the display can graph the following values related to laser power. The values are reported as an absolute value in mw. The Laser Power measures the intensity of the beam before it enters the cell. This value is not used in calculations on the Möbiu. The FM Amplitude enables the Möbiu to measure transmitted light through the flow cell and sample. The value of the forward monitor (FM) amplitude indicates whether the instrument is properly aligned and whether the cell is properly installed. For absorptive samples, the FM amplitude can also be used to gauge the absorption of the sample. The Alarms panel indicates whether the laser is on and stable. Möbiu User s Guide (M3001 Rev. B) 29

30 Chapter 4: Instrument Components Alarms The Möbiu will sound an audible alarm when a potential hazard is reported. Hazards include: vapor or liquid leak sensor activates cell protection thermostat activates (for over temperature conditions) external alarm input activates When a potential hazard is detected, the alarm output on the back panel also activates. This is so that this signal can be used to control other instruments. For example, this signal can be used to turn off a pump. For details about the alarms conditions detected by the Möbiu, see Alarm Panel on page 42. Vapor Sensor The Möbiu has a vapor sensor to aid in the safe operation of the instrument, especially at high temperatures. The vapor sensor is not intended as a protection device but as a convenience to alert the operator to the possibility of flammable liquid or vapor inside the instrument. The alarm activates within 15 to 30 seconds after vapor is present. The alarm should reset within 30 seconds after all solvent disappears from the flow cell cavity. The sensitivity of the vapor sensing device is different for each solvent. The sensor is set to a sensitivity level that works for both toluene and tetrahydrofuran. You can use the Alarm Out connector to shut down the pump system or activate an external alarm if a leak is detected. See Connecting Auxiliary Devices on page 23. Liquid Level Leak Sensor The Möbiu also has a liquid level leak sensor to detect leaks from the flow cell or the front panel. This sensor detects both aqueous and organic leaks. However, approximately 2 ml of liquid must leak into the reservoir before the liquid level leak sensor will activate. The alarm will reset after all liquid is removed from the leak reservoir. Turning Off the Alarm When either the vapor or liquid sensor activates, there is an audible alarm and the alarm button on the Main page (and the Alarm page) turns red. You can turn off the audible alarm from the Alarms page on the front panel. See Audio Alarm on page 33. Note: Even when the audible alarm is turned off, the back panel alarm output will remain active and the red indicator will remain lit. 30 Möbiu User s Guide (M3001 Rev. B)

31 5 Using the Instrument Display This chapter describes how to navigate and change settings in the Möbiu Display Window. CONTENTS Navigating the Display Panels...32 Front Panel Buttons 32 Warning Lights and Alarms...33 Hazards 33 Audio Alarm 33 Main Panel...34 Selecting Display Settings for the X and Y Axes 35 Adjusting the Display Range 36 Setting the Cell Temperature 37 Laser 37 Mobility Panel...38 QELS Panel...39 Alarm Panel...42 System Panel...45 Comm Panel...47 Möbiu User s Guide (M3001 Rev. B) 31

32 Chapter 5: Using the Instrument Display Navigating the Display Panels You navigate through the display panels using the buttons on the keypad to the right of the display window. Instruments with model number 4 and higher, which have touch screens, allow you to navigate through the display panels by touching the appropriate fields. Panel tabs Keypad Figure 5-1: Display panel Front Panel Buttons ESC: Use the escape key to navigate through the different front panel tabs. Press ESC to select the current page. Then use the left and right arrows to navigate from one tab to the next. You may also select a specific tab using the number key. For example, ESC+1 selects the Main tab, as shown in Figure 5-2. Figure 5-2: Display panel tabs Tab cycles through various fields in the current panel. Enter displays the options for the current field (such as a drop-down list). The currently selected option is highlighted. Use the arrow keys to change the selection, and then press Enter to confirm that selection. If the field is a check box, Enter toggles the option. Tip: If you miss a field, press Esc and restart tabbing through the fields. 32 Möbiu User s Guide (M3001 Rev. B)

33 Warning Lights and Alarms Warning Lights and Alarms Both the Main and Alarm panels display colored indicators for alarm conditions that the Möbiu can detect. The colors used have the following meanings: Yellow Green Red Color Meaning Not ready Ready Potential hazard For example, the bottom of this Main panel shows that there are no current alarm conditions: Figure 5-3: Main panel showing no alarms Hazards The Möbiu sounds an audible alarm when a potential hazard is detected. Hazards include: a vapor or liquid leak is detected an over temperature condition is detected an external alarm input is activated (signal from associated equipment) Audio Alarm Note: Even when the audible alarm is turned off, the back panel alarm output will remain active. To turn off the audible alarm: Display the Alarm panel. Tab to the Audio Alarm check box and press Enter to uncheck the Audio Alarm box. To enable the audible alarm: Press Enter again to check the Audio Alarm box. Möbiu User s Guide (M3001 Rev. B) 33

34 Chapter 5: Using the Instrument Display Main Panel The Main panel lets you perform the most common Möbiu actions. The display shows a graph of two of the data streams collected by the instrument. One data stream is displayed in blue and labeled on the left axis; the other is displayed in red and labeled on the right axis. Figure 5-4: Main panel While the front panel display on the Möbiu lets you set some values related to collecting data with the instrument, you will need to use the DYNAMICS software to set more detailed parameters. For example, use DYNAMICS to set the types of data to collect (PALS and/or DLS), the electric field frequency, the electric field amplitude in volts, and the collection period for mobility data. See the DYNAMICS User s Guide for more about instrument and experiment settings. 34 Möbiu User s Guide (M3001 Rev. B)

35 Main Panel Selecting Display Settings for the X and Y Axes You can select the data channel you want displayed on each axis. Left Y-axis Selector X-axis Selector Right Y-axis Selector Figure 5-5: Main panel axis setting and scaling fields Left and Right Y-axis Selectors To change the data channel for the left or right Y-axis, follow these steps: 1. Tab to the selector field and press Enter to open the list of data channels. 2. Use the up and down arrow keys to scroll through the items. 3. Press Enter to select an item. The left Y-axis data channel is shown in blue; the right Y-axis data channel is shown in red. The data channels available for display on the Möbiu are: AUX Input 1 AUX Input 2 Cell Temperature Bench Temperature N2 Pressure FM Amplitude (forward monitor) Laser Power LS Amplitudes 1 through 31 X-axis Selector The X-axis for this graph is the instrument time, which can be set on the System panel. The X-axis selector lets you set the time range from 1 minute to 2 hours. 1. Tab to the Time field and press Enter to open the list of time ranges. 2. Use the up and down arrow keys to scroll through the list. 3. Press Enter to select time range. To change the instrument s time clock, see the Set Time field under System Panel on page 45. Möbiu User s Guide (M3001 Rev. B) 35

36 Chapter 5: Using the Instrument Display Adjusting the Display Range You can adjust the Y-axis range displayed by the graph in by autoscaling, using the zoom and pan buttons, or setting a numeric range. To autoscale 1. Tab to the Auto Scale box for the left or right Y-axis. 2. Press the Enter button on the keypad to changes the scaling for this axis to fill the display. To use the zoom and pan buttons: 1. Tab to the Set Scale button for the left or right Y-axis. You see the zoom/pan buttons display. Use the keypad buttons on the front panel of the instrument to zoom and pan the graph. Press the left keypad arrow to zoom in. Pan up Press the right keypad arrow to zoom out. Zoom in Zoom out Press the up keypad arrow to pan up. Press the down keypad arrow to pan down. Pan down To change the scale numerically: 1. Tab to the Set Scale button for the left or right Y-axis. 2. Press Enter. This opens the Set Scale window. 3. To change values, tab to the Max field and enter a value. Tab to the Min field and enter a value. 4. To toggle between Figure 5-6: Setting the scale numerically positive and negative values, tab to the +/- button and press Enter. 5. Tab to the Set button and press Enter. 36 Möbiu User s Guide (M3001 Rev. B)

37 Main Panel Setting the Cell Temperature You can set the cell temperature by tabbing to the Set field and using the numeric keypad to enter the value. The temperature range supported by the Möbiu is 4 70 C. The Möbiu adjusts to within 0.05 C of the set temperature. Figure 5-7: Main panel cell temperature and laser on/off fields The thermocontrollers are programmed to change the temperature at a rate of 1 C per minute to ensure that the flow cell glass does not crack due to thermal stresses. For example, if you wish to operate your system at 70 C, and your system is initially at 25 C, it will take about 45 minutes for the temperature to reach 70 C. Laser To turn the laser on or off, tab to the Laser button and press Enter. When the laser is off, the button is yellow with the word OFF to denote that the system is not ready to take data. When the laser is on, the button is green with the word ON to denote normal operation. Möbiu User s Guide (M3001 Rev. B) 37

38 Chapter 5: Using the Instrument Display Mobility Panel The Mobility panel shows the mobility graph and values being collected by the Möbiu. This graph shows the electrophoresis per unit electric field in m cm/v on the Y-axis and time on the X-axis. This panel does not continuously update like the Main or QELS panels. The Mobility panel only updates when DYNAMICS is actively collecting PALS data. When the instrument is idle, this panel displays the last V- graph measured. The Mobility field shows the calculated average electrophoretic mobility (in m cm/v s), which is the ratio of a particle s velocity during electrophoresis to the applied electric field. This is a widely accepted proxy for molecular charge and interfacial potential, also known as the zeta potential. The Conductivity field shows the ability of a solution to conduct electricity. Electrolytic conductivity is determined by measuring the resistance of the solution between two electrodes. It is shown in units of milli-siemens per centimeter (ms/cm). Use the DYNAMICS software to set measurement parameters. For example, DYNAMICS lets you set the types of data to collect (PALS and/or DLS), the electric field frequency, the electric field amplitude in volts, and the collection period for mobility data. See the DYNAMICS User s Guide for more about mobility settings and graphs. 38 Möbiu User s Guide (M3001 Rev. B)

39 QELS Panel QELS Panel The QELS panel displays dynamic light scattering (DLS) data and is not present if the DLS option has not been added to the Möbiu. Figure 5-8: QELS Panel showing Correlation Function Y-Axis Selector 1. Tab to the drop-down list at the top of the panel. Press Enter to open the list of data channels. 2. Use the up and down arrow keys to scroll through the items. 3. Press Enter to select an item. Möbiu User s Guide (M3001 Rev. B) 39

40 Chapter 5: Using the Instrument Display You can choose whether to view the Correlation Function data or the Count Rate. Count Rate: This data set contains the photon count rate for the DLS detector. The X-axis for this graph is the instrument time, and the scale can be adjusted in the same way as the scale on the Main panel (see Selecting Display Settings for the X and Y Axes on page 35). Correlation Function: This data set displays the intensity correlation curve for a single DLS acquisition, from which the hydrodynamic properties of diffusion coefficient and hydrodynamic radius are calculated. The X-axis for this graph is the Delay Time. DLS measures the correlation function, which is a statistical measurement of how the scattered intensity fluctuates. It is a function of, which is a time difference. For large values of, the correlation function approaches 1.0, indicating that the light intensity at time t is uncorrelated to the intensity at time t +. For smaller values of, the correlation function increases, indicating that the scattered intensity is correlated. The time difference at which the correlation function transitions from being correlated to being uncorrelated is related to the molecular diffusion coefficient. Small particles diffuse rapidly; this leads to rapid fluctuations of the scattered light and a short correlation time. Large particles diffuse slowly and have a long correlation time. See the DYNAMICS User s Guide for a more detailed explanation of the physics of DLS. See Adjusting the Display Range on page 36 for how to adjust the Y-axis scaling. Integration Time The Integration Time field lets you set the DLS acquisition time, in seconds, for each DLS measurement. The minimum time is seconds. Integration times of up to 3600 seconds can be set, but are rarely used. Typical values range from 1 to 5 seconds. The DYNAMICS User s Guide has more information about optimizing the DLS acquisition time. The instrument will round off the time you specify to the nearest multiple of sec. As an aid to assessing DLS data, intermediate results are displayed on the QELS panel auto-correlation function graph in red every one second. After the measurement is complete, it is plotted in blue, and new intermediate results are plotted. The slider on the bottom shows the percent complete for the current measurement. 40 Möbiu User s Guide (M3001 Rev. B)

41 QELS Panel The delay time is used on the horizontal axis of the correlation function graph. It is always less than the integration time. APD Status The avalanche photodiode (APD) contains an internal Peltier cooler that cools the active element to provide improved performance. When it is first powered on, the detector is especially susceptible to damage from overillumination. The APD is extremely sensitive to light and must be protected at all times. Never expose it to room light with the power on. It must have either the dust cover or light fiber connected to it at all times. The DLS detector is equipped with a protection circuit that will shut off the APD in the event of over illumination, but it is intended as an emergency shutoff. Power There is no DLS power shutoff on the Möbiu front panel. The instrument protects the DLS detector sufficiently from room light. Caution! If you are connecting the DLS fiber as described in Appendix C, Adding DLS to Instruments, be sure the Möbiu is powered off. The DLS detector can be damaged during the connection process if the detector is powered on. Möbiu User s Guide (M3001 Rev. B) 41

42 Chapter 5: Using the Instrument Display Alarm Panel The Alarm panel displays sensor information and lets you adjust alarm settings. An alarm history is shown with the last few alarms and the time at which they occurred. Figure 5-9: Alarm panel You can Tab to the following fields. Toggle the setting of the selected field by pressing Enter. Audio Alarm: If this box is checked, the Möbiu sounds an audible alarm when a potential hazard is detected. If this box is unchecked, the Möbiu makes no sound in response to an alarm state. The audio alarm is triggered if any alarm state turns red. Yellow alarms do not trigger the audio alarm. To turn off the audio alarm when it is sounding, go to the Alarm panel, tab to the Audio Alarm checkbox, and press Enter. Note: Even when the audio alarm is turned off, the back panel alarm output will remain active. 42 Möbiu User s Guide (M3001 Rev. B)

43 Alarm Panel Alarm IN: If you check the Active Low box, the instrument detects an Alarm In event if the signal on this line transitions from 5 V to 0 V. When an Alarm In event occurs, the Alarm signal flashes on the display, and an Alarm Out signal is transmitted (see Alarm Out). If you don t check the Active Low box, the instrument detects an Alarm In event if the signal on this line transitions from 0 V to 5 V. Alarm OUT: If you check the Active Low box, instrument keeps this signal at 5 V for no alarm state, and brings the signal to 0 V in the event of an alarm state. In this context, an alarm state occurs if the internal liquid leak sensor detects liquid, or the internal vapor alarm detects organic solvent vapors, or the rear panel connector Alarm In signal is active (see Alarm In). If you don t check the Active Low box, the instrument keeps this signal at 0 V for no alarm state, and brings the signal to 5 V in the event of an alarm state. The Alarm panel also lists the current nitrogen gas pressure, bench temperature (measured just below the cell cavity), and cell temperature. Caution! If you set the cell temperature to 20 C or less, the nitrogen pressure should be at least 20 psi to prevent condensation from damaging the optics. You will not be able to set the temperature below 20 C if the nitrogen connection is not made or if the tank is empty. Here is a list of alarms and the meanings a red or yellow condition for each alarm: Alarm Out: The alarm output on the back panel is activated. This alarm turns red when activated. Alarm In: The external alarm input on the back panel is activated. This alarm turns red when activated. Overheat: Triggered if the read head ever exceeds 100 C. This alarm turns red when activated. Laser Detected: This alarm turns yellow if the laser is off. Liquid Leak: The liquid sensor detected a leak. This alarm turns red when activated. See Liquid Level Leak Sensor on page 30. Auto Inject: An auto inject signal was detected from the device connected to the Auto Inject IN port on the back panel of the Möbiu. This alarm turns yellow when activated. Vapor Sensor: The vapor sensor detected a leak. This alarm turns red when activated. See Vapor Sensor on page 30. Door Interlock: Indicates that the top panel door is open. This alarm turns yellow when activated. Laser Stable: The laser power is not stable. This generally means the instrument is still starting up. This alarm turns yellow when activated. Möbiu User s Guide (M3001 Rev. B) 43

44 Chapter 5: Using the Instrument Display Cell Temperature Lock: The cell temperature is not stable. This generally means the temperature is still adjusting to a new set point. This alarm turns yellow when activated. EField Low Mode: If the sample is electrically conductive for example if you are using a saline aqueous solution the Möbiu caps the maximum output voltage at 10 V. Otherwise, the maximum voltage is 100 V. This alarm turns yellow if the user chooses to apply voltages higher than 10 V for non-conductive samples. (You can set the voltage in the Parameters Instrument node of the DYNAMICS software. See the DYNAMICS User s Guide for advice about voltage settings.) Conductivity Too High: This alarm turns yellow to indicate that an attempt was made to set an applied voltage higher than 10 V for a conductive sample. Nitrogen Pressure: An alarm message is shown if the temperature is set below 20 C, but the nitrogen pressure is less than 20 psi. In this case, the alarm activates briefly and resets the system temperature to 20.5 C. This prevents condensation from damaging the optics if the nitrogen connection is not made or if the tank runs empty. The maximum allowable nitrogen pressure is 80 psi. 44 Möbiu User s Guide (M3001 Rev. B)

45 System Panel System Panel The System panel contains additional options for some of the selections on the Main panel. Figure 5-10: System panel This panel provides the following fields and buttons: Language: You can use this drop-down list to set the language of the user interface to English or another supported language. Attenuator: The intensity of the collected scattered light for DLS measurements needs to be adjusted so that optimal signal levels reach the photodetectors. This is done by an adjustable attenuator. Minimum attenuation is used with weakly scattering samples to provide the best sensitivity. You can use automatic attenuation, manually set an attenuation percentage, or set absolute attenuation levels numerically. Additionally, there is a Low power laser mode that you can set with the DYNAMICS software. Auto Attenuate: This option is on by default. Check this box to automatically attenuate the light that reaches the detector to ensure that the signal is on scale. The target count rate for auto attenuation is set to 2 million counts/second in the firmware. Attenuation (%): This field shows the current percentage by which the light intensity is attenuated before it reaches the detector. If you are not using auto attenuation, you can type a percentage here. Möbiu User s Guide (M3001 Rev. B) 45

46 Chapter 5: Using the Instrument Display Configure Auto Attenuation: If you click this button, you see the following dialog, which lets you set target and threshold values (in counts per second) for the laser intensity at the detectors. The attenuator is then adjusted to achieve the levels you specify. Typically, levels of up to 8 million counts per second can be used. Set Time: The field displays the current instrument time. This value is used on the X-axis of graphs. To change the current time, select the Set Time button and modify the time. Load Factory Default: Select this button to reset the instrument to the settings installed when the instrument was shipped. Restart Instrument: Select this button to reboot the Möbiu. This is typically used only when installing a firmware update. The System panel also shows the instrument s serial number and the current firmware version, which will be needed if you contact Wyatt Technical Support. 46 Möbiu User s Guide (M3001 Rev. B)

47 Comm Panel Comm Panel The Comm panel allows you to connect the Möbiu to a computer network. Figure 5-11: Comm Panel This panel shows the instrument s name, IP address, subnet mask, and IP address mode. You may need to refer to this information when connecting to the instrument over the network with the DYNAMICS software. There are two choices for how the IP address and subnet mask of the instrument are determined: Obtain an IP address automatically: Once the instrument is connected to a computer or LAN, the IP address and subnet mask will be assigned automatically. This option requires that the network has a DHCP server. When using DHCP, it may take several minutes for the IP address to be assigned. During this time, the IP address and subnet mask will read Once the IP address and subnet mask have Möbiu User s Guide (M3001 Rev. B) 47

48 Chapter 5: Using the Instrument Display been assigned, both will be automatically updated, and should no longer read At this point, it should be possible to connect to the instrument from a computer where DYNAMICS is installed. Use the following IP address: If you wish to use a static IP address and subnet mask, please contact your IT department to obtain a valid address and mask. Enter the information into the IP address and subnet mask fields. Remote Access PIN: Sets the PIN for users wanting to use the Aurora mobile app to monitor the front panel. 48 Möbiu User s Guide (M3001 Rev. B)

49 6 Plumbing and Introducing Sample This chapter discusses the different configurations for plumbing and introducing sample and solvent into the Möbiu. These include manual injections directly into the flow cell, manual injections using an HPLC pump and injector valve, manual measurements with the optional Dip Cell assembly, and automated injections using an HPLC pump and autosampler. In addition, DLS measurements may be made with a disposable semi-micro cuvette and the provided cuvette adapter or the reusable quartz DLS cell and Dip Cell adapter. CONTENTS PAGE Sample Preparation...50 Fluid Cells...51 Flow Cell 51 Optional Dip Cell Assembly 52 Disposable Cuvette (DLS Only) 52 Measuring Single Samples...53 Performing Manual Injections into the Flow Cell 53 Using an HPLC Pump and Manual Injector Valve 57 Using the Quartz Cuvette and Dip Cell 58 Making DLS-Only Measurements with Disposable Cuvettes 61 Automated Injections with an Autosampler...62 Terminology 62 Autosampler Sequence 63 Example Event Schedule and Data 64 Möbiu User s Guide (M3001 Rev. B) 49

50 Chapter 6: Plumbing and Introducing Sample Sample Preparation For accurate determination of hydrodynamic radius and electrophoretic mobility, samples and solvents should be free of particles and contaminants. All solvents should be HPLC grade and filtered to 0.2 µm (preferably 0.1 µm). Dust particles, large aggregates, and other contaminants should be removed from samples via filtration or centrifugation. Protein samples should be filtered to the smallest pore size possible (typically 0.1 µm) using a syringe-tip filter. For larger particles, use a filter with an appropriate pore size. The volume of sample required for measurements depends on the sample delivery method. The minimum sample concentration required for mobility measurements is 1 mg/ml of lysozyme (M = 14 kda). Larger molecules require proportionally lower concentrations. For example, the minimum concentration for a typical IgG antibody (M = 150 kda) is 0.1 mg/ml. The minimum sample concentration for DLS-only measurements with either the flow cell or optional quartz cuvette is 0.1 mg/ml lysozyme. The minimum sample concentration for DLS-only measurements with the disposable cuvette is 1 mg/ml lysozyme. As with mobility measurements, larger molecules require proportionally lower concentrations. 50 Möbiu User s Guide (M3001 Rev. B)

51 Fluid Cells Fluid Cells Flow Cell The following types of fluid cells are available for the Möbiu. The reusable PEEK (polyetheretherketone) flow cell supports simultaneous mobility and DLS measurements. It comes with replaceable platinumcoated electrodes and optical-quality windows. This cell can be used with aqueous and organic solvents. Samples and solvents may be introduced manually with a syringe, manually using an HPLC pump and injector loop or automatically using an HPLC pump and autosampler. Inlet Outlet To instrument front The total volume of the flow cell from the manifold inlet to the manifold outlet is ~180 µl. The actual scattering volume the illuminated part of the sample that is viewed by the detectors is less than 1 µl. See Flow Cell Basic Maintenance on page 70 and Disassembling and Cleaning the Flow Cell on page 73 for more information about using the flow cell. Möbiu User s Guide (M3001 Rev. B) 51

52 Chapter 6: Plumbing and Introducing Sample Optional Dip Cell Assembly The reusable Dip Cell accessory is available for manual, low-volume mobility and DLS measurements. A 45 µl quartz cuvette, adapter, and electrode assembly comprise the Dip Cell, which can be used as an alternative to the standard Möbiu flow cell for appropriate samples. The Dip Cell does not seal and cannot be used with the Atlas accessory, therefore it should only be used for aqueous samples (that is, samples with non-organic solvents) with low conductivity (~7 ms/cm, or approximately 75 mm NaCl equivalent ionic strength). The 45 µl quartz cuvette may also be used for DLS only measurements. Disposable Cuvette (DLS Only) A cuvette adapter and disposable cuvettes are provided in the Möbiu hardware kit. The minimum sample volume is 100 µl. Disposable cuvettes may only be used to measure dynamic light scattering (DLS). Mobility measurements may not be performed with the disposable cuvette. The disposable cuvette is compatible with aqueous solvents only. It may not be used with organic solvents, including alcohols and other polar solvents. 52 Möbiu User s Guide (M3001 Rev. B)

53 Measuring Single Samples Measuring Single Samples Single samples can be directly injected into the Möbiu flow cell or through the Möbiu front panel prior to each measurement. The injection can be performed by hand (page 53) or using an HPLC pump and manual injector valve (page 57). The optional Dip Cell assembly offers a low volume option for measuring mobility and hydrodynamic radius (page 58). Hydrodynamic radius by DLS can also be measured with a disposable cuvette (page 61). To measure a series of different samples, automated injections may be made to the Möbiu flow cell using an HPLC pump and autosampler (page 62). Performing Manual Injections into the Flow Cell Samples can be directly injected into the Möbiu flow cell (page 54) or through the Möbiu front panel (page 55). Both these flow paths represent open systems, and great care must be taken to avoid introducing bubbles, particles, and other contaminants, especially when changing fluid connections. Injecting directly into the cell requires a smaller fluid volume and enables bubbles to be released more easily. See Removing Bubbles from the Flow Cell on page 56. Injecting through the Möbiu front panel is required when using the Atlas accessory (see the Atlas Hardware User s Guide). Möbiu User s Guide (M3001 Rev. B) 53

54 Chapter 6: Plumbing and Introducing Sample Injecting Directly into the Flow Cell This setup requires as little as 200 µl of sample. To inject samples directly into the flow cell, follow these steps: 1. Disconnect the flow cell, and plug the inlet and outlet fluid ports inside the Möbiu with 1/4-28 plugs. Remove the cell through the top panel door. 2. Remove the cone plugs from the provided union fittings in the Möbiu hardware kit (Wyatt part P ). Install the unions on the inlet and outlet ports. See Figure 6-1. Outlet port Inlet port Flow cell key Union fitting (left: exterior with cap, right: interior) Figure 6-1: Fluid connections for direct injections into the flow cell 3. Fill the cell through the inlet union with a syringe (smaller than 3 ml) fitted with an appropriate syringe-tip filter. Let some sample overflow through the outlet tube to make sure the entire volume is full. Avoid getting sample on the exterior of the flow cell windows. 4. Look through the cell windows, and inspect the cell carefully for bubbles. If there are any bubbles, continue to inject sample and let it overflow through the outlet until the bubbles are gone. See Removing Bubbles from the Flow Cell on page Thread the cone plug into the outlet fitting. 6. Remove the syringe from the inlet fitting and dribble sample into the fitting to avoid introducing bubbles when you cap the inlet. 7. Thread the cone plug into the inlet fitting. 54 Möbiu User s Guide (M3001 Rev. B)

55 Measuring Single Samples 8. Reinstall the flow cell into the top panel. You can fold the fittings down so that they fit inside the instrument as shown in Figure 6-2. Alternatively, you can use the Möbiu Removable Door Kit, which allows you to collect data with the top panel door open. Figure 6-2: Flow cell plugged and installed for manual measurements 9. Between samples, remove the flow cell, and flush it with solvent and filtered dry air. Injecting through the Möbiu Front Panel In this configuration, the Möbiu flow cell is connected to the detector inlet and outlet, as shown in Figure 6-3, and sample is introduced through the front panel, as shown in Figure 6-4. This configuration requires as little as 250 µl of sample. Ensure the inlet connector is threaded onto the left fitting (white side) and the outlet connector is threaded onto the right fitting (blue side). Figure 6-3: Flow cell connected to front panel Möbiu User s Guide (M3001 Rev. B) 55

56 Chapter 6: Plumbing and Introducing Sample On the front panel, plumb the OUT port to a waste reservoir using 0.030" ID tubing. Plumb the IN port with an appropriate luer adapter and short piece of 0.01" ID tubing, as necessary. If you have an Atlas accessory, you may plumb the front panel of the Möbiu to the Atlas and introduce sample through the Inject port of the Atlas, as described in the Atlas User s Guide. Use a syringe (smaller than 3 ml) fitted with an appropriate syringe-tip filter to inject sample into the instrument. IN/OUT Fluid Connectors Figure 6-4: Möbiu front panel fluid connections Removing Bubbles from the Flow Cell Bubbles in the Möbiu flow cell can disrupt mobility and DLS measurements. Bubbles may be introduced when you change fluid connections, and can be detected by observing the forward monitor (FM) signal. If the signal decreases substantially (even dropping to 0), there is most likely a bubble in the flow cell. Pressurization via the Atlas accessory can shrink or dissolve bubbles so that they will not affect the measurement. For more information about manual injections through the Atlas, see the Atlas Hardware User's Guide. When using manual injections without the Atlas, eliminate bubbles as follows: 1. Remove the flow cell from the Möbiu and observe the bubble. 2. While fluid is flowing, block the outlet tubing of the Möbiu (e.g., with a gloved hand). 3. After a few seconds, pressure will build up inside the flow cell and shrink or re-dissolve the bubble. 4. Release the pressure to free the fluid and bubble from the flow cell. 5. Once the bubble has been removed, return the flow cell to the Möbiu. You should see the FM signal return to its normal maximum value. 56 Möbiu User s Guide (M3001 Rev. B)

57 Measuring Single Samples Using an HPLC Pump and Manual Injector Valve As an alternative to direct injections into the flow cell, you can deliver samples to the Möbiu using a manual injection valve, such as the Wyatt Injection System for High pressure (WISH). This configuration provides a closed system, reducing the risk of introducing bubbles and contaminants. However, you must start and stop the HPLC pump flow manually and carefully manage the flow rate and flow time to ensure that the sample is properly delivered to the cell for measurements. Note: In order to make mobility measurements with the Möbiu, the fluid flow must be stopped. When using a chromatography pump to deliver samples and solvent to the Möbiu, ensure the flow cell is installed as shown in Figure 6-3 with the inlet connector threaded onto the left fitting (white side) and the outlet connector threaded onto the right fitting (blue side). These lines are plumbed to the fluid connectors behind the door on the front panel of the Möbiu. Fluid connections between the HPLC pump and Möbiu are described below. Modifications to the fluid flow path for use with the Atlas accessory are described in the Atlas Hardware User s Guide. Figure 6-5: Möbiu plumbed with HPLC pump injector (manual injector valve or autosampler) In this configuration, the HPLC pump delivers sample and solvent through the Möbiu. The sample is injected into the sample loop while the loop is bypassed from the HPLC flow path. After the loop is filled with sample, the valve is switched, and the flow path is changed so that the solvent pushes the sample from the loop into the Möbiu flow cell. Möbiu User s Guide (M3001 Rev. B) 57

58 Chapter 6: Plumbing and Introducing Sample A large injection volume (greater than the volume of the sample loop) is required for each sample injection. The WISH accessory is equipped with 1 ml sample loops. Using the Quartz Cuvette and Dip Cell This configuration (shown in Optional Dip Cell Assembly on page 52) requires as little as 45 µl of sample per measurement. The quartz cuvette can be used by itself for DLS-only measurements or with the reusable electrode assembly for mobility measurements (with or without simultaneous DLS). To use the Dip Cell assembly for mobility measurements, follow these steps: 1. Place the cuvette into the cuvette holder. 2. Load the sample into the cuvette using a pipette or syringe. Place the pipette tip or syringe needle all the way to the bottom of the cuvette and dispense µl of sample. As with all cuvettes, always handle the cuvette from the top and take care to avoid scratching the sidewalls with the pipette. 3. Carefully insert the electrode assembly into the cuvette. (The electrode assembly is keyed so that it cannot be oriented incorrectly.) Cuvette holder Electrode assembly Front window of cuvette 4. Check to make sure the electrodes are visible at the sides of the sample volume when viewing through the front window of the cuvette. The sample meniscus should not be visible through the front window. Occasionally fluid may wick up the sides of the electrode assembly and a meniscus will be visible. This is usually the result of dirty surfaces, and if a meniscus is observed, you must remove the sample and thoroughly clean and dry the cuvette and electrode assembly prior to continuing. 5. Remove the standard flow cell from the Möbiu and replace it with the assembled Dip Cell. Measurements can now be made using standard procedures. 58 Möbiu User s Guide (M3001 Rev. B)

59 Measuring Single Samples Cleaning the Dip Cell Cuvette Prior to each measurement, the quartz cuvette must be thoroughly cleaned. The cleaning procedures described in this section are based on the assumption of an aqueous sample. For samples in organic solvents, the cuvette and electrodes must first be rinsed with a miscible intermediate solvent before using aqueous-based cleaning agents. For example, you could proceed from toluene to ethanol to water. Whenever handling cuvettes, make sure to touch only the upper part of the cuvette to avoid smudges on or near the optical surfaces. The materials listed below are required for a standard cuvette cleaning. If more aggressive cleaning is needed, the cuvette may be first be cleaned with 1% Hellmanex, 10%, nitric acid, aqua regia, or an appropriate organic solvent prior to the standard cleaning described below. Distilled water Liquinox cleaning detergent for general cleaning or Tergazyme cleaning detergent for biological samples. Both detergents are prepared in a 1% solution. Polyurethane foam swabs (from Hardware or Cuvette kit) Reagent alcohol (ethanol) Squeeze bottles or plastic transfer pipettes Filtered nitrogen or compressed air Caution! Ultrasonic cleaners can operate at frequencies that are resonant with the cuvette, which may cause it to break. Wyatt Technology Corporation does not recommend the use of ultrasonic cleaners and will not warranty cuvettes that have been cleaned in ultrasonic cleaners. 1. Rinse cuvette thoroughly with distilled or deionized water 3 5 times. With each rinse, grasp the cuvette tightly and with a flicking motion, shake the water out of the cuvette into the sink. 2. Rinse the inside and outside of the cuvette with Liquinox (or Tergazyme for biological samples) solution 3 5 times. 3. To aid in the removal of any residue, use the polyurethane foam swabs to gently wipe the inside of the cuvette when using the cleaning solutions above. 4. Fully rinse the cuvette with distilled water 3 5 times while flicking the water out of the cell after each rinse. 5. With the cuvette in hand, rinse the inside and outside with ethanol 3 5 times, shaking out the ethanol after each rinse. Möbiu User s Guide (M3001 Rev. B) 59

60 Chapter 6: Plumbing and Introducing Sample 6. If the cuvette will be used immediately after cleaning, turn it upside down and dry with filtered air (or nitrogen). Dry the outside first, directing the gas flow from the bottom of the cuvette toward the top. Dry the inside of the cuvette by flushing with filtered air using a slight swirling motion for approximately 5 seconds. 7. If the cuvette will not immediately be used, carefully place it upside down on a drying rack, taking care not to touch the optical surfaces. Prior to use, inspect the outer surfaces to ensure that no smudges are present. If necessary, rinse once more with an even spray of ethanol over the entire outer surface and allow it to air dry at least 15 minutes before use. A jeweler's loupe (included in the instrument or cuvette kit) may be used to inspect the cleanliness of the optical surfaces of the cuvette. Streaks and residue on the outside or inside of the cuvette indicate that the cleaning detergent has not been fully removed. In this case, more thorough rinsing with distilled water is advised. Cleaning the Dip Cell Electrodes The Dip Cell electrodes should be cleaned between samples and before storage. It is not necessary to disassemble the electrode assembly prior to cleaning, although care must be taken to thoroughly dry all components prior to resuming data collection. To clean the Dip Cell electrodes, follow these steps: 1. Rinse all wetted-surfaces under a stream of distilled or deionized water. 2. Rinse the inner surfaces of the electrodes with Liquinox or Tergazyme. 3. Rinse all wetted-surfaces thoroughly with distilled or deionized water to ensure that all detergent residue is removed. 4. Thoroughly dry the entire assembly under a stream of filtered air or nitrogen. It is usually not necessary to scrub the electrodes; rinsing is sufficient for routine cleaning. However, if any discoloration is visible on the inner electrode surfaces, they can be scrubbed using the polyurethane foam swabs provided in the ship kit. Gently swab the inner electrode surfaces after first wetting with detergent and follow with a thorough rinse with distilled or deionized water as above. 60 Möbiu User s Guide (M3001 Rev. B)

61 Measuring Single Samples Making DLS-Only Measurements with Disposable Cuvettes The disposable cuvette can be used only for DLS measurements. It cannot be used to measure mobility. To use the cuvette, remove the PEEK flow cell, and insert the DLS-only batch adapter into the Möbiu. Load the sample into the cuvette using a pipette or syringe. Place the pipette tip or syringe needle all the way to the bottom of the cuvette and dispense ml of sample. Be careful not to scratch the sidewalls of the cuvette with the pipette tip or needle. Handle the cuvette only from the top. Keep the cuvette capped to avoid contamination from dust and other particulates. Möbiu User s Guide (M3001 Rev. B) 61

62 Chapter 6: Plumbing and Introducing Sample Automated Injections with an Autosampler Sample and solvent may be delivered to the Möbiu using a liquid chromatography pump and autosampler. This automated setup enables you to characterize electrophoretic mobility and hydrodynamic radius for multiple samples with minimal setup and intervention. The autosampler sequence automatically alternates buffer and sample injections to allow adequate cleaning of the Möbiu flow cell, the needle, and the sample loop to reduce cross-contamination. This configuration is a closed system, reducing the risk of introducing bubbles, particles, or contaminants to the Möbiu flow cell. At least 500 µl is injected for each sample. Thus, thus a large sample loop (for example, 900 µl) must be installed in the autosampler. DYNAMICS can interface directly with Agilent pumps and autosamplers to control sample and solvent injections. Instructions for installing the autosampler control services and connecting to the autosampler hardware are given in the DYNAMICS User s Guide. Other pump systems may be programmed using their manufacturer s software, with measurements triggered in DYNAMICS by an Auto-Inject signal (see Appendix B, Using an Alternative Autosampler ). Terminology The following are important terms for automating mobility measurements with an autosampler: Sample flow rate: Pump flow rate during sample injections; typically ml/min. Sample injection volume: Volume of sample to be injected; typically 500 µl. Sample pump run time: Amount of time the pump flows to deliver sample to the Möbiu before stopping the flow. Calculated as: Sample pump run time = (Sample injection volume) / (Sample flow rate) Wash flow rate: Pump flow rate during wash injections; typically ml/min. Wash injection volume: Volume of wash buffer to be injected; typically ~ µl greater than the sample injection volume. Wash pump run time: Amount of time the pump flows to deliver wash buffer and additional solvent, flushing out the autosampler sample loop and Möbiu flow cell; typically 2 5 ml solvent must be flushed through the Möbiu between injections. Calculated as: Wash pump run time = (Desired wash volume) / (Wash flow rate) 62 Möbiu User s Guide (M3001 Rev. B)

63 Automated Injections with an Autosampler Autosampler Sequence The autosampler sequence is controlled by the DYNAMICS software and consists of alternating sample and wash buffer injections. These are described in detail in the sections that follow. For information about setting up an Event Schedule, including moving to particular autosampler vials, pressurizing/depressurizing the Möbiu, and assigning labels to each measurement, see the DYNAMICS User s Guide. Sample Injection For each sample measurement, the autosampler and HPLC pump deliver sample to the Möbiu, the flow is stopped, the cell is pressurized with the Atlas (optional), and a measurement is taken with DYNAMICS. The sample injection volume, flow rate, and pump stop time are controlled by the DYNAMICS Event Schedule. These parameters should be set such that the flow is stopped immediately after all the sample has cleared the sample loop and entered the flow stream, as described in the preceding Terminology section. For each sample injection, perform 3 5 PALS and DLS measurements. Wash Injection Install one or more vials of wash buffer in the autosampler and use them to wash the needle and sample loop between injections. Leave these vials uncapped to facilitate washing the outside of the needle. The wash buffer may be the same as the mobile phase or another miscible solvent, such as a high salt buffer or surfactant-containing solution, to provide adequate cleaning. The flow rate and injection volume for the buffer injections need not be the same as for the sample injections. The buffer injection volume should be at least as large as the sample injection volume. The wash pump run time should be long enough to allow at least 2 5 ml of mobile phase to flow through the entire system, including the sample loop. It is recommended that wash data be collected between sample measurements to assess the condition of the flow cell, electrodes, and windows. Note: Since the wash pump run time is longer than the time required to deliver the wash injection volume, the wash data represent measurements of the mobile phase being delivered by the HPLC pump, not the particular wash buffer in the autosampler vial. Möbiu User s Guide (M3001 Rev. B) 63

64 Chapter 6: Plumbing and Introducing Sample Example Event Schedule and Data An example Event Schedule is shown in Table 6-1. This example experiment has four sample injections delivered to the Möbiu by an Agilent autosampler. The Event Schedule includes commands to pressurize and depressurize the flow cell using the Atlas hardware accessory. To perform this experiment as written, load nine autosampler vials starting with a vial of wash buffer in position #1. Alternate wash and sample vials, ending with a final wash vial in position #9. For each wash (blue) and sample (green) injection, the Möbiu will collect five measurements. You can monitor the progress of each experiment from the Möbiu front panel to verify proper sample delivery throughout the autosampler sequence. Light scattering data for one detector and the forward monitor (FM) signal are shown in Figure 6-6. Figure 6-6: MP-PALS data for an auto-sampler sequence as viewed from the Möbiu front panel For this example, an auto-inject cable was connected from the autosampler to the Möbiu, although this is not required for Agilent pumps, and the green lines indicate where the auto-inject signal occurred and the injection began. The DLS count rate and autocorrelation function are also good measures of the presence of sample or contaminant at any time during the experiment. The time at which the flow was stopped and measurements were performed are indicated below the chart. Example data for automated Möbiu experiments are given in Wyatt Application notes, such as Automated Measurements of Electrophoretic Mobility with the Möbiu ( Mobius_Autosampler.pdf). 64 Möbiu User s Guide (M3001 Rev. B)

65 Automated Injections with an Autosampler Table 6-1: Example Event Schedule Set sample and wash injection parameters: Command Parameter Description Set sample flow rate (ml/min) 0.5 Set sample injection volume (µl) 500 Set sample pump run time (min) 1 Time required to inject 500 µl at 0.5 ml/min Set wash flow rate (ml/min) 0.5 Set wash injection volume (µl) 700 Set wash pump run time (min) 5 Time needed to inject 700 µl of wash buffer and allow an additional 1.8 ml solvent to flush through the sample loop and Möbiu Move to vial 1 Appropriate autosampler vial number Injections and measurements for each wash-sample pair: Command Parameter Description Do 4 Number of sample and wash vials Inject wash Wait (min) 0.2 Short equilibration time prior to pressurizing Pressurize Optional Do 5 Collect Wash data to assess cleanliness. Collect data Label measurement (formatted) Wash Loop Depressurize Move to next vial Inject sample Wait (min) 0.2 Short equilibration time prior to pressurizing Pressurize Optional Do 5 Collect 3 5 measurements for each sample injection. Collect data Label measurement (formatted) Sample Additional hardware and userdefined parameters can be included in the Label. Loop Depressurize Move to next vial Loop Möbiu User s Guide (M3001 Rev. B) 65

66 Chapter 6: Plumbing and Introducing Sample Final wash injection: Command Parameter Description Inject wash Wait (min) 0.2 Short equilibration time prior to pressurizing Pressurize Optional Do 5 Collect Wash data to assess cleanliness. Collect data Label measurement (formatted) Wash Loop Depressurize 66 Möbiu User s Guide (M3001 Rev. B)

67 7 Maintenance Procedures The Möbiu photometer requires little maintenance. When you remove parts for cleaning, you will find they are easy to access and disassemble. This chapter gives guidelines for keeping the instrument clean and in good working order. It also has the procedures for cleaning the flow cell and converting from the flow cell to a dip cell or DLS-only batch mode measurements. Note: Only trained personnel are authorized to perform work inside the instrument. These authorized individuals are those responsible for the safe use and maintenance of the equipment. Authorized personnel must have been trained by a Wyatt representative. Training may be achieved at Light Scattering University, as part of on-site instruction during installation or an on-site visit, or through other instruction provided by a Wyatt representative. Please contact Wyatt Technology at support@wyatt.com with any questions. CONTENTS PAGE General Maintenance...68 Outer Case Maintenance...68 Air Filter Maintenance...68 Changing a Fuse...69 Flow Cell Basic Maintenance...70 Regular Maintenance 70 On-line Cleaning 70 Protease Cocktail Rinse 72 Disassembling and Cleaning the Flow Cell...73 Preparation 74 Part List 75 Removing the Flow Cell 77 Disassembling the Flow Cell 77 Cleaning the Flow Cell Housing 80 Cleaning and Reinstalling the Electrodes 80 Cleaning and Reinstalling the Cell Windows 82 Cleaning and Reinstalling Inlet and Outlet Tubing 84 Reinstalling the Flow Cell 84 Möbiu User s Guide (M3001 Rev. B) 67

68 Chapter 7: Maintenance Procedures General Maintenance DANGER! The Möbiu has no user serviceable parts. For your safety, do not dismantle internal assemblies except for the flow cell assembly. Do not bypass any of the safety systems and interlocks that are in place for your health. If the instrument is not functioning properly, do not apply power. Outer Case Maintenance Keep the Möbiu on a flat, clean surface, with space behind and standing on its feet to allow proper air ventilation. Periodically wipe down the outside case of the instrument with a clean, moist cloth to keep it free from dust or surface stains. Keep the instrument cover on at all times with the cell door closed. Air Filter Maintenance Check the air filter every month or so. When the air filter gets dusty, pull the air filter cover off and remove the filter. Then gently clean it with warm soapy water. Make sure to let it dry completely before reinstalling. Spare filters are included in your hardware kit, and you can also order replacement filters from If you are in a dusty environment, clean the filter more often than monthly. Failure to keep the air filter clean will cause the instrument to heat up and will decrease the ability of the fan to blow dust particles out of the instrument. 68 Möbiu User s Guide (M3001 Rev. B)

69 Changing a Fuse Changing a Fuse What you need to change a fuse: Tool for prying the AC Power module cover off, such as a small-bladed screwdriver. Fuses from the spares supplied in the accessory kit. To replace the fuses, do the following: 1. Disconnect the power cord. 2. Open the cover of the AC Power module using a small blade screwdriver or similar tool. 3. Replace the burned out fuse with a 4 A, 250 V slow blow fuse. The fuse block contains two fuses. Both of them must be Figure 7-1: Fuseholder and Fuses installed for the instrument to operate correctly. 4. Replace the cover of the AC Power module and reconnect the power cord. Möbiu User s Guide (M3001 Rev. B) 69

70 Chapter 7: Maintenance Procedures Flow Cell Basic Maintenance The Möbiu reusable PEEK (Polyetheretherketone) flow cells come with replaceable platinum-coated electrodes and optical quality windows. This cell can be used with aqueous and organic solvents. A clean flow cell is critical to the operation of the Möbiu. In general, the Möbiu flow cell will need to be cleaned more often than flow cells for other Wyatt Technology instruments. Regular on-line cleaning will reduce, but not eliminate, the need to disassemble and clean the flow cell (see Disassembling and Cleaning the Flow Cell on page 73). Regular Maintenance To keep the flow cell free of contaminants, we recommend regular maintenance as described here. Always use solvents, including water, that are HPLC grade and filtered to <0.1 m. When not in use, store the flow cell filled with filtered, salt-free solvent, such as ethanol. Keep the cell inlet and outlet sealed when the flow cell is not in use to prevent solvent evaporation or introduction of particles. In addition to the on-line cleaning instructions in the next section, you will need to follow certain procedures for cleaning the flow cell and electrodes, as described in Disassembling and Cleaning the Flow Cell on page 73. On-line Cleaning Before and after completing an experiment, follow these cleaning instructions: Typical online cleaning can be performed with a mild detergent solution (for example, 1% w/v Tergazyme, 1% v/v Liquinox, or 2% v/v Contrad 70) and water. For more aggressive cleaning, 10% nitric acid may be used. If the flow cell contains organic solvents, first flush the fluid path with a miscible intermediate solvent before using aqueousbased cleaning agents. After performing the online cleaning procedure, check all fluid connections for leaks and salt or other deposits. After cleaning with nitric acid, remove and clean the flow cell as described in Disassembling and Cleaning the Flow Cell on page 73. To perform manual on-line cleaning: 1. Disconnect the Möbiu from your plumbing system. 70 Möbiu User s Guide (M3001 Rev. B)

71 Flow Cell Basic Maintenance 2. Inject 5 10 ml pure, filtered (0.02 µm) solvent through the IN port to flush the cell. Note: Do not flush the cell from outlet to inlet. Back-flushing the cell can cause particles to become lodged in the inlet tubing, which has a smaller inner diameter than the outlet tubing. 3. If necessary, flush with one or more miscible solvents before flushing with water. For example, if the flow cell previously contained toluene, flush with ethanol or isopropanol before flushing with water. 4. Flush with pure, filtered (0.02 µm) water. 5. Inject a mild detergent or protease cocktail rinse (page 72) to clean the flow cell. Allow it to soak for 30 minutes or as long as overnight. 6. Flush the cell with 100 ml pure water. The on-line cleaning process can be automated using an autosampler as described below. Injections of cleaning solution and water are performed with the autosampler, followed by thorough flushing with clean, filtered water. If necessary, modify the procedure for non-agilent HPLC pumps, as described in Appendix B, Using an Alternative Autosampler. 1. Install a vial of cleaning solution (for example, 2 5% v/v Contrad 70 in water) and a vial of double-distilled water (ddh 2 O) in the autosampler. Installing the vials without caps helps clean the outside of the autosampler needle. 2. Load fresh, filtered ddh 2 O onto the HPLC pump, and set it to flow at ml/min. 3. Set up an Event Schedule in DYNAMICS that includes the following actions: a. Perform two sample injections of µl each. b. If an Atlas is present, pressurize the cell for two minutes after each injection, and then depressurize. c. After the second injection, wait minutes with the flow stopped. This allows the flow cell to soak in the cleaning solution. d. Perform two wash injections with pure water and a Wash pump run time command to allow 3 5 ml water to flow through the detector. 4. Continue flushing with the HPLC pump until a clean baseline is reached, as measured by the PALS amplitude and the DLS autocorrelation function. Möbiu User s Guide (M3001 Rev. B) 71

72 Chapter 7: Maintenance Procedures Protease Cocktail Rinse Some users have found that a simple protease cocktail rinse is effective in removing protein deposits from glass flow cell surfaces. You might be able to use this rinsing treatment rather than disassembling the flow cell: Ingredients for 3 ml of protease cleaning solution: All enzymes are sequencing grade preparations from either Boerhringer Manheim or Roche. Trypsin, modified 25 g, lyophilized Chymotrypsin 25 g, lyophilized Pepsin 25 g, lyophilized Note: You might be able to get away with just pepsin alone, as it s non-specific. Procedure: 1. Reconstitute each enzyme with 1 ml of PBS (25 mm Na phosphate / 150 mm NaCl, ph 7.25). 2. Mix the three solutions and vortex. Load a syringe fitted with a 0.02 m filter. 3. Flush the detector with 20 ml of pure water, then infuse ~ 1 ml of cocktail via a syringe pump. 4. Stop the flow and leave it for a few hours or overnight 5. The following morning, remove the syringe, flush the cell with 20 ml of HPLC grade water, then mobile phase. 72 Möbiu User s Guide (M3001 Rev. B)

73 Disassembling and Cleaning the Flow Cell Disassembling and Cleaning the Flow Cell Light scattering measurements are very sensitive to stray light, which can arise from various sources such as dirt and microbubbles in the scattering volume, particulate deposits on the windows, and improperly cleaned or dried flow cell windows. A moderate amount of stray light may not matter if strongly scattering samples are being measured, but such stray light will affect data quality and consistency for weakly scattering samples. You should clean the flow cell as described in this section if the results are showing data anomalies. A flow cell cleaning video can be found in the Wyatt Support Center ( hardwaresupport/mobius-support-center.html and select the Tutorials tab). Checking the Windows To check to see if the flow cell windows are dirty, follow these steps: 1. Flush the cell with filtered (< 0.1 m) deionized water. 2. Perform a measurement. Collect both PALS and DLS data, as appropriate. 3. Use the DYNAMICS software to check the baseline value in the PALS Amplitude data set. If the baseline value is greater than or equal to 2 times the baseline shown on the Certificate of Performance that came with your instrument, the flow cell windows need to be cleaned for optimal sensitivity. Checking the Electrodes In addition, with the Möbiu, the electrodes become coated with sample and need cleaning or replacement on a regular basis. To check to see if the flow cell electrodes are dirty, follow these steps: 1. Visually inspect the electrodes. If there are visible deposits on an electrode s surface, it needs cleaning. 2. You can also use the KCl (potassium chloride) standard provided with your Möbiu to check the conductivity reported by the electrodes. If the conductivity is significantly lower than that listed for the standard, the electrodes need to be cleaned or replaced. 3. Run an experiment with 2.0 mg/ml bovine serum albumin (BSA) in the cell. The BSA should be dissolved in 10x diluted PBS buffer with a ph value between 6.8 and Compare the mobility V-graph on the Certificate of Performance to the V-graph for your BSA sample. If the graph differs substantially, visually inspect the electrodes in the flow cell to determine whether they need cleaning. Möbiu User s Guide (M3001 Rev. B) 73

74 Chapter 7: Maintenance Procedures Caution! The flow cell you are about to remove constitutes a substantial amount of the purchase price of the Möbiu. Its parts are carefully machined and are expensive. If you have any doubts whatsoever about the safest procedure for handling the cell structure, do not hesitate to call Wyatt Technology. Cleaning Procedure Overview The flow cell cleaning procedure can be broken down into the following major steps: 1. Removing the Flow Cell on page Disassembling the Flow Cell on page Cleaning and Reinstalling Cleaning the Flow Cell Housing on page 80 Cleaning and Reinstalling the Electrodes on page 80 Cleaning and Reinstalling the Cell Windows on page 82 Cleaning and Reinstalling Inlet and Outlet Tubing on page Reinstalling the Flow Cell on page 84 All steps described here are based on the assumption of an aqueous based sample. You may need to perform additional steps for organic based samples. Cleaning should be performed according to miscibility of sample. Preparation What you will need for flow cell cleaning: A sheet of clean white paper taped down to your work surface Möbiu cell installation tool (WTC #169121) Filtered (< 0.1 m) deionized water Detergent, such as 1% w/v Tergazyme or 1% v/v Liquinox Reagent alcohol (ethanol) Squeeze bottles or plastic transfer pipettes Filtered nitrogen gas Nylon brush (WTC # ) Lint-free gloves 74 Möbiu User s Guide (M3001 Rev. B)

75 Disassembling and Cleaning the Flow Cell Part List The Möbiu flow cell assembly is Wyatt P/N The flow cell contains the following parts. The item numbers in the left column correspond to numbers in Figure 7-2 and the numbers in parentheses in the instructions. Table 7-1: Flow cell assembly, parts list Item Qty P/N Description Möbiu flow cell Assembly, Möbiu Electrode Fused Silica, One side AR coated, Flow Cell Window Electrode Retainer - Möbiu Cell Board Bottom Cover - Möbiu Cell PCB Assy, Electrode Heater Board, Field Only 7 2 P Retaining Ring for Ø1/2" Lens Tubes and Mounts 8 2 P Nut, Super Flangeless, 1/4-28, PEEK, Blue 9 2 P HL Nut, Flangeless, Short Headless, 1/16", 1/4-28, PEEK 10 2 P Ferrule, 1/16 tubing flat bottom 1/4-28 fitting 11 2 P S Ferrule, Super Flangeless Tefzel, SS Ring, 1/16 inch 12 2 P O-Ring, Kalrez, 2006, Compound P O-Ring, Kalrez, 2010, Compound S Flat-head machine screw M3x0.5x Assembly, Möbiu cell luer plumbing: IN Assembly, Möbiu cell luer plumbing: OUT 17 2 P Female to 1/428 Adapter and Fitting (batch use, not shown) Möbiu User s Guide (M3001 Rev. B) 75

76 Chapter 7: Maintenance Procedures The following diagram shows the parts of the Möbiu flow cell. The numbers in this diagram correspond to the numbers in the first column of the part list and the numbers in parentheses in the instructions Figure 7-2: Flow cell exploded 76 Möbiu User s Guide (M3001 Rev. B)

77 Disassembling and Cleaning the Flow Cell Removing the Flow Cell 1. Disconnect and seal the inlet and outlet ports on the front panel of the Möbiu. 2. Open the top door of the Möbiu and unscrew the two nuts (8) securing the inlet and outlet tubing. 3. Lift out the flow cell. 8 Disassembling the Flow Cell Note: The Möbiu Cell installation tool (WTC #169121), as shown below, is three tools in one. One end of the tool has two hex head wrenches, one for the electrode retainers and one for the M3x0.5x6 flat-head machine screws. The other end has the spanner wrench for the window retainer. Spanner wrench Electrode retainer wrench Machine screw wrench Caution! As you disassemble the cell, be careful not to damage or scratch the cell s optical surfaces. 1. Prepare a clean surface on which to place disassembled components. Möbiu User s Guide (M3001 Rev. B) 77

78 Chapter 7: Maintenance Procedures 2. Begin the flow cell disassembly by unscrewing the two nuts (9) that secure the tubing to the flow cell. 3. Remove the tubing (with attached nuts and ferrules) and set them aside. Optionally, you may leave the tubing attached to the flow cell so long as you flush the tubing with clean buffer or deionized water to remove any leftover sample that may contaminate the next batch. 4. Using the Möbiu Cell tool, remove the flat-head machine screw on the bottom board cover of the cell (14). Then remove the bottom board cover (5). This exposes two retainer screws (14) for the electrode heater boards on the sides. 5. Using the Möbiu Cell tool, remove the two 5 flat-head machine screws (14) that secure the electrode heater boards (6). Remove one board from each side of the flow cell Using the Möbiu Cell tool, unscrew the two electrode retainers (4). 78 Möbiu User s Guide (M3001 Rev. B)

79 Disassembling and Cleaning the Flow Cell 7. Using the center threaded driver of the Möbiu Cell tool, remove the two flow cell electrodes (12 and 2) WTC # Rotate the flow cell to see the sides containing the flow cell windows. Using the Möbiu Cell tool spanner wrench, remove the two window retaining rings (7) Using a rubber Q-tip or a piece of PEEK tubing, gently tap the flow cell windows around the outer edges to loosen the two flow cell windows (3) from the housing. If necessary, gently tap the entire housing on the lab bench to shake out the windows from the housing. 10. Remove the two window O-rings (13) from the housing, and inspect them to ensure there are no cracks or tears in the surface of the O- rings. If the O-rings stick to the flow cell, gently blow nitrogen gas on the assembly to loosen the O-rings. At this point, all parts should have been removed from the flow cell housing. Möbiu User s Guide (M3001 Rev. B) 79

80 Chapter 7: Maintenance Procedures Cleaning the Flow Cell Housing Good lab practice and common sense should be applied in the following procedure when handling and cleaning the flow cell parts. Streaks and particles left on the flow cell windows or inside the cell cavity can introduce stray light and distort the measurements. 1. Have a clean surface ready to place all cleaned dry components. 2. Wear clean lint-free gloves. As you clean the cell, be careful not to damage or scratch the cell s optical surfaces. 3. Rinse the Möbiu flow cell housing thoroughly with deionized water. 4. Using a small nylon brush, scrub the surface of the flow cell housing while rinsing the inside and outside of the cell with detergent. 5. Fully rinse the flow cell housing with deionized water. Then flush the inside and outside of the cell with ethanol to speed the drying process. 6. Blow dry the housing with filtered nitrogen, and place the housing on a clean surface. Cleaning and Reinstalling the Electrodes 1. Rinse the two cell electrodes (2) with deionized water. Then flush the surface of the electrodes with detergent using your fingers to scrub the surface. 2. Fully rinse the electrodes with deionized water. Then flush the electrodes with ethanol to speed the drying process. 3. Blow dry the electrodes with filtered nitrogen, and place the electrodes on a clean surface. 4. Examine each electrode. A worn electrode has an uneven color or retains sample deposits after cleaning. Peeling and pitting are also signs of a worn out electrode. In the picture below, the electrode on the left is unused. The electrode in the center has been used with a number of samples in one orientation. The deposits may be cleaned with a soft toothbrush and toothpaste. The electrode on the right is still worn, despite multiple cleanings and installation at multiple orientations, and needs to be replaced. New Electrode Used Electrode Needs Replacing Note: Failure to replace worn out electrodes is likely to result in unreliable data. 80 Möbiu User s Guide (M3001 Rev. B)

81 Disassembling and Cleaning the Flow Cell 5. The electrode heater boards (6) have a layer of conformal coating. If they are dirty, they can safely be rinsed with deionized water and then blown completely dry. 6. If you are replacing the electrodes (2), install new O-rings (12) onto the electrodes, and insert the electrodes into the flow cell. 7. Using the Möbiu Cell tool, secure the electrodes (2) by tightening the electrode retainers (4) Make sure the electrode heater boards (6) are completely dry. Blow dry them with filtered nitrogen, even if you did not get them wet. 9. Reinstall the electrode heater boards (6) by tightening the flat-head machine screws (14) with the Möbiu Cell tool. Make sure that the screws are secure in place; there is no need to over-tighten the screws Möbiu User s Guide (M3001 Rev. B) 81

82 Chapter 7: Maintenance Procedures 10. Reinstall the board bottom cover (5) using the remaining flat-head machine screw (14) and the Möbiu Cell tool. Make sure the screws are securely tightened Cleaning and Reinstalling the Cell Windows Perform the following steps for both sets of flow cell O-rings (13), windows (3), and retainer rings (7): 1. Rinse the window O-rings (13) with deionized water. Then flush them with detergent and scrub them with your fingers. Blow dry the O-rings with dry nitrogen. 2. Install the window O-rings (13) into the O-ring grooves. Be sure not to introduce droplets of liquid because they can leave streaks on the windows later. 3. Rinse one of the flow cell windows (3) with deionized water. Then flush it with detergent and gently scrub both surfaces with your fingers to ensure that any deposits or streaks have been removed. Blow dry the window with dry nitrogen. Be sure not to leave streaks. 82 Möbiu User s Guide (M3001 Rev. B)

83 Disassembling and Cleaning the Flow Cell 4. Determine which surface of the window is coated and which is uncoated as follows. The uncoated side must be on the inside of the flow cell in contact with the fluid sample for optimal instrument performance. Coated Side Must face outside of cell Stays dry during experiments Arrow on window edge points to this side Smaller bevel At a glancing angle, reflection off surface has blue tint Uncoated Side Must face inside of cell Contacts fluid during experiments Arrow points away from this side Larger bevel Reflection off surface has no tint. Uncoated surface: larger bevel. Faces inside of cell. Coated surface: smaller bevel, pointed to by arrow on side. Faces outside of cell. 5. Install the cleaned window with the uncoated surface in contact with the O-ring. Secure the window with the retaining ring (7) using the Möbiu Cell tool spanner wrench. 6. Repeat steps 3 through 5 for the other flow cell window Note: You may choose to clean the windows without removing the electrodes. In this case, you should flush the flow cell housing with detergent and then deionized water. We recommend that you remove the electrode heater boards before flushing the cell housing and reinstall them after blowing the cell dry for optimal electrical contact. Möbiu User s Guide (M3001 Rev. B) 83

84 Chapter 7: Maintenance Procedures Cleaning and Reinstalling Inlet and Outlet Tubing 1. Rinse the tubing and its fittings with deionized water. Then flush them with detergent. 2. Fully rinse each part with deionized water. Then flush them with ethanol to speed the drying process. 3. Blow dry the tubing and fittings with filtered nitrogen Reinstall the fittings into the cell in the correct orientation. Reinstalling the Flow Cell Note: The Inlet tubing (white) and the Outlet tubing (blue) must be installed correctly into the Möbiu. The Inlet tubing has an inside diameter of 0.010". The Outlet tubing has an inside diameter of 0.020". The outer diameters are the same. 1. Replace the flow cell into the read head. The flattened flow cell key is toward the front of the Möbiu. 2. Hand tighten the nuts that secure the Inlet and Outlet tubing to their respective ports. Flow Cell Key White Inlet Blue Outlet 3. Connect to the plumbing system and check for leaks. 4. Close the instrument door. 84 Möbiu User s Guide (M3001 Rev. B)

85 8 Mobility Theory and Calculations The optical system of the Möbiu implements the massively parallel phase analysis light scattering (MP-PALS) technique, a first-principle mobilitymeasuring method. This technique makes speedy measurements through massive parallelism of detection, and it extends the measurable molecular size below 2 nm. A much shortened measurement time 30 seconds or less contributes to preservation of precious and fragile samples. CONTENTS PAGE Electrophoresis and Electrophoretic Mobility...86 Introduction to Mobility Measurement with Light Scattering...88 Massively-Parallel Phase Analysis Light Scattering (MP-PALS)...92 Parameters Derived from the Mobility...93 Möbiu User s Guide (M3001 Rev. B) 85

86 Chapter 8: Mobility Theory and Calculations Electrophoresis and Electrophoretic Mobility Electrophoresis is the migration of (macro-)ions under the influence of an electric field. For moderate field strengths (< 200 V/cm), the steady-state electrophoretic velocity 1 v e attained by the migrating macro-ions is proportional to the applied electric field E, where is the electrophoretic mobility, or velocity per unit electric field. v e = E (1) As shown in Figure 8-1, a positively charged macromolecule in the solution is subject to an electric field E. Figure 8-1: A positively charged macromolecule surrounded by excess counter-ions in solution. In the steady state, the electric force QE will be exactly balanced by the frictional force 6 R h v e, where is the solvent dynamic viscosity and R h is the molecule s hydrodynamic radius. Eq. (2) shows the mobility to be proportional to the molecular charge and inversely proportional to the viscosity. = v e / E = Q / 6 R h (2) However, this relationship is too simplistic for most cases, because the effects of the counter-ions have been ignored. The charged macromolecule is, in reality, surrounded by an excess of counter-ions, which inevitably 1. The time scale within which a steady-state electrophoretic velocity is attained is on the order of magnitude m/f, where m is the mass of the macro-ion and f = 6 R h is the associated frictional coefficient; stands for the solvent dynamic viscosity and R h is the molecule s hydrodynamic radius. For BSA molecules in an aqueous solution, the quantity m/f is approximately 0.2 nsec. 86 Möbiu User s Guide (M3001 Rev. B)

87 Electrophoresis and Electrophoretic Mobility screens out the electric field and reduces the electric field strength experienced by the macromolecule. As a result, the electrophoretic mobility is usually much lower than that predicted by the net charge. The value of electrophoretic mobility can be used to evaluate the amount of charge (Ze) carried by a macro-ion or its zeta potential, which is defined as the electrostatic potential existing at the hydrodynamic plane of shear of a solvated macro-ion. Several relationships have been derived between Ze, and, but none of them is rigorous enough to be applicable under all circumstances. Various approximations and parameters such as Debye length, solution ionic strength, dielectric constant, viscosity, particle size, and macro-ion surface conduction all contribute to the complexity. Furthermore, non-idealities such as the electrophoretic effect 1 and ion relaxation 2 render an exact analytical expression between and a formidable, if not impossible, feat. While it is often not possible to compute the zeta potential rigorously, it is always possible to make first-principle physical measurements of the (electrophoretic) mobility, which conveys important information regarding the dispersed macromolecules. 1. Macro-ions are necessarily surrounded by low-molar-mass counter-ions and co-ions, usually derived from the electrolyte. An excess of counter-ions exists in the vicinity of each of the macro-ions. The electric field that drives the macro-ions also acts on these counter-ions in the opposite direction. The moving, solvated counter-ions drag the solvent along with them, and the solvent in turn acts on the macro-ions. The net effect is a secondary force that has a retardation effect on the movement of macro-ions. This is called the electrophoretic effect. (Adapted from Physical Chemistry of Macromolecules by C. Tanford.) 2. Ion relaxation refers to the perturbation from equilibrium of the distribution of co-ions and counterions around the macro-ions due to the same electric field that drives the movement of the macroions. Möbiu User s Guide (M3001 Rev. B) 87

88 Chapter 8: Mobility Theory and Calculations Introduction to Mobility Measurement with Light Scattering Since the early 1970s, various techniques of laser light scattering have been developed for measuring electrophoresis. Light scattered from moving particles is Doppler-shifted and carries information about their movement. As shown in Figure 8-2, the incident light is scattered by particles within the scattering volume into a new direction with a scattering angle s. Figure 8-2: Phase diagram for light scattering. The applied electric field E along the X-axis drives electrophoresis. The scattering vector is defined as q s = k s k 0, the difference between the wave vectors of the scattered and incident light. Its direction is as shown in Figure 8-1 and it has a magnitude q s = 4 nsin( s /2)/, where n is the refractive index of the solution and is the wavelength of the light in vacuum. The existence of an electric field E along the x-axis results in electrophoresis in the x direction with a velocity v e = E. The optical phase s of the scattered light is related to the positions of the scattering particles. Considering only electrophoretic movement, the optical phase difference between time 0 and t later is s = q s v e t where v e t is the displacement of the particles due to electrophoresis. We obtain, after some straightforward algebraic operations, the Doppler shift: s = d s /dt = 2 n sin( s )E / (3) In general, the movement of particles can be collective (due to fluid flow, electrophoresis, etc.) and diffusional (due to Brownian motions). The diffusional component, being stochastic in nature, averages to zero over time, and the collective component can be revealed if enough measurement is available to average away the diffusional component. If one measures the average optical phase shift per unit time d s / dt due to the applied electric field, the electrophoretic mobility can be determined. 88 Möbiu User s Guide (M3001 Rev. B)

89 Introduction to Mobility Measurement with Light Scattering Figure 8-3 shows how the optical phase associated with particle diffusion can be averaged away given enough measurement time. Figure 8-3: Measured optical phase due to (a) particle diffusion alone and (b) electrophoresis and diffusion. For both (a) and (b), the sample is Titania (Anatase TiO 2 from Inframat Advanced Materials TM ) dispersed in ethanol. Figure 8-3(a) shows the measured optical phase due to particle diffusion alone. The sample is titanium dioxide (TiO 2 ) dispersed in ethanol, R nm. The phase has been measured and normalized to sin s. As h 100 the figure shows, this diffusional component is indeed a random walk. The average displacement due to particle diffusion happens to average to zero at t = 4 sec. The smaller the particles hydrodynamic radius, the more conspicuous the diffusion will be and the more time it will take to average this component away. In Figure 8-3(b), a 10 Hz, square-wave electric field with a magnitude of 18 V/cm is applied to drive electrophoresis. The scaled and offset electric field is plotted below the optical phase for visual reference. Here the optical phase consists of both an electrophoretic and a diffusional component. It is clear that the electrophoretic component reverses its direction every time the electric field switches polarity, while the diffusional component remains independent of the electric field and proceeds with its random walk. Since the diffusional component should average out in the order of 4 seconds, the electrophoretic mobility can be measured from Figure 8-3(b) and is computed m cm/v sec. Möbiu User s Guide (M3001 Rev. B) 89

90 Chapter 8: Mobility Theory and Calculations In general, light scattering (LS) can be carried out in two different ways: light scattering in the homodyne mode mixes only light scattered from particles, while light scattering in the heterodyne mode mixes light scattered from particles along with some unscattered light, often referred to as the local oscillator. To appreciate the distinctions between these two methods, we consider the photocurrent i(t) generated at the detector as shown in Eq. (4). (4) In Eq. (4), is the detector responsivity, is the laser angular frequency, P LO is the local oscillator power, P 0 is the power of the scattered light from each of the N particles in the scattering volume, and j (t) is the phase of the light scattered by the j th particle. Without loss of generality, we have assumed that all particles scatter equally efficiently; that is, they have the same polarizability. The phase j (t) is related to the position x j (t) of particle j by j (t) = q s x j (t). Because both diffusional and collective movements contribute to x j (t), we have the following relationship: j (t) = q s x j (t) = q s [x jd (t) + v c t] = jd (t) + c (t) (5) In Eq. (5) x jd (t) and jd (t) = q s x jd (t) are the displacement and phase components associated with the Brownian motion of the j th particle and c (t) = q s v c t is the phase component due to the collective velocity v c, which is common to all particles. Note that x jd (0) stands for the initial position of the j th particle. Expanding i(t), we get Eq. (6). (6) In the homodyne mode, P LO = 0 and we end up with: (7) Note that all of the collective phase components c (t) s cancel out in i(t) for homodyne detection. Measurements obtained at all times t have the information-carrying terms jd (t) kd (t) = q s [x jd (t) + x kd (t)]. It is now clear that LS in the homodyne mode is insensitive to collective particle movement since only diffusional information is available. This makes homodyne LS suitable for measuring hydrodynamic radii even when a flow is present, but it cannot measure electrophoretic mobilities. 90 Möbiu User s Guide (M3001 Rev. B)

91 Introduction to Mobility Measurement with Light Scattering On the other hand, signals obtained from heterodyne LS contain the terms cos[ jd (t) + c (t)] and carry phase information of both collective and diffusional components. Therefore, in order to measure electrophoresis with light scattering, optical heterodyning is necessary 1. Dynamic light scattering in the heterodyne mode had been used to measure the mobility of bovine serum albumin molecules (5% by weight, or about 50 mg/ml) in 4mM NaCl solution 2. Another method, laser Doppler electrophoresis (LDE), involves detecting light scattered from electric field-driven macro-ions moving within a stationary fringe pattern 3. The fringe pattern is generated by the interference of two light beams derived from the same laser to ensure a good contrast ratio 4. As macro-ions traverse the light and dark fringes, a temporally sinusoidal intensity signal is detected from the scattered light. Since the spacing between adjacent light and dark fringes is known from the angle of intersection between the interfering laser beams, the electrophoretic velocity can be measured and mobility can be obtained from the signal frequency. LDE has important advantages over earlier techniques. It greatly reduces the volume of sample required for mobility measurement (to milliliters or less) because the fringe pattern can be generated within the space between electrodes that are merely 1 2 mm apart. Since the electric field is the voltage difference between the electrodes divided by their spacing, the reduced electrode spacing decreases the magnitude of the voltage that must be applied to generate observable electrophoresis. In addition, a large number of macro-ions contribute to the detected signal, which produces statistically sound measurements. To avoid electrode polarization, whereby macro-ions accumulate near the electrodes and shield the bulk from the applied field, an oscillating (AC) electric field is preferable to a DC field. The frequency of the field reversal should be made much lower than the Doppler frequency and much higher than the reciprocal of the time it takes for electro-osmosis to develop 5. However, when it comes to measuring low-mobility species 6, LDE cannot satisfy both conditions without applying excessively high voltages and running into problems such as profuse electrolysis and fluid convection. Another limitation of LDE is that both positive and negative electric fields produce the same optical signal, so the sign of the mobility cannot be determined. 1. B. J. Berne and R. Pecora, Dynamic Light Scattering with Applications to Chemistry, Biology and Physics, John Wiley & Sons Inc. 79 (1976). 2. R. Ware and W. H. Flygare, J. Colloid Interface Sci. 39, 670 (1972). 3. E. E. Uzgiris, Prog. Surf. Sci. 10, 53 (1981). 4. The direction of the driving electric field is almost always made perpendicular to the phase fronts of the fringes. 5. The characteristic time for development of electro-osmosis is d 2 /, where d is the relevant linear dimension and is the kinematic viscosity of the solution. For aqueous solutions at 20 C, 1.0 mm 2 /sec, and the time it takes for electro-osmosis to develop is about 1 sec for a 1 mm electrode gap. 6. Species where the absolute value of the mobility is smaller than m cm/v sec. Möbiu User s Guide (M3001 Rev. B) 91

92 Chapter 8: Mobility Theory and Calculations Massively-Parallel Phase Analysis Light Scattering (MP-PALS) To address the limitations of LDE, phase analysis light scattering (PALS) 1 was developed in the late 1980 s and early 1990 s. PALS is derived from LDE; the major distinction between them is that in PALS a sweeping, instead of a stationary, fringe pattern is established in the scattering volume. Such fringe patterns can be generated by phase-modulating one or both of the interfering laser beams. A sweeping fringe pattern not only obviates the necessity of macro-ions having to traverse a few light and dark fringes during a single (electric) field period, but also enables the determination of the sign of mobility. Zero-mobility (and therefore stationary) particles give rise to a signal frequency equivalent to that of the sweeping fringes, and macro-ions migrating in the same/opposite direction of the sweeping fringes produce a signal frequency lower/higher than that of the sweeping fringes. There is another incarnation of PALS: instead of interfering the two laser beams at the scattering volume, the second laser beam (or local oscillator) is mixed/heterodyned directly on the detectors with scattered light from the macro-ions and acts both as an optical amplifier and as a phase reference. The local oscillator is phase-modulated for the same reason explained above. In this configuration, PALS is really an interferometric method in which one arm is the modulated local oscillator and the other is the scattered light. As the incident laser beam goes straight through the sample, the sample volume can be further reduced. The optical system of the Möbiu builds upon this technique and takes it a step further. The implementation of the PALS technique in the Möbiu enables multiple simultaneous measurements with an array of detectors. As long as the active area of each detector element is greater than or equal to the coherence area of the scattered light 2, the signals collected from different detector elements are truly independent. Since each detector channel makes an independent measurement, N channels will reduce the measurement time by a factor of N. In the Möbiu, N=31 (and can be increased in the future with available computation power). This parallelism is made possible by an innovative optical design, massively- 1. J. F. Miller, K. Schätzel, B. Vincent, The Determination of Very Small Electrophoretic Mobilities in Polar and Nonpolar Colloidal Dispersions Using Phase Analysis Light Scattering, J. Colloid Interface Sci. 143, (1991). 2. When scattered light impinges on a screen, a diffraction/speckle pattern is produced which depends, among other things, on the extent of the scattering volume. That is, the intensity maxima and minima depend on the dimensions of the scattering volume. On any given point P on the screen, one could define an area near the point P such that the signals at all points within this area are partially coherent with that at P. This area would be called the coherence area. A useful estimate of the coherence area for light scattering is A coh = 2 /, where is the solid angle subtended by the scattering volume at the detector(s). As grows smaller, A coh becomes larger. (Adapted from Dynamic Light Scattering with Applications to Chemistry, Biology and Physics by B. J. Berne and R. Pecora.) 92 Möbiu User s Guide (M3001 Rev. B)

93 Parameters Derived from the Mobility parallel PALS (MP-PALS), which facilitates free space multiplexing of the interfering beams. Moreover, the detectors used in the Möbiu are linear and robust over their excellent dynamic range (from nw all the way up to mw). This range ensures that the full advantage of the coherent amplification is fully utilized to achieve superior instrument sensitivity. Parameters Derived from the Mobility Several important molecular parameters can be derived from if appropriate models and/or theories are applied. Summarized below are the parameters the DYNAMICS software reports for mobility measurements: Net Charge If the hydrodynamic radius R h is known, the net charge (also known as the molecular charge or Debye-Hückel-Henry charge, Z DHH ) is computed through the relationship: (8) where Z is the valence of the macromolecules, e (~1.6 x coulombs) is the elementary charge, is the sample viscosity, is the Debye-Hückel parameter, and f( R h ) is Henry s function. Both the Debye-Hückel parameter and Henry s function are explained in the section on Henry s equation on page 94. Zeta Potential The DYNAMICS software provides the following three different formulas for calculating zeta potential. Researchers should choose the most suitable model for their applications. Smoluchowski s equation In the limiting case where the molecular hydrodynamic radius R h is much larger than the Debye length 1, Smoluchowski s equation can be used: (9) where 0 (~8.854 x F/m) is the permittivity of free space, and r (~80 for water at 20º C) is the solvent dielectric constant. Möbiu User s Guide (M3001 Rev. B) 93

94 Chapter 8: Mobility Theory and Calculations Hückel s equation In the other limiting case where the molecular hydrodynamic radius R h is much smaller than the Debye length 1,Hückel s equation can be used: (10) Henry s equation Henry s equation is the most general of the three formalisms. It includes both the Smoluchowski s and Hückel s equations as its limiting cases. Henry's equation is: (11) To utilize Henry s equation, researchers need to know the ionic strength I of their sample solution. The ionic strength is defined as: (12) where C i is the molar concentration of any mobile ion and z i its valence. The summation extends over all mobile ion species present. (As examples, the ionic strengths of 10 mm NaCl, CaCl 2 and MgSO 4 are, respectively, 10 mm, 30 mm and 40 mm.) In turn, the Debye-Hückel parameter can be determined from the ionic strength of the electrolyte, where N A (~6.02 x mol -1 ) is the Avogadro constant, k (~1.38 x10-23 JK -1 ) is the Boltzmann constant, and T is the absolute temperature in Kelvin. (13) The reciprocal of is defined as the Debye length, which corresponds to the double-layer thickness surrounding the molecules. The Debye length 1 of 10 mm NaCl is 3.05 nm, for example. 94 Möbiu User s Guide (M3001 Rev. B)

95 Parameters Derived from the Mobility Figure 8-4 shows a plot of Henry's function f( R h ) for reference: Figure 8-4: Plot of Henry s function As evident from the figure, when taken to one extreme, Henry s equation becomes Hückel s equation: (14) When taken to the other extreme, Henry s equation reduces to Smoluchowski s equation: (15) Colloidal Stability One of the most widely adopted uses for zeta potential is in the determination of colloidal stability. As the molecular charge increases, the probability of molecules aggregating and flocculating out of the solution decreases. Like charges repel each other. The widely accepted criterion for colloidal stability at room temperature is: (16) Using Smoluchowski s equation, a mobility of 1.00 m cm/v sec corresponds to a zeta potential of 12.7 mv at 25º C in aqueous solutions. Therefore most protein solutions, with < 2.00 m cm/v sec are considered borderline stable or unstable. Möbiu User s Guide (M3001 Rev. B) 95

96 A Connecting to a Network or PC These instructions contain a pictorial overview for connecting your Möbiu to a computer for data collection. The instructions are divided into seven sections: CONTENTS PAGE Components...97 Connecting to a LAN Connecting via USB Connecting via Ethernet Without a LAN Instrument Network Settings Troubleshooting and Diagnostics Please read over Components on page 97 to gain an understanding of the components to be used. Then read over either Connecting to a LAN on page 102, Connecting via USB on page 104, or Connecting via Ethernet Without a LAN on page 105 depending on your configuration. Finally, read over Instrument Network Settings on page 106 for instrument settings. If you experienced problems connecting to your instrument, please read Troubleshooting and Diagnostics on page 107 for diagnostics and trouble-shooting. Möbiu User s Guide (M3001 Rev. B) 96

97 Appendix A: Connecting to a Network or PC Components Instrument Connections Figure A-1 is a detail of the instrument back panel. The Ethernet port, designated with a yellow arrow, is to be used for all connections in these instructions. Please see Connecting via USB on page 104 for instructions on establishing a USB connection. Figure A-1: Detail of the back panel of the Möbiu. 97 Möbiu User s Guide (M3001 Rev. B)

98 Chapter A: Connecting to a Network or PC LAN Connection Figure A-2 shows a typical wall socket connection to a Local Area Network (LAN). If you are going to connect the instrument to a LAN, you will need access to this type of socket. Figure A-2: Wall socket LAN connections 98 Möbiu User s Guide (M3001 Rev. B)

99 Appendix A: Connecting to a Network or PC Computer Connections Computer connections can be made via either the Ethernet or USB port. Figure A-3 shows these ports on a standard laptop computer. Connecting to a LAN on page 102 and Connecting via Ethernet Without a LAN on page 105 describe instrument connections made via the Ethernet port. Connecting via USB on page 104 describes connections made via the USB port. Ethernet USB Figure A-3: Ethernet and USB ports on the computer 99 Möbiu User s Guide (M3001 Rev. B)

100 Chapter A: Connecting to a Network or PC Crossover Cable A crossover cable can be used to make a direct connection from the instrument to an Ethernet port on a computer or to an Ethernet to USB adapter. Please note that the crossover cable shipped with Wyatt Technology instruments is yellow to distinguish it from a standard Ethernet cable. Consult your IT department to determine if a yellow crossover cable is required. Figure A-4: The Ethernet crossover cable shipped by Wyatt Technology is yellow. Ethernet Cable A standard Ethernet cable is sometimes referred to as a patch cable, or a straight-through cable to distinguish it from the crossover cable in Crossover Cable on page 100. Ethernet cables provided by Wyatt Technology are black, blue, white, or gray, but never yellow (yellow is reserved for the crossover cable). For these instructions, the Ethernet cable will always be black. Figure A-5: Standard Ethernet cable. For these instructions, the standard cable is always black. 100 Möbiu User s Guide (M3001 Rev. B)

101 Appendix A: Connecting to a Network or PC Ethernet to USB Adapter This device can be used to connect an Ethernet cable to a USB port on the computer. Using this adapter, it is possible to have the computer connected to a LAN via the computer s Ethernet port, and the instruments connected to the computer via USB. The Ethernet to USB adapter supplied by Wyatt Technology will look similar to this. The first time you connect an Ethernet to USB adapter to your computer, you may be prompted to install USB drivers for the device. To do so, use the CD supplied with the Ethernet to USB adapter, and follow the Microsoft Windows instructions. Ethernet USB Figure A-6: Standard Ethernet to USB adapter. The Ethernet cable is plugged into the wall port, and the USB plug is plugged into a USB port on the computer. Ethernet Switch Ethernet switches are used to connect several Ethernet cables to one resource, such as the LAN socket in Figure A-2. The Ethernet switch supplied by Wyatt Technology will look similar to the five port switch shown below. However, different Ethernet switch models may be shipped by Wyatt Technology as they become available. Please note that Ethernet cables can be connected to the switch in any order or position. Also, the switch has an external AC adapter (not shown) to provide power to the switch. Figure A-7: Five-port Ethernet switch 101 Möbiu User s Guide (M3001 Rev. B)

102 Chapter A: Connecting to a Network or PC Connecting to a LAN If an instrument is connected to a LAN, it can be accessed by any computer plugged into the same LAN. Instrument to LAN Plug the instrument into a LAN wall socket using a standard Ethernet cable. The computer that is to communicate with the instrument must be on the same LAN. Figure A-8: Connection for instrument to LAN 102 Möbiu User s Guide (M3001 Rev. B)

103 Appendix A: Connecting to a Network or PC Instrument and Computer to LAN If there is only one LAN wall socket available for both the instrument and computer, it is necessary to use an Ethernet switch to connect both the computer and instrument to the LAN. In this configuration, the computer can access the LAN and the instrument, and the instrument can be accessed from any other computer on the LAN. Figure A-9: Instrument and a computer can be connected to the LAN using an Ethernet switch. Note that the actual Ethernet switch provided by Wyatt Technology may be a different model than the one pictured here. 103 Möbiu User s Guide (M3001 Rev. B)

104 Chapter A: Connecting to a Network or PC Connecting via USB If it is not possible or desired to have the instruments connected to a LAN, it is possible to connect to the instruments via USB. In this way, the instruments can be isolated from the LAN, even while the computer maintains its own Ethernet connection with the LAN. The instrument may be directly plugged into the USB-to-Ethernet adapter, or it may be connected through an Ethernet switch as shown in the figure below. You may be prompted to install drivers for the Ethernet to USB adapter the first time it is plugged into the computer. To install the drivers, insert the CD that came with the adapter and follow the Windows instructions. Figure A-10: Connecting instrument to USB using an Ethernet switch Note that the actual Ethernet switch provided by Wyatt Technology may be a different model than the one pictured here. 104 Möbiu User s Guide (M3001 Rev. B)

105 Appendix A: Connecting to a Network or PC Connecting via Ethernet Without a LAN If the computer is not on the LAN, it is possible to use the Ethernet port directly to connect to the instruments. You may connect the instrument directly to the computer's Ethernet port or using an Ethernet switch as shown below. Figure A-11: Connecting instrument to the computer using an Ethernet switch Note that the actual Ethernet switch provided by Wyatt Technology may be a different model than the one pictured here. 105 Möbiu User s Guide (M3001 Rev. B)

106 Chapter A: Connecting to a Network or PC Instrument Network Settings Figure A-12 shows the standard settings on the instrument front panel that will work with all of the above connection schemes. As shown in Figure A-12, to set the IP address there is a choice of Obtain an IP address automatically, to use an associated DHCP server, or Use the following IP address: to set a static IP address. In general, this setting can be left to DHCP. With DHCP, once the instrument is connected to a computer or LAN, the IP address and subnet mask will be assigned automatically. This will even work with the USB connections described in Connecting via USB on page 104. When using DHCP, it might take several minutes for the IP address to be assigned. During this time, the IP address and subnet mask will read Once the IP address and subnet mask have been assigned, both will be automatically updated, and should no longer read At this point, it should be possible to connect to the instrument from the computer. If you wish to use a static IP address and subnet mask, please contact your IT department to obtain a valid address and mask. Figure A-12: Standard settings on instrument front panel for instrument connectivity 106 Möbiu User s Guide (M3001 Rev. B)

107 Appendix A: Connecting to a Network or PC Troubleshooting and Diagnostics If you are experiencing instrument connectivity problems, please go over these steps. If you still cannot connect to your instrument after going over this section, please contact Wyatt Technology for assistance or visit for the latest troubleshooting guides. Verifying Instrument Connections Please verify that the instrument is communicating with the computer. Open a Windows command prompt, as shown in Figure A-13. At the command line, type ping plus the IP address of the instrument as shown on the instrument front panel (see Figure A-12). If the instrument is connected properly, the result should be similar to that shown in Figure A- 13. Figure A-13: Using ping to verify the instrument connection If the instrument is not connected properly, the result should be similar to that shown in Figure A-14. Figure A-14: Failure to connect to instrument using ping 107 Möbiu User s Guide (M3001 Rev. B)

108 B Using an Alternative Autosampler This appendix describes how to use an autosampler and HPLC pump other than that produced by Agilent Technologies to automate alternate injections of buffer and sample into the Möbiu. This configuration requires an appropriate auto-inject cable to be connected from the Autoinject Out port of the autosampler to the Auto-inject In port of the Möbiu. CONTENTS Overview Terminology Autosampler Sequence Möbiu User s Guide (M3001 Rev. B) 108

109 Appendix B: Using an Alternative Autosampler Overview If you are using the Automated Injections with an Autosampler on page 62 plumbing method, but are not using an Agilent pump system, you can still perform automated injections of buffer and sample into the Möbiu. A wait for auto-inject command in the DYNAMICS Event Schedule enables completely unattended data collection for each sample injection. Figure B-1: Concurrent autosampler sequence and DYNAMICS Event Schedule 109 Möbiu User s Guide (M3001 Rev. B)

110 Chapter B: Using an Alternative Autosampler Terminology The following are important terms for automating mobility measurements with an autosampler: Flow Rate (Q pump ): The rate at which the HPLC system is pumping mobile phase. This may differ for sample and wash buffer injections. Injection Volume (V inject ): The sample or buffer volume that is withdrawn into the sample loop and intended for injection into the Möbiu. Mobile Phase: The buffer or other solvent being pumped through the HPLC system and in which samples are formulated. This may or may not be the same as the wash buffer. Number of Measurements (n): The number of DLS and PALS measurements made for each injection. The flow should be stopped for the entire n measurements Pump Stop Time (t stop ): The amount of time that the HPLC pump will flow buffer through the sample loop, used to push fluid from the sample loop into the Möbiu and out to waste For each sample, the Stop Time should be set to the amount of time to empty the sample loop at a given flow rate without introducing additional mobile phase: t stop = V inject Q pump For each wash buffer step, the Stop Time should be long enough to allow 3 5 ml mobile phase to clean the sample loop. Wait Time (t wait ): Time set in the DYNAMICS Event Schedule to allow for sample or buffer to be injected and for the flow to stop completely, typically: t wait min ( ) = t stop Möbiu User s Guide (M3001 Rev. B)

111 Appendix B: Using an Alternative Autosampler Autosampler Sequence The autosampler sequence consists of alternating sample and wash buffer injections. These are described in detail in the subsections that follow. Diagrams use green for sample injection steps, blue for wash buffer injection steps, and pink for measurement steps. Sample Injection For each sample measurement, the autosampler and HPLC pump deliver sample to the Möbiu, the flow is stopped, and then a synchronized measurement is taken with DYNAMICS. Each sample injection should be set up so that the flow is stopped immediately after all the sample has cleared the sample loop and entered the flow stream. This is accomplished by setting the pump stop time (t stop ) to the amount of time required to flush the loop and then turning off the flow immediately after the injection sequence has finished. The Event Schedule in DYNAMICS is set up to wait for the auto-inject signal sent from the autosampler to the Möbiu as the sample is injected. An additional wait time (t wait ) is included in the Event Schedule to account for the time required to deliver the sample to the instrument and for the flow to stop completely. The flow should be stopped for at least as long as is required to take the desired number of measurements typically seconds per measurement. The DYNAMICS Event Schedule commands to wait for a sample injection before collecting data are as follows: 111 Möbiu User s Guide (M3001 Rev. B)

112 Chapter B: Using an Alternative Autosampler Wash Buffer Injection The flow rate and injection volume for the buffer injection need not be the same as for sample injections. The buffer injection volume should be at least as large as the sample injection volume. In this example, we performed 500 L sample injections and 700 L buffer injections. The flow rate was increased from 0.5 ml/min to 1.0 ml/min. The stop time should be long enough to allow at least 3 5 ml of mobile phase to flow through the entire system, including the sample loop. In this example, the stop time for wash buffer injections was 5 minutes, flushing 5 ml of mobile phase through the system. Although unnecessary, it may be beneficial to record data for the wash buffer injections to ensure adequate flushing of the autosampler and Möbiu as was done in the example that follows on the left. If you do not want to collect wash data, be sure to wait for auto-inject for both the buffer and sample injections before collecting data, as shown in the example that follows on the right. The DYNAMICS Event Schedule commands to wait for two auto-inject signals to account for the buffer and sample are as follows, depending on whether you are collecting data for the wash phase: 112 Möbiu User s Guide (M3001 Rev. B)

113 C Adding DLS to Instruments Dynamic light scattering (DLS, also known as quasi-elastic light scattering or QELS) is an internally installed option that measures timedependent fluctuations in the scattered light signal using a fast photon counter. DLS measurements can determine the hydrodynamic radius of macromolecules or particles. CONTENTS Requirements Connecting to a Wyatt HELEOS or TREOS Möbiu User s Guide (M3001 Rev. B) 113

114 Chapter C: Adding DLS to Instruments Requirements The DLS correlator in the Möbiu can be connected externally to another instrument such as Wyatt s DAWN HELEOS, DAWN HELEOS II, or minidawn TREOS to process the DLS data for the other instrument. If your situation meets all of the following conditions, you may use this feature: You have a Möbiu with the DLS option. You have a DAWN HELEOS, DAWN HELEOS II, or minidawn TREOS without the DLS option. You want to collect DLS data with the DAWN HELEOS, DAWN HELEOS II, or minidawn TREOS. You are not currently using the Möbiu to collect electrophoretic mobility data. Connecting to a Wyatt HELEOS or TREOS An optical fiber cable picks up light from the light scattering cell in the DAWN HELEOS, DAWN HELEOS II, or minidawn TREOS and conveys it to the APD inside the Möbiu. This information will be transmitted to the ASTRA software through the Möbiu s Ethernet connection. An example of the completed connection is shown in Figure C-1. Solvent reservoir Injector HELEOS, HELEOS II, or minidawn TREOS De-gasser and pump Waste Möbiu with DLS Figure C-1: DLS Correlator connected to a DAWN HELEOS II 114 Möbiu User s Guide (M3001 Rev. B)

115 Connecting to a Wyatt HELEOS or TREOS DLS Connections Notice that the Möbiu has only a fiber connection running between the instruments and an Ethernet connection. All sample fluid connections are to the DAWN HELEOS, DAWN HELEOS II, or minidawn TREOS. Before following this procedure, you will need to order an optical fiber cable and mount from Wyatt Technology. The part number is WDPC if you have a DAWN HELEOS and WDPC if you have a minidawn TREOS. The following instructions explain how to install the optical fiber connection on the Möbiu end of the fiber connection shown in Figure C-1: 1. Make sure that the power to both the HELEOS or TREOS and the Möbiu is off. Unplug both power cords. 2. Place the instruments side by side. This lets you remove the HELEOS or TREOS top cover to install and align the optical fiber receiver. 3. Use a flathead screwdriver to remove the two screws from the DLS connection cover on the back of the Möbiu. 4. Remove the green cap from the safety chamber. 5. Remove the Möbiu fiber from the FIBER port. Möbiu User s Guide (M3001 Rev. B) 115

116 Chapter C: Adding DLS to Instruments 6. Secure the Möbiu fiber to the safety chamber to keep the fiber tip safe while it is not in use. 7. Connect the HELEOS or TREOS fiber cable (P/N WDPC or WDPC-01-04) to the Möbiu FIBER port. An example of the completed connection is shown in Figure C-2. Figure C-2: DAWN fiber cable connected to the Möbiu 8. Install and align the optical fiber receiver onto the HELEOS or TREOS read head. Refer to the Removing and Installing the Optical Fiber Receiver and Aligning the Optical Fiber sections of your HELEOS or TREOS Hardware User s Guide. 9. Replace the cover on the DAWN HELEOS, DAWN HELEOS II, or minidawn TREOS instrument. 116 Möbiu User s Guide (M3001 Rev. B)

117 D Troubleshooting This appendix contains troubleshooting advice. CONTENTS PAGE Low Forward Laser Monitor Voltage Low Forward Laser Monitor Voltage There are several possible reasons you may see a low Forward Monitor signal compared to the value on the PALS Certificate of Performance for the instrument. We recommend that you first check all possibilities listed for Case A and Case B. If none of the suggestions fixes the problem, please contact Wyatt Technology Technical Support at support@wyatt.com for assistance. Case A: Sample Blocks Laser Transmission Potential causes are The laser may be blocked by an air bubble or air: If bubbles are present in the flow cell, gently tap the PALS cell while adding more fluid to the cell chamber to remove the air bubble. If an Atlas is installed, pressurize the cell to shrink and dissolve the bubble. If there is a visible meniscus in the dip cell, clean and thoroughly dry the quartz cuvette and electrodes. Refill the cuvette with µl of fluid and re-insert the electrodes. The sample absorbs light at 532 nm. Continue the measurement as long as the forward monitor signal is >0.2 V. Alternatively, dilute the sample to increase laser transmission. The sample is inhomogeneous or poorly mixed. Allow ample time for the sample to equilibrate after injection. Alternatively, fill the cell with buffer before each sample injection to eliminate refractive index inhomogeneity. The sample causes thermal lensing. Measure exclusively in low laser mode. Möbiu User s Guide (M3001 Rev. B) 117

118 Appendix D: Troubleshooting Case B: A Setting on the Instrument is Incorrect Potential causes are The laser is set to OFF. Using the Mobius front panel, turn on the laser. There is no measurement cell in the instrument. Insert the flow cell or dip cell to actuate the mechanical shutter. The bottom cover of the flow cell is installed backwards. This prevents the flow cell from being inserted properly. Inspect the bottom cover and install it properly if needed. If this still does not fix the situation, remove the bottom cover temporarily to test whether the bottom cover itself is the cause of the malfunction. The instrument is in low laser mode. Switch to normal mode in DYNAMICS, and make a measurement to reset the forward monitor. (The display on the front panel will only update after a PALS measurement has been made.) The instrument is set to DLS only mode in DYNAMICS. The forward monitor signal on the front panel and in DYNAMICS may show values close to zero (0 V). Switch to DLS and PALS (Simultaneous) or PALS only mode in DYNAMICS. Then, make a measurement to reset the forward laser monitor. 118 Möbiu User s Guide (M3001 Rev. B)

119 E Instrument Specifications Since MP-PALS is an interferometric technique, the coherence length (frequency stability) of the laser source plays a vital role in the performance of the Möbiu. The Möbiu uses a 50 mw diode-pumped sold-state (DPSS) singlelongitudinal-mode laser that emits at 532 nm (or 660 nm for the alternate model). The stable single-frequency operation ensures the superior performance. Of the 50 mw laser power, 45 mw is delivered to the cell and 5 mw is used as the reference beam. In addition to a stable frequency, the laser power is also stabilized to be better than 0.1%. CONTENTS PAGE Electrical and Optical Specifications Environmental Specifications Laser Safety Notes Electrical and Optical Specifications Table E-1: Electrical and optical specifications Power Output 50 mw Laser Operating Wavelength 532 nm or 660 nm Polarization Ratio 100:1 Coherence Length > 10 m Typical Optical Noise < 0.2% Environmental Specifications The ambient temperature required to operate the Möbiu is 15 C to 30 C. However above 28 C ambient, the temperature to which samples can be cooled is restricted to the ambient temperature minus 24 C. For example, at 30 C ambient, the bench can only be cooled to 6 C, instead of the low temperature specification of 4 C. These specifications are for 0 95% relative humidity (non-condensing). Möbiu User s Guide (M3001 Rev. B) 119

120 Appendix E: Instrument Specifications Laser Safety Notes The lasers used in the Möbiu are Class IIIb lasers. However the Möbiu itself is classified as a Class 1 Laser Product according to IEC :1993+A1+A2 and CFR Title 21 Subchapter J. Note these environmental specifications apply to the laser subsystem and not to the instrument as a whole. This means that under normal operation, no laser radiation should escape from the instrument, and no protective equipment must be worn. However the follow warning applies: WARNING! Use of controls or adjustment or performance of procedures other than specified herein may result in hazardous radiation exposure. The instrument also bears the following warning label: DANGER! Laser Radiation when open. Avoid direct exposure to beam. Note: Laser safety labels are in English. If you need safely labels in a language other than English, please contact Wyatt Technology 120 Möbiu User s Guide (M3001 Rev. B)