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Summary of Contents for Malvern Zetasizer uV

• Page 1 μV user manual Malvern...
• Page 3 Zetasizer μV User Manual MAN0428 Issue 1.0 December 2008...
• Page 4 We therefore disclaim all liability for any changes, errors or omissions after the date of publication. No reproduction or transmission of any part of this publication is allowed without the express written permission of Malvern Instruments Ltd.
• Page 5 Addendum to manual Addendum number ADD0076-1.0 Date 04/02/2009 Manual number MAN0428-1.0 Manual name Zetasizer μV User Manual This addendum presents new information for taking measurements at ambient temperatures while using the Flow-mode facility on the Zetasizer V instrument. μ The information below should be inserted as a third paragraph in the Sample - Temperature section on page 9.16, and as a fifth paragraph in the Flow-mode SOPs section on page 9.33.
• Page 6 Page 2 Addendum 0076-1.0...
• Page 7 Addendum to manual Addendum number: ADD0081-1.0 Date: 26/05/2010 Manual number: MAN0428-1.0 Manual name: Zetasizer μV User Manual This Addendum presents new information for operating the Zetasizer V instru- μ ment. The content should be read in-place of, or in conjunction with, the sections indicated in the respective manual.
• Page 8 Due to the risk of melting, polystyrene cuvettes must not be used for measurements above 70°C. Malvern offers a range of cells for performing measurements with the Zetasizer μ The choice of cell is dependent upon the type of measurement being performed and the sample that will be measured.
• Page 9 Specification - Read in place of relevant section on page A-1 of the Zetasizer μV User Manual. Specification Parameter Specification Size range maximum (radius) * 0.15nm to 1micron Molecular weight range ** 342Da to 2x10 (est from hydrodynamic diameter) Molecular weight range ** 980Da to 2x10 (calculated using Debye plot) Minimum sample volume...
• Page 10 Page 4 Addendum 0081-1.0...

• Page 11: Table Of Contents

  T able of contents Part 1 - Operator’s Guide Introduction to this manual ......... 1-1 Introduction .
• Page 12 Table of Contents Zetasizer μV ......4-13 The Measurement display Records and Reports - Viewing the Results .
• Page 13 Zetasizer μV Table of Contents ........9-37 Extracting an SOP .
• Page 14 Table of Contents Zetasizer μV ....... . . 12-11 Scattering functions Size theory .

• Page 15: Part 1 - Operator's Guide

  Part 1 - Operator’s Guide...

• Page 17: Introduction To This Manual Introduction

  Introduction to this manual Introduction This manual covers the operation and maintenance of the Zetasizer μV particle analyser. Note The Zetasizer model, Serial number, software and firmware version can be found by left-clicking the μV icon in the right corner of the status bar. The aim of this manual is to: Identify what the instrument is.
• Page 18 Chapter 1 Introduction to this manual We recommend reading both these manuals fully before starting the first measure- ment, though if more familiar with particle size analysers, jump straight to Chap- ter 4. This chapter gives practical details on making measurements. This manual is divided into three sections as follows.

• Page 19: Access To The Instrument

  Within this manual, reference is made to the various people who will have access to the instrument. Malvern personnel Malvern personnel (service engineers, representatives, etc.) have full access to the instrument and are the only people authorised to perform all service procedures that may require the removal of the covers.

• Page 20: Where To Get Help

  Chapter 1 Introduction to this manual The combination of the Zetasizer μV instrument, the computer and Zetasizer soft- ware may be referred to as the “the system”. Cells and Cuvettes Any device for holding and measuring the sample in the instrument will generally be referred to as a “cell”.
• Page 21 Maintenance – for both the optical unit and accessories. Help desk All queries regarding the system should initially be directed to the local Malvern representative. Please quote the following information: Model and serial number of the instrument (located on the rear panel).
• Page 22 Chapter 1 Introduction to this manual Page 1-6 MAN 0428...

• Page 23: What Is The Zetasizer μv

  What is the Zetasizer μV? Introduction This chapter gives a brief overview of the Zetasizer μV instrument and simple explanations about the measurement technique. What does the Zetasizer μV do? The Zetasizer μV instrument provides the ability to measure two characteristics of particles or molecules in a liquid medium.

• Page 24: What Are Particle Size And Molecular Weight

  Chapter 2 What is the Zetasizer μV? What are Particle Size and Molecular weight? This section describes the basics of Particle Size and Molecular weight are and why they are important. Greater detail on the instrument’s measurement techniques is given in the chapters that cover theory (Chapter 13 and Chapter 14). What is Size? The Particle Size measured in a DLS instrument is the diameter or radius of the sphere that diffuses at the same speed as the particle being measured.
• Page 25 With the Zetasizer μV instrument, the molecular weight can now be determined by use of Static Light Scattering (SLS) measurement techniques. This technique is explained in Chapter 14. Note Malvern uses Daltons as the unit of molecular weight. Results are dis- played in kiloDaltons (kDa). Zetasizer μV Page 2-3...
• Page 26 Chapter 2 What is the Zetasizer μV? Why do we want to know it? We need to know the molecular weight, so we can determine how many grams there are in 1 mole of a substance. The mole is the chemistry standard term for ‘1 molecular weight’, e.g.

• Page 27: How Does The Zetasizer μv Work

  How does the Zetasizer μV work? Introduction Previously we identified the instrument and described the various measurement processes that can be performed. This chapter introduces the hardware and software features that the instrument incorporates. The initial section, “How is a Zetasizer measurement performed?”, will briefly describe the following: What is involved in making a measurement? What are the major components of the system?

• Page 28: How Is A Zetasizer Measurement Performed

  Chapter 3 How does the Zetasizer μV work? How is a Zetasizer measurement performed? The Zetasizer system comprises the Zetasizer instrument and a computer with the Zetasizer software installed . A cell is filled with the sample and loaded into the cell area on the top of the instrument ill 6744 The software is used to control the measurement of the sample, there are two basic ways to make a measurement:...
• Page 29 Measurement display (below). Once the measurement is complete the results can be viewed; either in a Record view , by selecting one of the Malvern pre-set reports , or a user defined report Zetasizer μV Page 3-3...
• Page 30 Chapter 3 How does the Zetasizer μV work? UPDATE ill 8300 The measurement results will be automatically saved to a measurement file. Note The measurement file must be selected before the measurement is started as the measurements will be saved in the file currently open. Page 3-4 MAN 0428...

• Page 31: What Does The Zetasizer Consist Of? - Identifying The Hardware

  How does the Zetasizer μV work? Chapter 3 What does the Zetasizer consist of? - Identifying the hardware The diagram below shows a typical system with its key modules, the Zetasizer instrument and a computer system with the Zetasizer software installed. It is preferable the computer is dedicated to just running the Zetasizer software.
• Page 32 Chapter 3 How does the Zetasizer μV work? The Zetasizer μV instrument Optical unit Positioned on the cover is a label that identifies the instrument model. ill 8307 Rear Panel The rear panel provides all the connections. These are identified below. ill 8308 Power input socket Mains power input socket for the instrument.
• Page 33 Pressure = 100 kPa g For connection purposes, the purge input uses a push-fit connector. An optional purge connection kit is available from Malvern instruments if this facility is to be used. When performing a purge measurement the following rules should be taken into account: The flow rate will be preset at 1.5ltr/min at 1 bar.
• Page 34 Serial number and Model number label Identifies the actual Zetasizer μV model and its serial number. Please quote all numbers in any correspondence with Malvern Instruments. Power light Illuminates blue when the system is powered on.
• Page 35 How does the Zetasizer μV work? Chapter 3 Refer to Appendix A for a list of the cell area components that may come into contact with any spilt sample. Features of the cell area Other important features of the cell area are described below. ill8310 Cell area lid Press down on top of the cell area lid (where it is marked “Push”) to unlock the...
• Page 36 Chapter 3 How does the Zetasizer μV work? Flow tube channel Channel provides access for a tube when an optional flow cell is connected. Flow tube notch Notch provides access for a tube when an optional flow cell is connected. Cell lid catch Mechanism to keep the cell closed during a measurement.

• Page 37: Navigating The Software

  Chapter 3 Navigating the software The Malvern Zetasizer software controls the system during a measurement and then processes the measurement data to produce either a size or Molecular weight result. It displays the results and allows reports to be printed.
• Page 38 Chapter 3 How does the Zetasizer μV work? Menu bar The menu bar contains the main menu headings for all software functions. Items which end with a row of dots (...) will cause dialogue boxes to appear. Similarly any items which end with an arrow ( ) will cause a second menu to appear.
• Page 39 How does the Zetasizer μV work? Chapter 3 As a shortcut, a list of the most recently used measurement files are shown at the bottom of the menu for immediate opening. Exit will close down the software. Edit Menu This allows movement and manipulation of records in the Measurement file window(s).
• Page 40 Chapter 3 How does the Zetasizer μV work? View Menu The View menu allows the selection of which reports will be shown in the measurement file window and those Toolbars to be displayed. To see the Standard deviation, the Relative standard deviation, the minimum, the maximum or the mean of a selected number of records, a Statistics bar can be displayed.
• Page 41 How does the Zetasizer μV work? Chapter 3 Note A combination of the Edit and View menus will appear when the right mouse button is clicked anywhere in the measurement file window. Measure Menu Select this menu when ready to perform a measurement. There is a choice of using an existing measurement SOP (Measure-Start SOP...) or manually setting up the measurement and sample details (Measure-Manual).
• Page 42 Count rate meter section at the end of Chapter 6. The Macros submenu will show additional available programs. These are programs written by Malvern to enable the system to operate in a mode required for a specific application. These can be installed and selected using the Options feature.
• Page 43 Security Menu To prevent unauthorised changes, the Malvern software can be configured to limit each user’s access to various functions - e.g. modifying an SOP. Users are assigned operating permissions that allow, or restrict, access; refer to Chapter 7 for details.
• Page 44 Help Menu Help Topics... give access to the help files. Links to the Malvern website... including downloads and consumables. Tip of the day... will give hints on how to use the Zetasizer software; a different tip will appear each time this is selected. An option is given for turning off the Tip of the day dialogue that appears when the software starts.
• Page 45 How does the Zetasizer μV work? Chapter 3 Toolbars The toolbars contain a selection of tools that can be used to perform the most popular operations. Each tool will have its equivalent commands within the menu bar. For example, using the Open tool is equivalent to using the File-Open menu item.
• Page 46 Workspace dialogues. Selecting a report tab will display a predefined report as selected by the Report pages tab in the workspace dialogues. Malvern supplies several reports that give different views of the measurement settings and results, whilst custom reports can be generated using the Report Designer.
• Page 47 How does the Zetasizer μV work? Chapter 3 Measurement display When a measurement is being performed a measurement display will appear. The Measurement display shows the progress of the measurement. The screen display shown changes depending on the type of measurement being performed and the view tab selected.
• Page 48 Malvern logo ( ) or an (M) alongside. This identifies it as Malvern defined and cannot be overwritten. The Malvern defined parameters can be used as a template that can be altered and saved under a different name. Units Conversion...

• Page 49: A Tutorial - Making A Measurement

  A Tutorial - Making a measurement Introduction After reading this chapter a user should be able to make simple measurements. The chapter goes through the basics from turning on the system to displaying the results of a measurement. The first section, “Quick guide to making a measurement” will run through the basic steps, giving an overview of the measurement process.

• Page 50: Quick Guide To Making A Measurement

  Chapter 4 A Tutorial - Making a measurement Note that most of the settings and dialogues used for a manual measurement are the same as those used in an SOP measurement. The sections that follow, Quick guide to making a measurement, etc, will focus on SOP measurements.

• Page 51: Sample Preparation

  Due to the risk of melting, polystyrene cuvettes must not be used for measurements above 50°C. Malvern offers a range of cells for performing measurements with the Zetasizer μV. The choice of cell is dependent upon the type of measurement being performed and the sample that will be measured.
• Page 52 All the cells mentioned below are available from Malvern and should be used with the supplied cell caps. Using the caps will ensure greater thermal stability of the sample, as well as preventing dust introduction and possible spillage.
• Page 53 A Tutorial - Making a measurement Chapter 4 Disposable low volume Glass - round aperture polystyrene (ZEN0117) (PCS8501) Typical solvent Water, Water/alcohol Water, most organic and inorganic solvents Optical quality Good to very good Excellent Minimum 100μl Sample volume Advantages Low cost Highest optical quality Low volume...
• Page 54 Chapter 4 A Tutorial - Making a measurement Low volume Glass flow Low volume quartz cuvette (ZEN0023) (ZMV1008) Typical solvent Water, most organic and Water, most organic and inorganic solvents inorganic solvents Optical quality Excellent Excellent Minimum 75μl plus tubing 8μl Sample volume Advantages...

• Page 55: Filling The Cell

  A Tutorial - Making a measurement Chapter 4 Filling the cell When filling the cell there are several actions to consider; some that apply to all cells and others that are only applicable to the measurement type and the cell chosen.
• Page 56 Chapter 4 A Tutorial - Making a measurement Do not overfill the cell, use about 15mm maximum, as this can produce thermal gradients within the sample that will reduce the accuracy of the temperature control. When filling, tilt the cuvette and allow it to fill slowly (see above). To stop bubbles forming let the sample flow down the inside.

• Page 57: Inserting The Cell

  A Tutorial - Making a measurement Chapter 4 Inserting the cell In the status bar, the software will prompt when the cell needs to be inserted. This will always be after the SOP has been started - see the next section. When and how the cell is inserted will depend on the application, and the measurement choices selected.
• Page 58 Chapter 4 A Tutorial - Making a measurement optical surface must be inserted so that the “light path” indicator arrow in the cell enclosure passes through it - i.e. facing the front of the instrument. Most cells have a small triangle at the top to indicate the side that faces the front. This is especially critical for Molecular weight measurements.

• Page 59: Making An Sop Measurement

  A Tutorial - Making a measurement Chapter 4 Making an SOP measurement If a measurement is being made using an SOP, then all the hard work has already been done. The instrument has been turned on and the software started; the sample has been prepared and added to the cuvette.
• Page 60 Chapter 4 A Tutorial - Making a measurement Note All measurement files have the extension .dts. This is added automatically to all new files. Starting an SOP measurement Everything should now be ready to make the actual measurement. To start an SOP measurement, select Measure-Start SOP. The Open SOP dialogue will appear.

• Page 61: Making A Manual Measurement

  A Tutorial - Making a measurement Chapter 4 Making a manual measurement Making a manual measurement is essentially the same as making an SOP measurement, except that, where as in an SOP measurement all the measurement options are pre-specified, here it will be necessary to set them immediately. Instead of starting an SOP, select Measure-Manual.
• Page 62 Chapter 4 A Tutorial - Making a measurement sequence. The dialogues displayed depend upon the measurement type selected. The diagram below shows the display for a size measurement. The features of the measurement display are: Button bar The button bar provides the control for the measurement operation. The Settings button opens the measurement settings dialogue.
• Page 63 A Tutorial - Making a measurement Chapter 4 Progress meter The progress meter shows how far the measurement has progressed plus the number of measurements performed and the measurement runs completed. Also shown are the temperature, measurement position and attenuator settings. Tab views The Tab views enable the progress and results of the measurement to be viewed.
• Page 64 Chapter 4 A Tutorial - Making a measurement Count rate Displays the number of photons detected per second. The count rate is useful for monitoring the sample quality. Normal count rate Normal count rate display. ill 8376 If dust is present Dust present then sharp spikes will be observed.
• Page 65 A Tutorial - Making a measurement Chapter 4 Correlation function The correlation function helps the experienced user to interpret any problems with the sample. Small size Large size sample sample Time Time ill 6760 Contaminated Noisy data sample = variable results Time Time ill 6779...
• Page 66 Chapter 4 A Tutorial - Making a measurement Refer to the Expert Advice section in Chapter 10 for a description. Molecular weight measurements Result tab (1st tab) Please also refer to size description above. For Molecular weight measurements the default result view shown is a Debye plot.
• Page 67 A Tutorial - Making a measurement Chapter 4 The Result tab is named after the result view chosen, the result view shown above is Flow trace vs Time. Different views can be selected by right-clicking on the graph and selecting from the list displayed. The views available are: Count Rate, Correlation Function, Flow trace vs Volume and Flow trace vs Time and Monitor.
• Page 68 Chapter 4 A Tutorial - Making a measurement Measurement sequence Note The status bar will prompt for certain actions during the course of the measurement. Before the measurement sequence begins the cell temperature will change to the starting temperature requested in the SOP. The measurement will then continue with an optimisation or initialisation stage where the cell positions, compensation and attenuator settings for the cell, sample and measurement type will be determined.
• Page 69 A Tutorial - Making a measurement Chapter 4 Press Start to begin the dark count measurement. The laser is turned off and a measurement is taken of the background light level. Insert the scattering standard cell (i.e. Toluene) and press Start when ready. The measurement will measure the scattering intensity of the scattering stand- ard used.
• Page 70 Chapter 4 A Tutorial - Making a measurement Page 4-22 MAN 0428...

• Page 71: Records And Reports - Viewing The Results

  Records and Reports - Viewing the Results Introduction Once a measurement has finished the results will need to be reviewed. This chapter details how to display the final measurement result. The final result is displayed in the measurement file window as either a measurement record or report.
• Page 72 Chapter 5 Records and Reports - Viewing the Results Melting point records To view a sequence of Melting point records, select the first record in the sequence; all subsequent records in that sequence will then automatically be selected. Outliners For Melting point records, outliners are employed. This enables the main ‘parent’ record to be shown without the ‘child’...
• Page 73 Records and Reports - Viewing the Results Chapter 5 The Outlined record view can be turned off and on by selecting View-View type- Sorted or Outlined or by right-clicking the mouse on the table, as shown above. Statistics bar Use this to display the Standard deviation, the Relative Standard deviation, the minimum, the maximum or the mean of a selected number of records.
• Page 74 Zetasizer, the filename and record number of the measurement and the date printed, plus the Malvern contact phone number. Each measurement type has a ‘standard’ report associated to it. The same result information is present in both the computer and printed versions, except for the diagnostic reports which contain extended information on the printed version.
• Page 75 Records and Reports - Viewing the Results Chapter 5 Note To show multiple results on one report, hold down Shift or Ctrl and select the required records and then click the required report tab. Units conversion As in the Records view, within the results section of each report, it is possible to change the units used to define some of the parameters displayed.
• Page 76 Chapter 5 Records and Reports - Viewing the Results Size measurements - standard report The standard report for Size measurements is Intensity PSD (M). (PSD stands for Particle Size Distribution). ill 8375 The report is divided into four areas; these are described below. Sample details This section gives details of parameters relating to the sample.
• Page 77 Records and Reports - Viewing the Results Chapter 5 Cell type This displays the cell type selected. Results This section gives the results of the measurement. The values given here will be based either on intensity, volume or number, depending on which view tab is selected.
• Page 78 Chapter 5 Records and Reports - Viewing the Results Intercept This is the amplitude of the G correlation function at time 0. For a good meas- urement it will be 0.85 to 0.95. Lower values will be obtained for measurements on weaker scattering sample, or in turbid sample where background scattering is significant.
• Page 79 To print out a different graph a new report will have to be created using Report designer. Other Size reports Other Malvern views available for size measurements include: Intensity Statistics (M) Volume psd (M) Volume Statistics (M)
• Page 80 This report is described below. Size quality report (M) This is a summary report detailing whether the result meets the Malvern Instruments quality criteria for a size measurement. The report indicates Result meets quality criteria, or Result does not meet quality criteria in which case a list of parameters that do not meet the criteria, and probable reasons will be displayed.
• Page 81 Records and Reports - Viewing the Results Chapter 5 Count rate is out of range (too high) Attenuator not set to automatic Sample contains large particles/aggregates/dust Sample is not stable during measurement Insufficient signal collected Measurement duration not set to automatic Filter factor in research software not set to default (50%) Cumulant fit error high Data quality too poor for cumulant analysis...
• Page 82 Chapter 5 Records and Reports - Viewing the Results Molecular weight measurements - standard report The standard report for Molecular weight measurements is Molecular weight report (M). ill 7795 The report is divided into five areas; these are described below. Sample and measurement details These two sections show measurement details and details of parameters relating to the sample.
• Page 83 Records and Reports - Viewing the Results Chapter 5 System details The System section gives details on settings configured during the measurement process. Please see the size description for details about Temperature, and Cell type. Results The results section gives four pieces of information: Molecular weight Shows the measured weight of a molecule within the measured sample expressed in atomic mass units;...
• Page 84 Chapter 5 Records and Reports - Viewing the Results Flow-mode measurements - Standard report The Standard Flow-mode report, Chromatogram Summary (M), gives the same information as seen in a standard size report, plus additional information relating to the flow duration and rate used. The three main peaks in the measurement will also be shown, displaying the sample intensity, width, Molecular weight and start and end flow volumes.
• Page 85 Records and Reports - Viewing the Results Chapter 5 Trend measurements (including Protein Melting point) Trend measurements can be viewed using the reports: Trend, Trend v Custom 1 and Trend v Custom 2 reports. Melting point trend measurements can be viewed using the Melting point (M) report.
• Page 86 Chapter 5 Records and Reports - Viewing the Results Page 5-16 MAN 0428...

• Page 87: Sample Preparation

  Sample Preparation Introduction Before filling the cell or cuvette that will be used in the measurement, the sample will need to be prepared. To ensure reliable and accurate measurements proper sample preparation is extremely important. Preparation of the sample for the different measurement types will involve specific preparation techniques.

• Page 88: Preparing The Sample - Molecular Weight

  Chapter 6 Sample Preparation Considerations for small particles Minimum concentration For particle sizes smaller than 10nm, the major factor in determining a minimum concentration is the amount of scattered light that the sample generates. In practice, the concentration should generate a minimum count rate of 10,000 counts per second (10kcps) in excess of the scattering from the solvent.
• Page 89 Sample Preparation Chapter 6 Very small samples such as proteins in aqueous solutions will often require filtering. A number of concentrations of the sample must be prepared (typically 0.25 to 1 ). The polymer must be fully soluble and dust must be Solvent excluded.

• Page 90: Count Rate Meter

  Chapter 6 Sample Preparation Count rate meter After preparing the sample, the count rate can be checked by using the count rate meter. Selecting Tools-Count rate meter will display the following dialogue. The Count rate is displayed in Kcps (Kilo counts per second), and enables the sample quality and concentration to be checked without having to start a measurement.
• Page 91 Sample Preparation Chapter 6 Drifting count rate This may indicate that thermal gradients are present in the sample, and further time is required for temperature equilibration. Increasing/Decreasing count rate A steadily increasing count rate will indicate an aggregating sample, while a decreasing count rate will indicate a sedimenting sample.
• Page 92 Chapter 6 Sample Preparation Page 6-6 MAN 0428...

• Page 93: Part 2 - Supervisor's Guide

  Part 2 - Supervisor’s Guide...

• Page 95: Security

  SOPs, deleting records and editing results can be reserved for specific users only. Within the Malvern Security system, one or more persons are set up as the admin- istrator. The administrator then controls access to the instrument by defining “User groups”...

• Page 96: Initial Start-up - Set Up The Administrator

  Chapter 7 Security Initial start-up - set up the administrator Note It is vital that at least one user has access to the configuration of the security system. Select Security-Configure Security. The first time the system is run, there will be just one user (Administrator) and one group (Administrators).

• Page 97: Enabling Security

  Security Chapter 7 Note It is preferable to have at least two users assigned to the Administrators group. The users user name and password should be stored in a secure location. This is to safeguard against accidental lockout or deletion of per- missions that may prevent the security settings being available.

• Page 98: User Groups

  Chapter 7 Security User groups Note Only users assigned to the Administrator group can add or edit the user group properties. Adding/editing a group Select Security-Configure security to enter the Security configuration dia- logue. Select User-New Group... to display the Group Properties dialogue, alternatively double-click on an empty row.

• Page 99: Users

  Security Chapter 7 allocated to that group will be shown. Selecting one or more users (hold down the control key to select multiple users) and press OK to add those users to the group. To remove a member, simply select the member and press the Remove button. Note that members are only removed from the group, they are not removed from the system.
• Page 100 Chapter 7 Security Username is commonly an abbreviated form of the individual name or a unique identifier such as an employee code. The Full Name is used to hold the full printed name of the individual and can be used on reports to identify the user if an employee code is used as a Username. The Description field is optional and is just used to add some descriptive text to the user details.
• Page 101 Security Chapter 7 To change the user password, enter the current password and specify a new pass- word with confirmation. Pressing OK makes the change to the security settings. User can not change password This option prevents a user from changing a password once it has been initially set.
• Page 102 Chapter 7 Security Page 7-8 MAN 0428...

• Page 103: Measurement File Window - Workspaces

  Measurement file window - Workspaces Introduction The Measurement File window is where all results (or records), and reports for each measurement file can be viewed, moved and analysed. Depending upon the Window options set, multiple measurement files can be viewed at the same time. Once a result has been calculated, the data can be displayed as either a record or report - by selecting the appropriate tab.

• Page 104: Measurement File Window

  Chapter 8 Measurement file window - Workspaces Malvern provides several default reports (identified with (M) in the name) that will be sufficient for most users. The user also has the ability to create their own custom reports to satisfy their individual needs. Details on creating custom reports (using Report Designer) are given in Chapter 11.
• Page 105 Measurement file window - Workspaces Chapter 8 What measurement information is presented in the Report and Record tabs is controlled by the Workspace selection dialogues (below). The tabs can be configured to display either all the measurement file records and associated reports (i.e.
• Page 106 Weight measurement records will be displayed. Use the Record view parameters to specify what parameters will be viewed as the column headings. Report pages allows selection of both Malvern created and customised reports. When selected, click OK. Once completed the new workspace can be selected using the View menu or Workspace toolbar.
• Page 107 Measurement file window - Workspaces Chapter 8 Alter the settings as required and press OK; the new settings will be saved. Deleting a Workspace Select <Delete workspace> from the toolbar or the Tools-Settings- Configure Workspace menu. From the list, select the workspace to be deleted and click OK. Exporting and Importing Workspaces Workspaces can be exported to, and imported from, folder locations by using the <Export workspace>...
• Page 108 Chapter 8 Measurement file window - Workspaces Parameters can be added or removed from this list by using the Add and Remove keys Change the order in which the parameters are displayed by selecting a parameter, and pressing the Move up or Move Down buttons.
• Page 109 Measurement file window - Workspaces Chapter 8 Report tabs The report tabs show every report that has been selected for a workspace. With the Summary workspace selected the default reports are Intensity PSD (M) and Molecular weight (M). These and other reports can be added or removed using the workspace dialogues.
• Page 110 Selecting Edit-Delete will instantly remove any selected records from the measurement file. They will not be deleted from the software, but be stored in a .del file in the My DocumentsMalvern InstrumentsZetasizerMeas- urement Data directory. This file has the same name as the original measure- ment file but with a .del extension.

• Page 111: Using Sops

  Using SOPs Introduction An SOP measurement uses pre-set parameters, to ensure that measurements made on the same type of sample are made in a consistent way. SOPs are ideal if repeatedly measuring the same type of sample. Having to set the same parameters each time a measurement is made is tedious and also adds the risk of introducing errors into the settings - using SOPs avoids this.
• Page 112 Chapter 9 Using SOPs Once a measurement has been completed it will be added to the list in the Records view tab of the Measurement file window. The SOP used can be viewed by right-clicking over the measurement record and selecting Extract SOP. In each of the SOP dialogues there are Help or Advice buttons that will advise on how to complete the SOP.

• Page 113: The Sop Editor

  Using SOPs Chapter 9 Alternatively if Browse for SOP... is selected and the icon is pressed. The SOP directory will be displayed where an SOP can be manually selected. The title of the SOP shown is that typed into the Sample name entry box in the Sample SOP dialogue.
• Page 114 Chapter 9 Using SOPs Tree menu view On opening the SOP editor the window below will be displayed; the exact list displayed will depend upon the Measurement type selected. The SOP dialogues available for each measurement type will be displayed down the left hand side of the window, with the respective highlighted SOP dialogue shown alongside.
• Page 115 Using SOPs Chapter 9 Measurement type Allows selection of the type of measurement that the SOP will perform, i.e. Size, Protein melting point, Molecular weight, Flow or Trend. Note It is important that the Measurement type is the first parameter that is selected, as all subsequent settings will be related to this measurement type.
• Page 116 Chapter 9 Using SOPs Sample - Temperature This enables selection of the temperature at which the measurement will be performed. This is a common dialogue for all SOPs except Trend and Protein melting point. Sample - Cell This dialogue allows selection of the cell to be used. This is a common dialogue for all SOPs.
• Page 117 Using SOPs Chapter 9 Data Processing - Export Enables the measurement results to be exported, as a text file, to third party software packages such as Excel or Wordpad. This is a common dialogue for all SOPs. Trend - Sequence Enables the selection of the temperature trend parameters, i.e.

• Page 118: Creating An Sop

  Chapter 9 Using SOPs SOP Editor Menu and Toolbar SOP file management, saving and associated software functions are handled using either the Menu bar or the Toolbar. The functions are described in the following table. Toolbar Menu item icon Description of function File-New Opens a new SOP File-Open...

• Page 119: Measurement Type Selection

  Using SOPs Chapter 9 Measurement type selection The measurement type must first be selected before entering the settings on the subsequent SOP dialogues. To Select the Measurement type Left click on Measurement type and select the measurement type from the pull down menu.

• Page 120: Size Sops

  Chapter 9 Using SOPs Size SOPs Though the majority of the Sample and Measurement dialogues described below are unique to Size measurements; this section also includes SOP dialogues that are common to all SOPs. The Size SOP Editor menu is: Sample The components of the dialogue are: Sample name:...
• Page 121 Using SOPs Chapter 9 Notes: The general notes area allows comments about the measurement to be entered. Note that any comments written during the SOP creation will appear in all measurement records that use the SOP unless the Show this page when the SOP is started...
• Page 122 Chapter 9 Using SOPs Sample - Material The Zetasizer software requires certain information about the physical properties of the dispersant that is used in the sample that is to be measured. Selecting the Sample - Material dialogue and pressing the button, displays the Material properties manager where these properties can be defined.
• Page 123 Using SOPs Chapter 9 The Refractive index and Absorption are only needed if the result is required as a volume distribution. The Refractive index can be obtained from a number of sources and is only required to two decimal places. Note that the refractive index is wavelength dependent.
• Page 124 Propanol 25C. Note Viscosity is temperature dependent. The Malvern defined dispersants include a built in viscosity calculation that determines the correct viscosity of the dispersant at any sample temperature.
• Page 125 Using SOPs Chapter 9 Sample - General Options Mark-Houwink parameters. Selecting the Define button displays the Mark-Houwink parameters dialogue. This allows the Mark-Houwink parameters to be entered to enable a Molecular weight to be calculated based on the dynamic light scattering data. a –...
• Page 126 Chapter 9 Using SOPs If the viscosity of the sample is known only at certain temperatures select the Prompt operator to enter viscosity button. This allows the user to enter both the viscosity and the temperature, prior to the measurement being started. Sample - Temperature Note The Zetasizer is capable of heating or cooling the sample to allow the...
• Page 127 Using SOPs Chapter 9 Cell Select a Cell type from the drop down menu that matches the cell in which the sample is prepared. If required, select the Show cell type... check box to display a message to the operator reminding them of this cell type when the SOP is run. Measurement This dialogue allows the duration of the measurement to be defined, and to make multiple measurements of the same sample.
• Page 128 Chapter 9 Using SOPs default. Automatic measurements will be divided into a number of runs of at least 10 seconds in length. Using Manual the time may be reduced for the measurement of a latex standard, or increased to improve the repeatability of the measurement of particularly polydisperse samples.
• Page 129 Using SOPs Chapter 9 Measurement - Instructions This dialogue allows instructions to the user to be displayed both before and/or after running an SOP. This dialogue will not appear when doing a manual measurement. Display the following instructions…before… This area is useful to give details that will allow measurements to be made correctly and consistently;...
• Page 130 Chapter 9 Using SOPs Measurement - Advanced The Advanced dialogue contains functions to alter the cell and attenuator position settings. For normal operations both will be at the default setting. Data Processing If characteristics are already known about the sample being measured, this dialogue will allow an appropriate analysis model to be applied and so optimise the measurement calculation.
• Page 131 Using SOPs Chapter 9 Analysis Models Protein Analysis The default setting, select this model when undertaking a protein analysis. General Purpose Select this model if the characteristics of the sample to be measured are unknown. Multiple Narrow Modes If it is known that the sample to be measured will give a distribution of one or more narrow peaks (i.e.
• Page 132 Chapter 9 Using SOPs If it is known that all particles within the sample will fall within a certain size range, then the size Display range can be set to ignore data or artifacts at each end of the distribution. The transformations to volume and number will then ignore the data outside the set limits.
• Page 133 Using SOPs Chapter 9 Data Processing - Reports The Reports dialogue enables various reports to be selected and then printed automatically once the measurement has finished. Two selection boxes are available. One to enable a report to be printed after each measurement is completed and the other to print out a report after all measurements have been completed.
• Page 134 Chapter 9 Using SOPs Data Processing - Export The Export dialogue enables the measurement results to be exported to third party software packages such as Excel or Wordpad. With the Export results check box selected measurement parameters can be defined and exported on completion of the measurement. Select the required Export template from the list;...

• Page 135: Molecular Weight Sops

  Using SOPs Chapter 9 Molecular weight SOPs The majority of the Sample and Measurement dialogues described below are unique to Molecular weight measurements, however when indicated please refer to the description in the Size SOP section. The Molecular Weight SOP editor menu is shown right: The dialogues are described in turn below.
• Page 136 Chapter 9 Using SOPs Solvents Manager Selecting this dialogue enables solvents to be added, modified and deleted. To define a new Solvent, press the Add... button and choose from defining either a Simple dispersant or solvent or a Complex solvent. Please refer to the Sample - Dispersant description in the Size SOP section for details.
• Page 137 Using SOPs Chapter 9 Standards Manager Input and define the scattering “standard” properties of the liquid to be used in the measurement: Temperature, Refractive index and Rayleigh ratio. This will nearly always be Toluene. The standard properties can be added, modified and deleted in the same way as detailed for the Solvent Manager above.
• Page 138 Chapter 9 Using SOPs novel combinations the dn/dc can be measured by use of a sensitive differential refractometer. Note This will require a refractometer capable of measuring to an accuracy of six decimal places. Sample - Temperature Please refer to the Sample - Temperature description in the Size SOP section. Sample - Cell Please refer to the Sample - Temperature description in the Size SOP section.
• Page 139 Using SOPs Chapter 9 Shape correction model This option allows selection of the shape correction model used for the measurement. With knowledge of the sample structure it is possible to improve the result of the measurement by adding the value that most closely corresponds to the probable sample shape;...
• Page 140 Chapter 9 Using SOPs These systematic variations can occur for a few reasons; possibly thermal drifting of the sample, poorly mixed sample, or instrument and laser stabilisation after initial switch on. (Wait 30 minutes after switch on for the laser to stabilise). If any variations are detected that are greater than the set values, then the measurement will be repeated until a result below the thresholds is achieved.
• Page 141 Using SOPs Chapter 9 Size Measurement With the Perform a size measurement check box selected a size measurement will be performed simultaneously with Molecular weight. Measurement Duration The Measurement Duration setting can affect the accuracy and repeatability of the size results. Please refer to the Measurement description in the Size SOP sections, for details of the Measurement duration settings.
• Page 142 Chapter 9 Using SOPs Size Measurement - Advanced Measurement settings Please refer to the Measurements - Advanced description in the Size SOP section for a description of the Measurement Position and attenuator functions. Data Processing If characteristics are already known about the sample being measured, this dialogue will allow an appropriate analysis model to be applied and so optimise the measurement calculation.

• Page 143: Flow-mode Sops

  Using SOPs Chapter 9 Flow-mode SOPs The Flow SOP editor menu is: The Flow-mode SOP enables measurements to be performed on a flowing sample stream. The Scattered light intensity and Hydrodynamic diameter can be plotted as a trend, and, with the addition of optional external input dialogues, information from external measuring sources, such as a refractive index detector, can be monitored and displayed.
• Page 144 Chapter 9 Using SOPs Measurement Measurement Duration The measurement duration setting may affect the accuracy and repeatability of the results. In Measurement duration, input the total measurement time or volume amount required, and adjust the units to suit: Time or Volume. The Run duration value determines the length of each individual measurement within the experiment.

• Page 145: Trend Sops

  Using SOPs Chapter 9 Trend SOPs The Trend SOP Editor menu is: The Trend SOP allows size and Zeta measurements to be performed and plotted as a function of temperature. Trend SOPs are similar to the standard size and Zeta SOPs, the differences being the inclusion of a trend and altered measurement dialogues.
• Page 146 Chapter 9 Using SOPs Trend - Sequence The Trend - Sequence dialogue enables the selection of the temperature trend parameters, i.e. start and end temperatures, and intervals between measurement steps. Start Temperature Input the temperature that the first measurement is to be made at. The instrument will initially cool, or heat, to this temperature before starting the measurement.

• Page 147: Extracting An Sop

  Using SOPs Chapter 9 Size Measurement The trend and melting point measurement dialogues are generally the same as those used with either a size or Zeta measurement SOP. The additional features are: At Each step gives the option to control the Equilibration time and Number of measurements performed.

• Page 148: Modifying An Sop

  Chapter 9 Using SOPs Modifying an SOP To modify an SOP, select File-Open-SOP. The SOP dialogues will be displayed, allowing any settings to be changed. Alter the settings within the dialogues, and then save (select File-Save or File- Save as... as appropriate).

• Page 149: Advanced Features

  Advanced features Introduction This chapter contains the following advanced features and capabilities of the Zetasizer μV: Solvent builder SOP player Averaging results Editing the result Exporting the results Flow-mode capability Options dialogue Expert advice Zetasizer μV Page 10-1...

• Page 150: Solvent Builder

  Chapter 10 Advanced features Solvent Builder The Solvent builder is a database of components that can be added in different quantities for estimating the properties of solutions. The database is used for the calculation of Complex solvents; generally necessary when performing measurements involving proteins.
• Page 151 A dispersant, or solvent, can be deleted by selecting the dispersant from the list and pressing the Delete... button. Note that it is not possible to modify or delete any dispersants or solvents that were specified by Malvern. Zetasizer μV Page 10-3...

• Page 152: Sop Player

  Chapter 10 Advanced features SOP Player The SOP player is a stand alone dialogue that enables a sequence of actions - SOPs, measurements, pauses or macros - to be constructed and then played. This sequence is called a playlist. This is useful for performing different measurement types consecutively without any user interaction;...
• Page 153 Advanced features Chapter 10 The SOP player controls are: Button Description Select this tab to add new items (or actions) to a playlist. Choices are SOP, Macro, Temperature and Pause. These are described below. Input the properties for the selected action. Creates a new ‘blank’...
• Page 154 Chapter 10 Advanced features Construct the playlist by selecting and configuring the Actions in the left pane; These are described in the following sections. The list can be arranged in order as required by dragging and dropping an action at the desired point. For each action, enter the action properties as required.
• Page 155 Advanced features Chapter 10 Selecting the SOP in the Playlist will display both the SOP properties and the sample details. Select each parameter to edit as required. The SOP used can be changed by selecting ; this will open the dialogue above. If the SOP is to be altered, this must be done using the SOP Editor.
• Page 156 Chapter 10 Advanced features Note The Macros available will be supplied by Malvern. Pause Playlist To add a pause (or time-delay) between each playlist action, select the Pause playlist icon. This will add a pause action into the playlist, with the properties displayed alongside.

• Page 157: Result Averaging

  Advanced features Chapter 10 If 10°C is entered, the instrument will cool, or heat, until that temperature is achieved. The temperature range available is that of the instrument: 2°C to 90°C. The details entered will be shown alongside the temperature action in the playlist. Note Any temperature requested in the Set temperature action will override the temperature setting requested in any previous SOP action within the...

• Page 158: Editing The Result

  Chapter 10 Advanced features Editing the result It is possible to re-analyse a measurement record using different measurement parameters. The re-analysed record will be added to end of the current file. Comments on the reasons for editing can be added and viewed in the report views. This option allows measurements to be reanalysed without the need for the instrument to be connected.
• Page 159 Advanced features Chapter 10 Note Each edit result dialogue will be slightly different depending upon the measurement type originally performed. The picture above shows the size view. Multiple results can be edited by selecting the records required before selecting the edit result dialogue.

• Page 160: Exporting Results

  Chapter 10 Advanced features Exporting results The Zetasizer software allows any of the parameters from one or more records, as well as graph and table data to be exported to another application such as Excel, Word or Wordpad. There are several ways to export the required information: One or more records can be selected in the Records view tab and moved via “drag and drop”...
• Page 161 Advanced features Chapter 10 File tab The data can be either Exported to file or Exported to Clipboard by selecting the appropriate radio button. To Export to file, type the destination filename into the File options selection box and decide whether to Append (add) to the existing file or Overwrite it.
• Page 162 Chapter 10 Advanced features Settings tab This tab determines how the data are exported. The Format options specifies the separator character that is used between each parameter. Different spreadsheet packages require different field separation characters to correctly space the data output. Commas are the most usual but these should not be used if they are numerical separators in the localised Windows version.
• Page 163 Advanced features Chapter 10 Settings tab The settings tab works in the same way as previously described. Parameters tab The list on the left displays all available parameters. The list on the right displays all parameters that will be included in the export template.

• Page 164: Flow-mode

  Chapter 10 Advanced features Flow-mode The Flow-mode option allows the Zetasizer μV to be connected, using a flowcell, to a flowing sample stream, such as the output of a chromatographic column, and measurements made without interrupting the flow. In a flow-mode measurement, both the scattered light intensity and hydrodynamic diameter are plotted as a trend.
• Page 165 Advanced features Chapter 10 Editing and inspecting a Flow-mode result A flow-mode result can be edited by right-clicking on the measurement record and selecting Edit-Result (see the Editing a Result section earlier in the chapter). This will display the dialogue below. ill 8209 The dialogue shows the following: Flow-mode plot...
• Page 166 Chapter 10 Advanced features The traces shown can be chosen by right-clicking on the y-axes or by using the Trace tab. Generally the Z-average mean values are shown as dots on the plot, whilst the Intensity is shown as a continuous line. Peaks The peaks detected by the analysis are indicated by the blue vertical bars on the plot.
• Page 167 Advanced features Chapter 10 Baseline pointer The Baseline pointer is used to set the datum level for the plot. When the measurement is originally performed the software will attribute an appropriate baseline level and use this as a filter to remove unwanted and erroneous data. All measurement data below this level will be removed from the measurement, plus any data above it that the software considers to be uncharacteristic of the measurement, in effect an “outlier”.
• Page 168 Chapter 10 Advanced features Note that the default External Inputs values are defined in the Options dialogue, explained later in this chapter. Peaks tab The Peaks tab displays all the details about the calculated peaks shown in the flow- mode plot. To display a peak's details either select a blue peak bar in the plot, or select one of the peaks identified at the top the peak tab;...

• Page 169: Options Dialogue

  Advanced features Chapter 10 Options dialogue The Options dialogue enables extra features and programs for the Zetasizer soft- ware to be installed, and to configure the default folder for saving of data. The Options dialogue enables extra features and programs for the Zetasizer software to be installed, and to configure the default folder for saving of data.
• Page 170 Macros option. This dialogue allows new macros programs (.zmac files) to be installed and selected. The Macro programs are written by Malvern to enable the system to operate in a mode required for a specific application. Highlighting the Macro in the left box will display the Macro details in the right box.
• Page 171 Advanced features Chapter 10 to the zetasizer/macros directory and add the macro to the list of available programs. To display the Macro in the Tools-Macros menu, select the check box alongside the macro and press OK. When the Tools-Macros list is next viewed the macro will be available to use.
• Page 172 Chapter 10 Advanced features Style 2 Style 3 Results This dialogue contains a check box that will allow measurements results to be saved even if the measurement is aborted before the measurement has finished collecting and analysing the data. Instrument type This dialogue provides the option to select which instrument type is currently in use.

• Page 173: Expert Advice

  Advanced features Chapter 10 Expert Advice Expert Advice enables a quality check to be performed on one or more completed measurement records. This will show how good the measurements are and whether they display any unwanted attributes, such as aggregation. To enable the Expert System, choose one or more records, right-click on the records and select Expert Advice, or select View-Expert Advice.
• Page 174 Chapter 10 Advanced features arrows to step through each record in turn. The record being inspected will then be indicated in the measurement type title line; e.g. Record 39. Poor or unacceptable Measurement Good Measurements Measurements type (green circle) (Amber triangle) Sizing Molecular weight...

• Page 175: Creating Custom Reports

  These can display parameters which are not on the default reports. Note The Malvern Instruments default reports described in Chapter 5 will be sufficient for most users. These are identified with (M) appended to the name - e.g. Intensity PSD (M))

• Page 176: Overview

  Chapter 11 Creating Custom Reports Overview This section introduces the Report Designer. Views Two views must be created due to the different aspect ratios of the printed page and computer screen: Screen Layout – shows the computer screen version. Page Layout – shows the printed version. This tailors the same contents to the dimensions of the printed page.

• Page 177: Opening An Existing Report

  Creating Custom Reports Chapter 11 Getting started To open the Report Designer select Tools-Report Designer. The Report Designer opens in a separate window as shown below. In this example the small Control palette has been dragged from the toolbar to make it easier to use.

• Page 178: Adding Elements To The Report

  Chapter 11 Creating Custom Reports Adding elements to the report This section shows how to add elements to a report, select any of these elements and move or align them on the screen. Add all the required elements to the report then move them to the correct positions before going on to the next stage.

• Page 179: Setting Up The Report Elements

  Creating Custom Reports Chapter 11 When the mouse button is released, the marquee receives the selected element. This will be marked initially by a hatched border like those shown below. This shows that the element is currently selected: Use the cursor to drag the selected element or the black resize handles around its border to resize the element if necessary.
• Page 180 Chapter 11 Creating Custom Reports Picture The Picture tool is generally used for company logos, etc. All the main graphics file formats are supported. With the element selected, use its Properties dialogue to browse for the picture to insert. Use the Keep Aspect ratio check box if required to maintain the aspect ratio of the original image.
• Page 181 Creating Custom Reports Chapter 11 Parameters with arguments Certain parameters allow specific values (or arguments) to be added. A Dv(90) percentile can be viewed by double-clicking the cursor on Measurement-Size-D(v) and then typing 0.90 as the Argument value. Parameter titles can be altered by double-clicking on them e.g. double-click on D(v) and alter to Dv(90) to reflect the particular percentile size viewed.
• Page 182 Chapter 11 Creating Custom Reports Calculation Editor Use the Calculation Editor to load an existing calculation for editing or to create a new calculation. The language used, Sax Basic, is similar to Microsoft Visual Basic for Applications (VBA). Select the browse icon to choose functions from the library lists, which can be used to build up calculations.
• Page 183 Creating Custom Reports Chapter 11 Graphs A number of data graphs can be added to a report using the Report Designer con- trol palette. Select the graph icon and then choose one of the following graphs from the drop- down menus. The menus are split into Trend, Titration, Size and Molecular weight with accompanying graph choices.
• Page 184 Chapter 11 Creating Custom Reports function that is back calculated from the size distribution displayed. The closeness of the fit is an indicator of the success of the algorithm used and the quality of the measurement. Cumulants fit Plots the difference between the correlation data and the polynomial that is the best fit to that data.
• Page 185 Creating Custom Reports Chapter 11 Trend graph properties For a Trend graph the Properties dialogue has a Properties tab for selecting the parameters to view: The Trend plots selection area is used for selecting the Y axes parameters. The Y1 parameter column is used to select the axis on the left of the graph and Y2 to select the axis on the right.
• Page 186 Chapter 11 Creating Custom Reports Trend tables Trend tables allow the measurement data from multiple records to be compared to investigate any trends in the information. Any two numerical parameters can be compared. Size tables The following size tables can be inserted. Result This shows the size result of the measurement.

• Page 187: Selecting Elements

  Creating Custom Reports Chapter 11 Any available parameter can be selected and inserted into the report. The parameter value displayed is taken from the selected measurement record. Selecting elements When selecting elements: To select one element, click on it. To select multiple elements, hold down the Shift or Ctrl key and click the mouse button on each element in turn.

• Page 188: Aligning And Sizing Elements

  Chapter 11 Creating Custom Reports Aligning and sizing elements Select two or more elements and use the following options of the Layout toolbar or menu to align/size them: Button Function Button Function Layout-Align-Align left Layout-Make Same Width Layout-Align-Align right Layout-Make Same Height Layout-Align-Align top Layout-Make Same Size Layout-Align-Align bottom...

• Page 189: A Finished Report

  Remember if the custom report is to be printed then a Page version must also be created. Both screen and page versions of each report will be saved in the My DocumentsMalvern InstrumentsZetasizerReports... directory. An easy way to create a screen report from a page report (or vice versa) is to use Edit-Select all from the menu, then copy and paste the complete page from the printed layout and directly paste into the screen version;...

• Page 190: Other Information Shown On A Report

  The printed copy of a report shows the software version number and instrument serial number at the bottom of the page. This is important information if a call is made to the Malvern Instruments help desk. The name of the measurement file the report is based on is also shown.

• Page 191: Protein Utilities

  Protein Utilities Introduction One of the basic functions of the Zetasizer μV is its ability to perform accurate measurements of a sample’s Molecular Weight. By measuring the sample scattering intensity over a range of concentrations and enter the necessary sample parameters, the Molecular weight can be determined.

• Page 192: Debye Plot

  Chapter 12 Protein Utilities Concentration Utilities. This dialogue contains features to establish the concentration and scattering levels that may be observed. Scattering Functions. A plot can be generated from entering the measurement data. In each case the effect of changing any of the input parameters can be seen instantly in all of the derived parameters.
• Page 193 Protein Utilities Chapter 12 Any of the parameters in the dialogue box can be changed; the other parameters will be instantly recalculated. This can be used to investigate the sensitivity of the result to changes in any parameter. For example, by first entering the concentration points from an existing meas- urement, a sample parameter - e.g.
• Page 194 Chapter 12 Protein Utilities To modify a concentration, select it from the list and press the Modify... but- ton. The Input values dialogue will appear, allowing the parameters to be changed. A concentration can be deleted by selecting the concentration from the list and pressing the Delete...
• Page 195 Protein Utilities Chapter 12 The parameters are described below. Data This indicates the data entries that have been entered into the debye plot table, see below. Sample Sample dn/dc (ml/g) This is the differential refractive index increment; the change in refractive index as a function of the change in concentration.
• Page 196 Chapter 12 Protein Utilities Toluene count rate (Kcounts) The scattering count rate of the toluene reference. Saving the Debye plot The parameters and data inputted to produce the plot can be saved by pressing the Save button, and then reviewed at a later stage by pressing the Load button. Copying the Debye plot The graph can be pasted into another application (such as Microsoft Word or Excel) by selecting the Copy button.

• Page 197: Molecular Weight Calculations

  Protein Utilities Chapter 12 Molecular weight calculations The hydrodynamic size measured by Dynamic Light Scattering (DLS) is defined as “the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured”. In practice though, macromolecules in solution are non-spherical, dynamic (tumbling), and solvated.
• Page 198 Chapter 12 Protein Utilities The Stokes-Einstein equation was developed using the assumption of hypothetical hard spheres. Perrin factor For non-spherical particles, the Perrin or shape factor (F) can be used to estimate particle shape. The Perrin factor is used to calculate the prolate and oblate axial ratios for ellipsoids with the same Perrin factor value.
• Page 199 Protein Utilities Chapter 12 If required, a solvent layer can be subtracted from the hydrodynamic radius when calculating the Perrin factor. To do this select the Subtract solvent layer check box. Molecular weight estimate Whilst the preferred method of measuring absolute Molecular weight is by performing concentration dependent light scattering measurements, this can be very time...

• Page 200: Concentration Utilities

  Chapter 12 Protein Utilities Concentration utilities Select the Concentration utilities tab to view concentration and scattering parameters. Concentration and Scattering This area of the dialogue contains features to establish the concentration and scattering levels that may be observed from the sample. Enter the values from the measurement into the table.

• Page 201: Scattering Functions

  Protein Utilities Chapter 12 Scattering functions A scattering function plot can be generated by inputting the measurement data in the list on the right, in this dialogue: Enter the values from the measurement into the table and press Plot - the graph will update to show the values entered.
• Page 202 Chapter 12 Protein Utilities Page 12-12 MAN 0428...

• Page 203: Size Theory

  Size theory Introduction The aim of this chapter is to describe the basic Size principles behind the Zetasizer μV. This will help in understanding the meaning of the results achieved. The chapter is divided into two major sections. What is Dynamic light scattering? and Operation of the Zetasizer μV - Size measurements.
• Page 204 Chapter 13 Size theory The speckle pattern will consist of areas of bright light and dark areas where no light is detected. What causes these bright and dark areas? The diagram below shows the propagated waves from the light scattered by the particles. The bright areas of light are where the light scattered by the particles arrives at the screen with the same phase and interferes constructively to form a bright patch.
• Page 205 Size theory Chapter 13 Interpreting scattering intensity fluctuation data We know that the Zetasizer measures the fluctuation in scattering intensity and uses this to calculate the size of particles within the sample - but how does it do this? Within the instrument is a component called a digital correlator. A correlator basically measures the degree of similarity between two signals over a period of time.
• Page 206 Chapter 13 Size theory Using the correlation function How does the correlation function relate to the Particle Size? We mentioned earlier that the speed of particles that are being moved by Brownian motion is related to the size of the particles (Stokes-Einstein equation). Large particles move slowly, while smaller particles move quickly.
• Page 207 Size theory Chapter 13 The X axis shows a distribution of size classes, while the Y axis shows the relative intensity of the scattered light. This is therefore known as an intensity distribution. Although the fundamental size distribution generated by DLS is an intensity distribution, this can be converted, using Mie theory, to a volume distribution.
• Page 208 Chapter 13 Size theory Operation of the Zetasizer μV - Size measurements A typical DLS system comprises of six main components. First of all a laser used to provide a light source to illuminate the sample particles within a cell or measurement area .
• Page 209 Size theory Chapter 13 become overloaded. To overcome this an “attenuator” is used to reduce the intensity of the laser and hence reduce the intensity of the scattering. For samples that do not scatter much light, such as very small particles or sam- ples of low concentration, the amount of scattered light must be increased.
• Page 210 Chapter 13 Size theory Page 13-8 MAN 0428...

• Page 211: Molecular Weight Theory

  Molecular Weight theory Introduction The aim of this chapter is to describe the basic Molecular weight principles behind the Zetasizer μV. This will help in understanding the meaning of the results achieved. The chapter is divided into two major sections. What is Static light scattering? and The Debye plot.
• Page 212 Chapter 14 Molecular Weight theory For samples where A >0, the particle ‘likes’ the solvent more than itself, and will tend to stay as a stable solution. When A <0, the particle ‘likes’ itself more than the solvent, and therefore may aggregate.
• Page 213 Molecular Weight theory Chapter 14 The standard approach for Molecular weight measurements is to first measure the scattering intensity of the analyte used relative to that of a well described ‘standard’ pure liquid with a known Rayleigh ratio. A common standard used in Static light scattering is Toluene, for the simple reason that the Rayleigh ratios of toluene are suitably high for precise measurements, are known over a range of wavelengths and temperatures and, maybe more importantly, Toluene is relatively easy to obtain.

• Page 214: The Debye Plot

  Chapter 14 Molecular Weight theory This phenomenon is known as Mie scattering, and it occurs when the particle size is of the same order as the wavelength. However when the particles in solution are much smaller than the wavelength of the incident light, multiple photon scattering will be avoided.
• Page 215 Molecular Weight theory Chapter 14 The weight-averaged Molecular weight (Mw) is determined from the intercept at zero concentration i.e. KC/R = 1/Mw (for c--> 0) where the Mw is expressed in θ Daltons (or g/mol). The 2 Virial Coefficient (A ) is determined from the gradient of the Debye plot.
• Page 216 Chapter 14 Molecular Weight theory Page 14-6 MAN 0428...

• Page 217: Part 3 - Appendices

  Part 3 - Appendices...

• Page 219: Specification Specification

  Specification Specification Parameter Specification Size range* 0.3nm to 0.5 microns Molecular weight range* 380Da to > 20MDa Minimum sample volume 2μL Sensitivity at minimum volume 0.1mg/ml lysozyme High concentration Yes - optically clear samples SEC detector mode 8 μl flow cell Laser 50mW, 830nm Temperature range...

• Page 220: Chemical Compatibility

  Appendix A Specification Recommended computer Contact the Malvern Helpdesk or website specification for the recommended computer specifica- tion, otherwise consult the Software Update Notification document supplied on the software CD. * sample dependent Chemical compatibility Components of the Zetasizer μV that may come into contact with the sample are manufactured from materials that are considered to give the widest protection from chemical attack.
• Page 221 Specification Appendix A Cells and Cuvettes Refer to the descriptions in Chapter 4 for the materials used for each of the availa- ble cells. Zetasizer μV Page A-3...
• Page 222 Appendix A Specification Page A-4 MAN 0428...

• Page 223: Unpacking Instructions

  Unpacking instructions Appendix B of the Zetasizer μV series User Manual MAN0425 Issue 3.1 Déballez l’instrument Auspacken des Gerates Desempaquetado del aparato Desempacotamento do instrumento Zetasizer μV Page B-1...
• Page 224 Appendix B Unpacking instructions If there is any sign of damage contact the freight carrier immediately. Si vous constatez des signes de dommages quelconques, contactez immédiatement le transporteur. Bei irgendwelchen Zeichen von Transportschäden den Spediteur sofort benachrichtigen. Si se advierte algún signo de deterioro, contactar inmediatamente con el transportista.
• Page 225 Unpacking instructions Appendix B Open the wings of the carton and remove the top foam insert. Ouvrez les rabats du carton et retirez les blocs de mousse du haut. Die Klappen des Kartons öffnen und die oberen Schaumeinlagen entfernen. Abrir los costados de la caja y extraer las piezas intercaladas de espuma de la parte superior.
• Page 226 Appendix B Unpacking instructions Never lift the instrument by its covers. Pour soulever l'instrument, ne le tenez jamais par ses couvercles. Niemals das Gerät an den Abdeckungen anheben. No levantar nunca el aparato por sus cubiertas. Nunca levante o instrumento pegando pelas suas tampas.

• Page 227: Installation

  Installation Introduction It should be noted that the Zetasizer μV system should initially be commissioned by Malvern trained personnel. This appendix will show how to re-install the system, typically required under the following circumstances: Moving the instrument. Should it become necessary to move the system from one laboratory to another, read the information in this appendix to correctly reconnect the system.

• Page 228: Installing The Zetasizer μv

  Appendix C Installation Installing the Zetasizer μV Installing the Zetasizer μV is very simple. To install the system: ill 7905 Connect the USB cable to the connector marked USB on the rear panel of the Zetasizer μV instrument. Connect the other end of the cable to the USB connection on the computer.
• Page 229 Installation Appendix C Note If the software is subsequently updated with a new version, any custom reports, parameter settings, SOPs, etc will be preserved. Zetasizer μV Page C-3...
• Page 230 Appendix C Installation Page C-4 MAN 0428...

• Page 231: Regulatory Statements

  For more detailed information about recycling of this product, please contact your local city office, your waste disposal service, or your Malvern representative. Zetasizer μV Page D-1...

• Page 232: Fcc Notice (us Only

  Note Changes or modifications not expressly approved by Malvern Instruments Limited could void the user’s authority to operate the equipment. Page D-2 MAN 0428...

• Page 233: Canadian Regulatory Information

  Canadian Department of Communications. Note that Canadian Department of Communications (DOC) regulations provide, that changes or modifications not expressly approved by Malvern Instruments Limited could void your authority to operate this equipment. This Class A digital apparatus complies with Canadian ICES-003.
• Page 234 Appendix D Regulatory Statements Page D-4 MAN 0428...
• Page 235 Index Attenuator index Symbols Audit trails .dass 9-21 Automatic duration 9-29 .del Avogadro’s constant 14-2 .pag 8-2, 11-2 .sop 9-38 .sopl 3-19 Baseline pointer 10-19 Batch print Numerics 3-12 Batch printing 21 CFR Part 11 7-1, 10-11 Branched polysaccharides 12-9 21 CFR Part 11 icon 3-22 Brownian motion...
• Page 236 Index Zetasizer μV Correlation function Editing a group (security) 13-4 Molecular weight Editing a result 4-18 10-11 Size Editing a user (security) 4-17 Correlator Editing the result 13-7 10-10 Count rate Electrode voltage interlock Molecular weight Engineering 4-18 3-16 Size Equilibration time 4-16 9-16...
• Page 237 Hydrodynamic diameter Macros (Options dialogue) 12-1, 12-7 10-22 Estimate Mains power input 12-9 Hydrodynamic radius Making a measurement 12-5 3-2, 4-2 Malvern logo 3-22 Malvern personnel Icon Malvern Website 21 CFR Part 11 Manual measurement 3-22 3-2, 4-1, 4-13 μV Mark-Houwink...
• Page 238 Index Zetasizer μV Measurement file window Navigation toolbar 3-19, 8-1 3-19 Measurement position New SOP 5-7, 9-20, 9-32 Measurement records Number distributions 3-20 13-5 Measurement sequence Number PSD 4-20 Molecular weight Molecular weight 4-20 4-18 Size measurements Size 4-20 4-17 Measurement tab 10-19 Measurement technique...
• Page 239 Zetasizer μV Index Sample - Standard (SOP) 9-26 Sample - Temperature (SOP) 9-16, 9-28 Quartz cuvettes 3-10 Sample (Size) 9-10 Sample (SOP) Sample concentration Rayleigh equation Sample name 14-2 9-10 Rayleigh ratio Sample preparation 9-27 4-3, 6-1, 6-2 Rayleigh scattering Sample viscosity options 14-3 9-15...
• Page 240 Index Zetasizer μV Solvent builder Sample - Material (Size) 9-13, 10-2 9-12 Solvents Manager Sample - Solvent (Molecular weight) 9-26 9-25 Sample - Standard 2-1, 3-2, 4-1, 9-1 Creating an... Sample - Standard (Molecular weight) 9-1, 9-8 9-26 Distributing an... Sample - Temperature 9-38 Existing...
• Page 241 2-1, 4-19 Exporting Trend measurements - Standard report 5-16 Importing Trend SOPs 9-35 Measurement files 3-20 Turning on Modifying www.Malvern.com Ultrasonics Units conversion 3-22, 5-3, 5-5 Z-average mean 10-17 Unpacking instructions Z-average Size USB cable Zetasizer model number User groups...
• Page 242 Index Zetasizer μV Page viii MAN 0428...
• Page 244 Printed in England MRK1132-01 Malvern Instruments Limited Enigma Business Park Grovewood Road, Malvern Worcs, WR14 1XZ, U.K. Tel: +44 (0) 1684 892456 Fax: +44 (0) 1684 892789...

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